Urinary circulating DNA detection for dynamic tracking of EGFR mutations for NSCLC patients treated with EGFR-TKIs.

PURPOSE: Changes in EGFR profiles of non small cell lung cancer (NSCLC) patients correlates to clinical outcome. Extracting quality tumor tissue remains a challenge for molecular profiling. Our study aims to ascertain the clinical relevance of urinary cell free DNA as an alternative tumor material source. METHODS: 150 patients with activating EGFR mutation and received EGFR-TKIs were recruited to participate in the serial monitoring study. Matched primary tumor samples were taken together with blood and urine specimens before the initiation of TKIs. The EGFR mutation testing was performed and quantified using ddPCR. For serial time point measurements, urine and blood samples were extracted at 1-month intervals for duration of 9 months. RESULTS: Urinary ctDNA yielded a close agreement of 88 % on EGFR mutation status when compared to primary tissue at baseline. Almost all samples detected via urine specimens were uncovered in plasma samples. Analysis of urinary cell free DNA at different time points showed a strong correlation to treatment efficacy. Interestingly, a secondary EGFR mutation T790M was detected for 53 % of the patients during monitoring. The results were corroborated with the plasma ctDNA analysis. The T790M+ group had a reduced median survival when compared to the wildtype group. CONCLUSION: Urinary cell free DNA may be a potential alternative to conventional primary tissue based EGFR mutation testing. Our findings showed that the assay sensitivity was comparable to results from blood plasma. Urinary samples being noninvasive and readily available have clinical utility for monitoring of EGFR TKI treatment.

A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma.

INTRODUCTION: In approximately 60% of patients with NSCLC who are receiving EGFR tyrosine kinase inhibitors, resistance develops through the acquisition of EGFR T790M mutation. We aimed to demonstrate that a highly sensitive and quantitative next-generation sequencing analysis of EGFR mutations from urine and plasma specimens is feasible. METHODS: Short footprint mutation enrichment next-generation sequencing assays were used to interrogate EGFR activating mutations and the T790M resistance mutation in urine or plasma specimens from patients enrolled in TIGER-X (NCT01526928), a phase 1/2 clinical study of rociletinib in previously treated patients with EGFR mutant-positive advanced NSCLC. RESULTS: Of 63 patients, 60 had evaluable tissue specimens. When the tissue result was used as a reference, the sensitivity of EGFR mutation detection in urine was 72% (34 of 47 specimens) for T790M, 75% (12 of 16) for L858R, and 67% (28 of 42) for exon 19 deletions. With specimens that met a recommended volume of 90 to 100 mL, the sensitivity was 93% (13 of 14 specimens) for T790M, 80% (four of five) for L858R, and 83% (10 of 12) for exon 19 deletions. A comparable sensitivity of EGFR mutation detection was observed in plasma: 93% (38 of 41 specimens) for T790M, 100% (17 of 17) for L858R, and 87% (34 of 39) for exon 19 deletions. Together, urine and plasma testing identified 12 additional T790M-positive cases that were either undetectable or inadequate by tissue test. In nine patients monitored while receiving treatment with rociletinib, a rapid decrease in urine T790M levels was observed by day 21. CONCLUSIONS: DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy" samples.

Urinary peptidomics in a rodent model of diabetic nephropathy highlights epidermal growth factor as a biomarker for renal deterioration in patients with type 2 diabetes.

Many diabetic patients suffer from declining renal function without developing albuminuria. To identify alternative biomarkers for diabetic nephropathy (DN) we performed urinary peptidomic analysis in a rodent model in which hyperglycemia and hypertension synergize to promote renal pathologic changes consistent with human DN. We identified 297 increased and 15 decreased peptides in the urine of rats with DN compared with controls, including peptides derived from proteins associated with DN and novel candidate biomarkers. We confirmed by ELISA that one of the parent proteins, urinary epidermal growth factor (uEGF), was more than 2-fold reduced in rats with DN in comparison with controls. To assess the clinical utility of uEGF we examined renal outcomes in 642 participants from the Edinburgh Type 2 Diabetes Study who were normoalbuminuric and had preserved renal function at baseline. After adjustment for established renal risk factors, a lower uEGF to creatinine ratio was associated with new-onset estimated glomerular filtration rate less than 60 ml/min per 1.73m(2) (odds ratio 0.48; 95% confidence interval, 0.26-0.90), rapid (over 5% per annum) decline in renal function (odds ratio 0.44; 95% confidence interval, 0.27-0.72) or the composite of both outcomes (odds ratio 0.38; 95% confidence interval, 0.24-0.62). Thus, the utility of a low uEGF to creatinine ratio as a biomarker of progressive decline in renal function in normoalbuminuric patients should be assessed in additional populations.

Urinary microprotein concentrations in the long-term follow-up of dilating vesicoureteral reflux patients who underwent medical or surgical treatment.

PURPOSE: This study examined the relationship between urinary microprotein concentrations and renal functional parameters in children with dilating (grade III-V) vesicoureteral reflux (VUR) who underwent either medical or surgical treatment. METHODS: All 44 dilating VUR patients who were followed for 4 years were screened for inclusion in this study. The patients' clinical features and clinical outcomes, as well as the urinary activities of albumin (ALB), transferrin (TRF), immunoglobulin G (IgG), alpha-1-microglobulin (α1-MG), and N-acetyl-ß-glucosaminidase (NAG), were retrospectively analyzed. RESULTS: High values of NAG, α1-MG, IgG, TRF, and ALB were noted in 73.33, 58.33, 43.33, 24.14, and 53.33 % of patients, respectively, at the first examination. Cystatin C, eGFR, and urinary microprotein levels were associated with a good prognosis after 4 years of follow-up. No differences in recurrent UTI, cystatin C concentration, most microprotein/creatinine (Cr) ratios, eGFR, or ΔGFR4 % were found between the groups. High levels of urinary proteins were found in 2.38-9.52 % of cases after 4 years of follow-up. ALB/Cr, IgG/Cr, and α1-MG/Cr levels were positively correlated with 99mTc-dimercaptosuccinic acid (DMSA) grade, and α1-MG excretion was inversely correlated with eGFR. CONCLUSIONS: The levels of microprotein were elevated at diagnosis in a higher proportion of patients than for the other markers examined. At long-term follow-up, the reflux level had decreased or completely resolved in all patients, and the proportions of microproteins that were elevated were significantly reduced. Renal impairment measured by eGFR and DMSA grade was related to increased urinary α1-MG levels.

Protein shedding in urothelial bladder cancer: prognostic implications of soluble urinary EGFR and EpCAM.

BACKGROUND: Better biomarkers must be found to develop clinically useful urine tests for bladder cancer. Proteomics can be used to identify the proteins released by cancer cell lines and generate candidate markers for developing such tests. METHODS: We used shotgun proteomics to identify proteins released into culture media by eight bladder cancer cell lines. These data were compared with protein expression data from the Human Protein Atlas. Epidermal growth factor receptor (EGFR) was identified as a candidate biomarker and measured by ELISA in urine from 60 noncancer control subjects and from 436 patients with bladder cancer and long-term clinical follow-up. RESULTS: Bladder cancer cell lines shed soluble EGFR ectodomain. Soluble EGFR is also detectable in urine and is highly elevated in some patients with high-grade bladder cancer. Urinary EGFR is an independent indicator of poor bladder cancer-specific survival with a hazard ratio of 2.89 (95% CI 1.81-4.62, P<0.001). In multivariable models including both urinary EGFR and EpCAM, both biomarkers are predictive of bladder cancer-specific survival and have prognostic value over and above that provided by standard clinical observations. CONCLUSIONS: Measuring urinary EGFR and EpCAM may represent a simple and useful approach for fast-tracking the investigation and treatment of patients with the most aggressive bladder cancers.

THE SIGNIFICANCE OF EPIDERMAL GROWTH FACTOR RECEPTOR AND SURVIVIN EXPRESSION IN BLADDER CANCER TISSUE AND URINE CYTOLOGY OF PATIENTS WITH TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER.

OBJECTIVE: To assess whether epidermal growth factor receptor (EGFR) and survivin immunostaining of tumour cells in urinary cytology and tissue of patients with bladder cancer has a prognostic significance. DESIGN: Prospective study SETTING: Department of Surgery (Division of Urology), Mubarak Al-Kabeer Teaching Hospital and Faculty of Medicine, Kuwait University, Kuwait SUBJECTS: Urine cytology smears obtainedpriorto cystoscopy in patients with transitional cell carcinoma (TCC) of the bladder were immunostained for EGFR and survivin. Bladder cancer tissue resected at surgery was also immunostained for EGFR and survivin expression. Tissue expression of EGFR and survivin in TCC of the bladder was compared to their expression in urine cytology and relationship to tumour grade and stage. RESULTS: 178 patients were studied (43 newly diagnosed bladder cancer, 58 with recurrent TCC and 77 in disease remission). Twenty five patients with normal urothelium served as controls. The mean sensitivity of urine cytology, tissue survivin immunohistochemistry (IHC) and tissue EGFR IHC was 30.5%, 62% and 59% respectively. The corresponding mean specificity was 95%, 79% and 38% respectively. For grades 1, 2 and 3 bladder tumors, tissue expression positivity for EGFR was 47.8%, 92.9%, 100% and for tissue survivin it was 27.8%, 18.2% and 33.3% respectively. For grades 1, 2 and 3 bladder tumors, urine expression positivity for EGFR was 35.7%, 40% and 67.7% and for urine survivin it was 8.3%, 42.9% and 33.3% respectively. CONCLUSION: Positive EGFR immunostaining of urine cytology specimen or tumour tissue increases with histological grade of TCC of the bladder. Survivin expression is less consistent in both urine cytology specimen and tissue samples. EGFR immunostaining may provide a useful tool in the grading of bladder TCC and aid in the selection of patients that may benefit from administration of EGFR inhibitors.

EGF receptor-binding activity in the urine of normal horses and horses affected by chronic laminitis.

A heterologous radioreceptor binding assay (RRA) has been developed capable of detecting nanogram amounts of epidermal growth factor (EGF) receptor-binding activity in equine urine. The binding parameters of [125I]mEGF (murine EGF) to EGF receptors on equine plasma membranes are in good agreement with values from other EGF-RRA systems. The dissociation constant estimated from equilibrium methods (KD = 4 X 10(-10) M) is in reasonable agreement with that determined from the rate constants (KD = 6 X 10(-10) M) and is in good agreement with values determined in other species. The assay is specific for equine EGF (eEGF) receptor-binding activity and capable of detecting less than 0.34 nM eEGF receptor-binding activity in urine. Equine EGF receptor-binding activity in equine urine form adult horses varied widely between samples (8.5 +/- 6.5 nM). This variability was somewhat reduced when values were adjusted for dilutional effects using urine creatinine as an indicator (3.6 +/- 2.0 nanomoles/g creatinine). No significant differences were demonstrated between the means of EGF binding activity concentrations in clinically normal horses and horses affected by chronic laminitis.