Non-invasive detection of bladder cancer through the analysis of driver gene mutations and aneuploidy

Current non-invasive approaches for bladder cancer (BC) detection are suboptimal. We report the development of non-invasive molecular test for BC using DNA recovered from cells shed into urine. This “UroSEEK” test incorporates assays for mutations in 11 genes and copy number changes on 39 chromosome arms. We first evaluated 570 urine samples from patients at risk for BC (microscopic hematuria or dysuria). UroSEEK was positive in 83% of patients that developed BC, but in only 7% of patients who did not develop BC. Combined with cytology, 95% of patients that developed BC were positive. We then evaluated 322 urine samples from patients soon after their BCs had been surgically resected. UroSEEK detected abnormalities in 66% of the urine samples from these patients, sometimes up to 4 years prior to clinical evidence of residual neoplasia, while cytology was positive in only 25% of such urine samples. The advantages of UroSEEK over cytology were particularly evident in low-grade tumors, wherein cytology detected none while UroSEEK detected 67% of 49 cases. These results establish the foundation for a new, non-invasive approach to the detection of BC in patients at risk for initial or recurrent disease.


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Much is now known about the genetic pathogenesis of BC. High rates of activating Carcinoma, non-invasive High Grade Papillary Urothelial Carcinoma and "flat" 96 Carcinoma in Situ (CIS), as well as in urinary cells from a subset of these patients [14]. 97 TERT promoter mutations have thus been established as the most common genetic 98 alteration in BC [14,30]. Other important oncogene-activating mutations include those 99 in FGFR3, RAS and PIK3CA, which have been shown to occur in a high fraction of non-100 muscle invasive bladder cancers [31,32]. In muscle-invasive bladder cancers, 101 mutations in TP53, CDKN2A, MLL2 and ERBB2 are also frequently found [33-40] 102 The current study assesses the performance of a massively parallel sequencing-based 103 assay, termed UroSEEK, for the detection of BC through the analysis of urinary cells.

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UroSEEK has three components: detection of intragenic mutations in regions of ten

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A schematic of the approach used in this study is provided in Figure 1A, and a flow 126 diagram indicating the number of patients evaluated in this study and the major results 127 is provided in Figure 1B.   145  146  147  148  149  150  151  152  153  154  155  156  157  158  159  160  161  162  163  164  165  166  167  168  169  170  171  172  173  174  175        on the basis for "false negatives", i.e., the reason that 21% of urine samples from 274 patients who developed BC had no detectable mutations in the 11 genes tested. The 275 reason could either have been that the corresponding BC did not harbor a mutation in 276 these 11 genes or that it did, but the fraction of neoplastic cells in the urine sample was 277 not high enough to allow its detection with the assays we used. We could identify a 278 mutation in at least one of the 11 genes in 62% of the primary tumors from patients with 279 false negative urine tests for mutations (Supplementary File 5 and 6). We conclude 280 that 38% of the 29 false negative tests for mutations were due to the fact that none of 281 the queried mutations were present in the tumor and that the other 62% of the false 282 negatives were due to insufficient amounts of cancer cells in the urine.

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UroSEEK: biomarkers in combination. As noted above, the ten-gene multiplex assay, 285 the TERT singleplex assay, and the aneuploidy assays yielded 68%, 57%, and 46% 286 sensitivities, respectively, when used separately (Supplementary Table 1 Table 4). Thus, when the three assays were used together (test termed "UroSEEK"), 294 and a positive result in either assay was sufficient to score a sample as positive, the 295 sensitivity rose to 83% (95% CI 76% to 88 %). Only one of the 188 samples from   cancer patients whose urines were positive by cytology and in 95% of the 23 cancer 338 patients whose urines were negative by cytology. Thus, in combination, UroSEEK plus 339 cytology afforded 95% (95% CI 83% to 99%) sensitivity, a 12% increase over UroSEEK

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Markers in combination. As noted above, the ten-gene multiplex assay, the TERT 395 singleplex assay, and the aneuploidy assays yielded 49%, 51%, and 30% sensitivities, 396 respectively, when used separately (Supplementary Table 2 423 The advantage of UroSEEK over cytology was particularly evident in low-grade tumors 424 (Papillary urothelial neoplasms of low malignant potential and non-invasive low grade 425 papillary urothelial carcinomas). There were a total of 49 low-grade tumors evaluated in 426 this study in whom cytology was available (six from the Early Detection cohort and 43 427 from the Surveillance cohort). None of these low-grade tumors were detected by 428 cytology (0% sensitivity; 95% CI 0.0% to 6.7%) In contrast, UroSEEK detected 67% 429 (95% CI 51% to 81%) of the low-grade tumors (identical rate of 67% in both cohorts;  Our purpose for developing UroSEEK was not to replace cytology but rather to augment 457 it. Cytology is a non-invasive test that is highly specific, and in expert hands nearly Although UroSEEK is more sensitive than cytology, it is less specific. In this study, we 480 were able to judge specificity in several independent ways. The first, and in some ways, identified in the urine was also present in the primary tumor, this was not true in 22% of 500 the cases in the Early Detection Cohort. In these cases, UroSEEK could be detecting 501 clonal proliferations in the bladder epithelium that did not progress to cancer, and such 502 proliferations may be more common in patients with BC than in the general population 503 [50, 51]. Because only one biopsy from the primary tumor was available for comparison, 504 it is also possible that intra-tumoral heterogeneity explains part of the discrepancies.  Our study lays the conceptual and practical framework for a novel test that could inform which is comparable to that of cystoscopy, but UroSEEK is non-invasive. for DNA purification, as previously described [14]. Electronic medical records were 574 reviewed to obtain medical history and follow up data in all patients.  primer was used to amplify a 73-bp segment containing the region of the TERT 587 promoter known to harbor mutations in BC [14]. The conditions used to amplify it were 588 the same as used in the multiplex reactions described above except that Phusion GC 589 Buffer (Thermo-Fisher) instead of HF buffer was used and 20 cycles were used for the 590 initial amplification. Note that the TERT promoter region could not be included in the 591 multiplex PCR because of the high GC content of the former. PCR products were 592 purified with AMPure XP beads (Beckman Coulter, PA, USA) and 0.25% of the purified 593 PCR products (multiplex) or 0.0125% of the PCR products (TERT singleplex) were then 594 amplified in a second round of PCR, as described in [57]. The PCR products from the 595 second round of amplification were then purified with AMPure and sequenced on an