Author's Accepted Manuscript “Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer” Ajjai Alva , Terence Friedlander , Melanie Clark , Tamara Huebner , Stephanie Daignault , Maha Hussain , Cheryl Lee , Khaled Hafez , Brent Hollenbeck , Alon Weizer , Gayatri Premasekharan , Tony Tran , Christine Fu , Cristian IonescuZanetti , Michael Schwartz , Andrea Fan , Pamela Paris

PII: DOI: Reference:

S0022-5347(15)03852-5 10.1016/j.juro.2015.02.2951 JURO 12553

To appear in: The Journal of Urology Accepted Date: 13 February 2015 Please cite this article as: Alva A, Friedlander T, Clark M, Huebner T, Daignault S, Hussain M, Lee C, Hafez K, Hollenbeck B, Weizer A, Premasekharan G, Tran T, Fu C, Ionescu-Zanetti C, Schwartz M, Fan A, Paris P, “Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer”, The Journal of Urology® (2015), doi: 10.1016/j.juro.2015.02.2951. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

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ACCEPTED MANUSCRIPT TITLE PAGE Manuscript submission to the Journal of Urology Article type: Research Article (New Technology and Techniques) Title: “Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer ” Running head: “Circulating tumor cells as bladder cancer biomarkers”

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Authors: Ajjai Alva1, Terence Friedlander2, Melanie Clark1, Tamara Huebner1, Stephanie Daignault1, Maha Hussain1, Cheryl Lee3, Khaled Hafez3, Brent Hollenbeck3, Alon Weizer3, Gayatri Premasekharan5, Tony Tran4, Christine Fu4, Cristian IonescuZanetti4, Michael Schwartz4, Andrea Fan4, Pamela Paris5 1

Hematology and Oncology, University of Michigan Comprehensive Cancer Center, MI Division of Hematology & Medical Oncology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 3 Department of Urology, University of Michigan, MI 4 Fluxion Biosciences, San Francisco, CA 5 Department of Urology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA

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Email: [email protected]

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Corresponding author: Ajjai Alva, MD MS Hematology/Oncology Department of Internal Medicine 7316 Cancer Center University of Michigan Ann Arbor, Michigan USA Phone: 734-936-0091 Fax: 734-615-2719

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MeSH Headings (Keywords): Urinary Bladder Neoplasms; Urinary Bladder; Neoplastic Cells, Circulating; Neoadjuvant Therapy; Pathology, Molecular

ACCEPTED MANUSCRIPT ABSTRACT Purpose: To explore the diagnostic use of circulating tumor cells (CTCs) in neoadjuvant bladder cancer patients using both enumeration and next-generation sequencing (NGS).

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Materials and Methods: Twenty bladder cancer patients eligible for cisplatin-based neoadjuvant chemotherapy were enrolled in an IRB-approved study. Subjects had blood draws at baseline and following 1 cycle of chemotherapy. Eleven metastatic bladder cancer patients and thirteen healthy donors were analyzed for comparison. Samples were enriched for CTCs using a novel microfluidic collection device (IsoFlux System). CTC counts were analyzed for repeatability and compared with the FDA-cleared CellSearch CTC test. CTCs were also analyzed for mutational status using NGS.

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Results: Median CTC counts were 13 (Neoadjuvant - baseline), 5 (Neoadjuvant - follow-up), 29 (Metastatic), and 2 (Healthy). Concordance of CTC level (Low30) across replicate tubes was 100% (n=15). In matched samples, the IsoFlux test showed 4/9 (44%) samples having 10 or more CTCs, while CellSearch showed 0/9 (0%). At time of cystectomy (4 months post-baseline), 3/3 patients (100%) with Medium/High CTC levels at both baseline and follow-up had unfavorable pStage (T1-T4 or N+). NGS analysis showed somatic variant detection in 4/8 patients using a targeted cancer panel; 8/8 (100%) of these were CTC Medium/High with a CTC fraction above 5% purity.

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Conclusions: This study demonstrates a potential role for CTC assays in management of bladder cancer patients. The IsoFlux method of CTC detection exhibits increased sensitivity compared with CellSearch. An NGS assay is presented with sufficient sensitivity to detect genomic alterations in CTCs.

ACCEPTED MANUSCRIPT Introduction Bladder cancer accounts for 15,500 deaths annually1. In the absence of metastases, radical cystectomy offers the possibility of cure but with limited success2. Pathologic stage, lymphovascular invasion, and histologic grade are prognostic factors for survival. Pathologic stage at cystectomy (pStage) associates with excellent 5-year disease-free survival (85%-90%)2,3,4.

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Perioperative chemotherapy has been investigated to improve survival in bladder cancer. Adjuvant cisplatin-based chemotherapy is only applicable to 20% of patients due to high incidence of renal insufficiency and perioperative complications. Patients generally have better performance status and organ function before cystectomy, providing the rationale for neoadjuvant therapy. Neoadjuvant chemotherapy prior to cystectomy results in both pathologic downstaging at surgery (38% frequency of pT0 compared to 15% in control patients not receiving neoadjuvant chemotherapy in a randomized phase III trial3) and an overall survival benefit2,5-7. Unfortunately, cisplatin-based chemotherapy is only feasible in 50% of bladder cancer patients due to inadequate renal, cardiac and neurologic function8. Cisplatin also carries a risk of kidney damage and neuropathy. Overall survival with cisplatin-based neoadjuvant chemotherapy is only 5% at 5 years. The critical unmet need in the management of muscle-invasive bladder cancer is the identification of minimally invasive biomarkers to stratify patients that would benefit from neoadjuvant chemotherapy while sparing others from needless toxicity9.

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Circulating tumor cells (CTCs) are detectable in most epithelial cancers and may enable early assessment to neoadjuvant chemotherapy in bladder cancer10,11,12, 13. CTC counts predict progression-free and overall survival in metastatic breast cancer14 and overall survival in colon cancer and castration-resistant prostate cancer15,16. CTCs were shown to be predictive biomarkers in castration-resistant prostate cancer patients treated with enzalutamide17,18. The CellSearch CTC Test (Janssen Diagnostics) detects CTCs in both non-metastatic and metastatic bladder cancer19-22. CellSearch has low sensitivity in localized bladder cancer, with only 17%-23% of patients having CTCs before cystectomy19,22. Nevertheless, the presence of CTCs pre-operatively was an independent adverse prognostic factor for cancer-free survival in patients undergoing cystectomy. One recent study showed CTCs remained an independent predictor of cancer-specific mortality in patients undergoing cystectomy without chemotherapy23.

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CellSearch’s low sensitivity limits its utility in bladder cancer. The IsoFlux System (Fluxion Biosciences) has previously been shown to increase sensitivity for CTC detection and molecular profiling in prostate and colorectal cancer24. This platform utilizes immunomagnetic isolation of CTCs in a microfluidic environment to enhance CTC capture and minimize white blood cell carry-over. The magnetic beads can be functionalized with multiple antibodies, although EpCAM alone was utilized for this study to directly compare with CellSearch. The resulting CTC samples are suitable for both enumeration and next-generation sequencing (NGS). This study explores the potential role of CTCs in identifying patients that might be better served to avoid additional chemotherapy by moving directly to cystectomy.

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Materials and Methods

Study aims – 1) explore use of CTCs as a bladder cancer biomarker, 2) compare assay performance of the IsoFlux System to CellSearch, 3) test repeatability of the IsoFlux assay, and 4) establish feasibility of using CTCs for NGS (Figure 1). Patient selection – Two prospective pilot studies totaling 30 bladder cancer patients were IRB-approved at the University of Michigan and the University of California, San Francisco. One cohort of 20 patients had locally-advanced resectable bladder cancer. These patients had blood collections before and during neoadjuvant chemotherapy. Eligible patients met these criteria: (1) histologic evidence of urothelial carcinoma invading the muscularis propria, (2) no radiologic evidence of metastases, (3) candidates for cisplatin-based chemotherapy, (4) candidates for curative-intent radical cystectomy. Two patients were lost to follow-up. Sixteen of these patients had pathological staging at time of cystectomy. A second cohort of 11 metastatic patients was used for comparison. Healthy donor samples (n=13) were collected as controls (AllCells, Alameda, CA). Sample collection – Neoadjuvant patient blood samples were collected at baseline and after the first cycle of chemotherapy. The second blood draw was taken at 3 weeks (cisplatin-gemcitabine) or 4 weeks (dose-dense MVAC

ACCEPTED MANUSCRIPT regimen, MVAC or classical cisplatin-gemcitabine) after baseline. In the metastatic and healthy groups, only one blood draw was taken. Samples were collected in 10mL EDTA tubes (Vacutainer, BD, Franklin Lakes, NJ) and shipped overnight to Fluxion for processing within 36 hours. Actual blood volumes were 5-10mL for IsoFlux enumeration tests. CellSearch CTC Test samples were collected in CellSave tubes and processed at the University of Michigan within 36 hours. All results were normalized to 7.5mL.

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CTC Enrichment and Enumeration –IsoFlux System samples were enriched using the IsoFlux CTC Enrichment Kits. CTCs were defined as morphologically intact, cytokeratin positive, CD45 negative, and nucleated (IsoFlux CTC Enumeration Kit). In a subset of samples (N=15), two tubes were collected at the same time point for repeatability testing. Samples sent for CellSearch enumeration followed the manufacturer’s instructions. This test relies on a similar definition for CTCs (CK+, CD45-, nucleated) but differs in exact reagents and equipment.

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NGS Analysis – A subset of samples (n=8) had a second blood tube drawn for NGS analysis using the IsoFlux NGS DNA Kit. This kit utilizes a column to further deplete leukocytes from the enriched CTCs, followed by Whole Genome Amplification based on the REPLI-g DNA Polymerase (Qiagen, Germany). The resulting gDNA went through target enrichment, library preparation, and sequencing using a targeted cancer panel (AmpliSeq CHPv2, Ion PGM, Thermo Fisher). NGS was done at the UCSF Genome Core and Thermo Fisher (South San Francisco). Final variants were filtered using IonReporter software and a specialized set of filters following the manufacturer’s instructions (IsoFlux NGS Kit) including percent mutant reads, mutant read counts, strand bias, homopolymer length, and exclusion of common germline mutations. Analytical validation of this filter set was performed using a model tumor cell line (MDA-MB-231, ATCC) with known mutations spiked into healthy whole blood. This cell line was chosen for its low EpCAM expression and mesenchymal-like properties to more closely approximate clinical samples. Statistical Analysis - CTC values and pathologic T/N Stage after cystectomy were analyzed in a 2X2 table. Sensitivity, specificity, positive predictive value, and negative predictive value are reported. Concordance is reported with the corresponding kappa statistic and 95% confidence interval.

Results

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Patient demographics Clinico-pathologic variables are presented for the three cohorts (Table 1): Neoadjuvant-N (n=21), Metastatic-M (n=11), and Healthy-H (n=13). Clinical staging in Neoadjuvnt group was 71% T2N0M0, 14% T3N0M0, 10% T4N0M0, and 5% T4N1M0. In the Metastatic group, 10% were chemo-naïve while the remaining 90% had all received one or more lines of chemotherapy, and 36% (4/11) had undergone prior cystectomy.

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CTC enumeration CTC counts are reported at baseline for all cohorts and following one cycle of chemotherapy (Neoadjuvant group only) (Figure 2a). For patients with multiple tubes tested at the same time point, CTC counts were reported as a mean of the two values. Median CTC values were 13 (Neoadjuvant – Baseline), 5 (Neoadjuvant – Follow up), 29 (Metastatic), and 2 (Healthy) (Figure 2b). There were low numbers of CTC-positive events in the Healthy group (median 2, range 0-8, 99% confidence interval of the mean [0.14,4.02]). To simplify the overall analysis, the CTC counts were grouped into Low (CTC 30) bins. In Neoadjuvant group, 60% of the baseline samples were CTC Medium or High. This fell to 33% after one cycle of chemotherapy. CTC counts are shown grouped by pStage at time of cystectomy (Figure 3) showing adverse pStage was associated with elevated CTCs (>10). The Metastatic cohort had 73% of samples at Medium or High, and the Healthy group had 0%. Assay reproducibility A subset of Neoadjuvant samples (n=15) had two blood tubes submitted at the same time point to assess reproducibility of the enumeration assay (Figure 4). When using the Low, Medium, High scheme, the concordance between Tube A and Tube B samples is 15/15 (100%). Comparison to CellSearch Five Neoadjuvant patients had blood tubes collected for parallel analysis between IsoFlux and CellSearch at baseline and follow up (Figure 5). This resulted in nine comparisons (one patient was lost to follow up). The IsoFlux test showed 4/9

ACCEPTED MANUSCRIPT (44%) samples having 10 or more CTCs. None of the 10 samples tested using the CellSearch system had a CTC count above 0.

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Correlation of CTCs to pathological staging A subset of Neoadjuvant patients (n=16) had pStage collected at time of cystectomy (4 months post-baseline). CTC levels (Low, Med, High) are shown at baseline and after one cycle of chemotherapy (Table 2). Analysis was performed to see how the pre- and post-chemo CTC counts associated with pStage. Nodal involvement (N1-N2) and tumor size (T1-4) both strongly correlate with poor prognosis. These parameters were used to assess if the tumor had responded well to the chemotherapy regimen. This data is presented to see if there is initial evidence that CTCs could predict which patients might benefit from proceeding straight to cystectomy rather than receiving further chemotherapy.

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Using baseline CTC with a cutoff of 10 CTCs in 44% (4/9) of the matched clinical samples tested, as compared to 0% for CellSearch, which identified no CTCs for the same 9 samples. This suggests a gain in sensitivity for detecting diagnosed bladder cancer patients as compared to healthy controls. This improvement is due to more uniform flow and magnetic forces applied in the microfluidic IsoFlux cartridge. The IsoFlux test was reproducible across the Low/Med/High classification with 100% (15/15) concordance in matched time-point samples. It demonstrated the ability to recover Medium/High levels of CTCs in 60% of neoadjuvant patients and 73% in metastatic patients (Figure 2b), the latter consistent with a likely higher tumor burden. The drop from 60% to

ACCEPTED MANUSCRIPT 33% in CTC High/Med patients following the first chemotherapy cycle was consistent with presumed cytolytic effect of chemotherapy. A matrix of associations were examined to determine if baseline or follow-up CTCs could predict pStage at time of cystectomy. The data suggested that baseline CTC levels was a better predictor of pStage (73% sensitivity), while followup CTC level after one chemotherapy cycle were not as indicative (30% sensitivity). While the small study size limits the statistical analysis of this association, this has helped refine the test plan for the planned expansion of this study.

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This study also explored the use of NGS for profiling somatic variants in CTCs. NGS has already shown benefit in tumor tissue samples by identifying patients likely to respond to the emerging class of molecular therapies. NGS samples must be of sufficient purity to detect somatic variants. This is readily achievable for tumor biopsies that routinely exceed 50% purity, but poses a challenge for CTCs that are often in the 5% purity in 85% of the patients tested and >10% purity for 71% of patients. For NGS analysis, both neoadjuvant and metastatic patients were selected (n=4 each). At purity levels near 5%, the known mutations in the analytical samples were detectable using a commercially available sequencer and targeted gene panel. For the clinical sample set, 7/8 samples had CTC purity above 5% and variants were detected for 4/8 samples. The same variants were detected at different NGS service labs thereby increasing confidence in the NGS workflow.

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An absence of detected mutations, however, cannot be equated with the lack of mutations in the patient’s tumor. Mutation detection is dependent on both the presence of CTCs in sufficient purity and the concordance between mutations harbored by CTCs and different parts of the tumor tissue. Although not performed in this study, comparison to the primary tumor tissue would add confidence that the mutations detected in CTCs are represented in the primary tumor. A recent study comparing single-cell CTC exome sequencing to data from multiple biopsy sites has demonstrated a high concordance between CTC mutations and metastatic trunk mutations25. This feasibility study establishes a workflow for detecting and analyzing CTCs without the need for single cell picking, a process that can be challenging for most clinical laboratories. Conclusions

Acknowledgments

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The study demonstrates a potential role for CTCs in management of neoadjuvant bladder cancer patients. The IsoFlux method increases sensitivity of CTC detection and enables molecular profiling of recovered cells via NGS. The authors have planned a larger prospective trial to explore these aims further.

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Supported by Michigan Institute for Clinical & Health Research and Clinical & Translational Science Award grant UL1TR000433. The authors thank UCSF (Kirsten Copren, Kathryn Thompson, Jennifer Dang) for their contributions to the sequencing.

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1. http://seer.cancer.gov/statfacts/html/urinb.html, Accessed on July 23, 2014. 2. Grossman HB, Natale RB, Tangen CM, et al. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 349:859-66, 2003. 3. Sonpavde G, Goldman BH, Speights VO, et al. Quality of pathologic response and surgery correlate with survival for patients with completely resected bladder cancer after neoadjuvant chemotherapy. Cancer 115:4104-9, 2009. 4. Stein JP, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol 19:666-75, 2001. 5. Parmar M K, et al. Neoadjuvant chemotherapy in invasive bladder cancer: a systematic review and meta-analysis. Lancet 361:1927-34, 2003. 6. Neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscle-invasive bladder cancer: a randomised controlled trial. International collaboration of trialists. Lancet 354:533-40, 1999. 7. Splinter TA, Scher HI, Denis L, et al. The prognostic value of the pathological response to combination chemotherapy before cystectomy in patients with invasive bladder cancer. European Organization for Research on Treatment of Cancer-Genitourinary Group. J Urol 147:606-8, 1992. 8. Galsky MD, Hahn NM, Rosenberg J, et al. A consensus definition of patients with metastatic urothelial carcinoma who are unfit for cisplatin-based chemotherapy. Lancet Oncol 12:211-4, 2011. 9. Kruk S, Gakis G, Stenzl A. Disseminated and Circulating Tumor Cells for Monitoring Chemotherapy in Urological Tumors. Anticancer Research June 2011 vol. 31 no. 6 2053-2057. 10. Gallagher D MM: Circulating Tumor Cells: A Potential Bladder Cancer Biomarker? European Journal of Clinical & Medical Oncology. 2:(1):105-110, 2010. 11. Friedlander TW, Premasekharan G, Paris PL. Looking back to the future of CTCs. Pharmacol Ther, 2014, 142:271280. 12. Ashworth T. A case of cancer in which cells similar to those in the tumors were seen in the blood after death. Aust Med J., 1869; 14. 13. Poste G, Fidler IJ. The pathogenesis of cancer metastasis. Nature 283:139-46, 1980. 14. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781-91, 2004. 15. Cohen SJ, Punt CJ, Iannotti N, et al. Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer. Ann Oncol 20:1223-9, 2009. 16. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 14:6302-9, 2008. 17. Scher HI, Beer TM, Higano CS, et al. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study. Lancet 375:1437-46, 2010. 18. Antonarakis ES, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014 Sep 11;371(11):1028-38. 19. Flaig TW, Wilson S, van Bokhoven A, et al. Detection of circulating tumor cells in metastatic and clinically localized urothelial carcinoma. Urology 78:863-7, 2011. 20. Gallagher DJ, Milowsky MI, Ishill N, et al. Detection of circulating tumor cells in patients with urothelial cancer. Ann Oncol 20:305-8, 2009. 21. Naoe M, Ogawa Y, Morita J, et al. Detection of circulating urothelial cancer cells in the blood using the CellSearch System. Cancer 109:1439-45, 2007. 22. Rink M, Chun FK, Dahlem R, et al. Prognostic role and HER2 expression of circulating tumor cells in peripheral blood of patients prior to radical cystectomy: a prospective study. Eur Urol 61:810-7, 2012. 23. Soave A, Minner S, Engel O, et al. Implications for circulating tumor cells on adjuvant chemotherapy decisionmaking in patients with urothelial carcinoma of the bladder treated with radical cystectomy. European Association of Urology Congress, 2014. Abstract ID 47554, 2014. 24. Harb W, Fan A, Tran T, et al. Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay. Transl Oncol 6:528-38, 2013. 25. Lohr JG, Adalsteinsson VA, Cibulskis K, et al. Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nat Biotechnol. 2014 May;32(5):479-84.

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Tables

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Table 1: Patient information. Patient demographics and clinico-pathologic variables are presented for the neoadjuvant bladder cancer patient cohort, metastatic bladder cancer cohort, and healthy donor controls.

Gender M M F M M M F M M M M

Chemotherapy History (naïve = 0, first = 1, second =2, more than 2 lines =3) 1 3 1 2 1 N/A 2 0 0 2 1

Metastatic sites (LN = 1, BONE =2, VISCERAL =3, BRAIN 4) 1,2,3 1, 2, 3 1, 3 1 1,2,3 N/A 1,2 3 1, 2 1,3 3

Prior Cystectomy No Yes No No Yes No Yes No No Yes No

Gender M M M M M M M M M M M M M

NAC regimen GC MVAC GC MVAC MVAC MVAC MVAC GC MVAC GC GC ddMVAC GC MVAC MVAC Split dose GC MVAC GC MVAC GC

eGFR by Cockroft Gault formula 72 99 81 86 71 103 62 61 97 73 >60 52 >60 >60 >60 58 >60 >60 59 59

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LVI in TURBT Present Absent Absent Absent Present Absent Absent Absent Absent Absent Not determined Absent Present Absent Absent Present Present Absent Absent Absent

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pStage pT3aN1Mx pT2N0Mx pT4aN2Mx pT0N0Mx pT1N0Mx pT2bN0Mx pT4N1Mx pT3aN0(0/17)Mx pT1N0(0/11)Mx pTisN0(0/11)Mx pT3aN0(0/9)Mx pT0N0(0/22)Mx N/A pT0N0 (0/10)M0 pT0N0(0/22)Mx pT3aN0(0/9)Mx pT4aN0Mx N/A N/A N/A

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cStage T2N0M0 T2N0M0 T4aN1M0 T2N0M0 T2N0M0 T2N0N0 T4aN0M0 T3N0M0 T2N0M0 T2N0M0 T2N0M0 T2N0M0 T2N0M0 T2N0M0 T2N0M0 T3N0M0 T4N0M0 T2N0M0 T2N0M0 T2N0M0

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Patient Identifier Age Met1 61 Met2 68 Met3 76 Met4 70 Met5 66 Met6 71 Met7 73 Met8 61 Met9 81 Met10 43 Met11 75 HEALTHY DONOR COHORT Patient Identifier Age HD1 22 HD2 22 HD3 44 HD4 44 HD5 26 HD6 30 HD7 30 HD8 54 HD9 27 HD10 26 HD11 24 HD12 36 HD13 36

Gender M M M M M M M M M M F F M F F M M M F M

EP

Age 82 58 79 59 50 49 62 62 65 73 71 55 76 76 52 69 62 82 65 57

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NEOADJUVANT COHORT Patient Identifier Neo1 Neo2 Neo3 Neo4 Neo5 Neo6 Neo7 Neo8 Neo9 Neo10 Neo11 Neo12 Neo13 Neo14 Neo15 Neo16 Neo17 Neo18 Neo19 Neo20 METASTATIC COHORT

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T0/Tis

T1-4 or N1/2

BASELINE - LOW

4 (57%)

3 (43%)

BASELINE – MEDIUM/HIGH

1 (11%)

8 (89%)

Kappa= 0.48 95% CI (0.05 – 0.9)

4 (36%%)

7 (64%)

Concordance 7 /15 = 46.7%

1 (25%)

3 (75%)

Kappa = 0.08 95% CI = (-0.3 – 0.4)

FOLLOW UP - LOW FOLLOW UP – MEDIUM/HIGH

Concordance: CTC LOW = T0/Tis CTC MED/HIGH = T1-4 or N1/2 Concordance 12/16 = 75%

3 (50%)

Low – Med/High

1 (100%)

0 (0%)

Med/High – Low

1 (20%)

4 (80%)

Med/High – Med/High

0 (0%)

3 (100%)

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Baseline vs. Follow Up paired samples Low – Low

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pStage

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pStage

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Table 2: Association of CTC counts with pathological staging at time of cystectomy. A subset of patients in the Neoadjuvant group had undergone cystectomy and had pathologic staging (pStage) available. Associations of CTC levels (LOW/MED/HIGH) and pStage (Nodal involvement-N, tumor size-T) are presented for Baseline (pre-chemo) and Follow-Up (after 1 cycle chemo, 1 month).

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Supplementary Table 1 – NGS Analytical Validation. A model tumor cell line (MDA-MB-231) was spiked into healthy donor blood, resulting in final CTC concentrations ranging from 2-8 CTC/mL blood. The cell line has 3 known somatic variants in the BRAF, KRAS, and TP53 genes. The NGS workflow was able to detect each of these variants in 23/24 attempts with only 1 false positive call made at the primary test site. Similar results were obtained at a secondary test site with 17/18 correct positive calls made and 2 false positives. The three false positive calls passing our filtering (VHL) across both runs were the same variant for all the affected samples. The variant location, ref and alternate allele and effect are also listed for the false positive call. Site 1 Results Site 2 Results Number of Number of MDA cells False False recovered PURITY BRAF KRAS TP53 BRAF KRAS TP53 Positives, Positives, (per ml) Genes Genes 100% MDA 100% 58% 68% 99% NA 58% 68% 99% NA cells 0 0 0 0 0 0 1, VHL 0 0 0

ND

ND

ND

0

ND

ND

ND

ND

ND

ND

0 0

Not Tested 5.0% 3.0% 6.0% 1.0% 2.0% 2.0% ND ND

7.0% 3.0% 6.0% 6.0% 2.0% 3.0% ND ND

11.0% 5.0% 11.0% ND 6.0% 5.0% ND ND

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44.0% 24.0% 10.0% 4.0% 12.0% ND 5.0% 5.0% ND ND

False Positive Loci, Effect

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22.0% 12.0% 6.0% 2.0% 6.0% 5.0% 2.0% 2.0% ND ND

0 0 0 0 1, VHL 1, VHL 0 0

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29.0% 21.0% 6.0% 3.0% 7.0% 1.0% 3.0% 2.0% ND ND

chr3:10188214 C>T, WT chr3:10188214 C>T, WT

Not Tested

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PBMC gDNA normal gDNA normal

22% 22% 15% 15% 8% 8% 5% 5% 0% 0%

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12 12 8 8 4 4 2 2 0 0

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Sample es

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Figure 1: Outline of neoadjuvant study endpoints. Twenty neoadjuvant bladder cancer patients were enrolled at time of transurethral resection of bladder tumor (TURBT). All patients had two tubes of blood drawn prior to the first cycle of neoadjuvant chemotherapy, and two tubes drawn after the first cycle (approximately one month later). The first 5 patients had one blood tube drawn and tested on the IsoFlux System, and a second blood tube tested on the CellSearch assay. The next ten patients had both tubes analyzed on the IsoFlux System to assess repeatability. The remaining six patients had both tubes processed on the IsoFlux System but one tube was retained for molecular profiling. Pathologic staging was available for the first 17 patients.

ACCEPTED MANUSCRIPT Figure 2: Enumeration results. (a) CTC counts are presented for patients across all 3 groups and reported as normalized to 7.5mL of blood. In the subset of neoadjuvant patients having multiple tubes tested at the same time point, the CTC results were averaged. (b) CTC counts for each cohort are grouped into bins of CTC Low (30) and reported as a percentage of each cohort. (a)

(b)

CTC 32 26 146 88 26 1 33 0 154 34 6

HEALTHY Patient Identifier HD1 HD2 HD3 HD4 HD5 HD6 HD7 HD8 HD9 HD10 HD11 HD12 HD13

CTC 1 0 2 0 3 4 4 8 2 1 2 0 0

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METASTATIC Patient Identifier Met1 Met2 Met3 Met4 Met5 Met6 Met7 Met8 Met9 Met10 Met11

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Follow Up CTC 60 N/A 7 2 4 2 750 0 6 3 0 0 3 0 34 9 33 100 N/A 14

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NEOADJUVANT Baseline CTC 50 10 60 8 0 0 11 2 19 1 179 128 88 1 3 15 358 183 233 8

Patient Identifier Neo1 Neo2 Neo3 Neo4 Neo5 Neo6 Neo7 Neo8 Neo9 Neo10 Neo11 Neo12 Neo13 Neo14 Neo15 Neo16 Neo17 Neo18 Neo19 Neo20

CTC Levels as a Percentage of Cohort 100%

100%

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90% 80%

67%

60% 50% 40%

40%

30%

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40%

28%

20%

20% 10%

55%

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70%

CTC LOW (30) 27% 18%

6% 0% 0%

0% NEOADJUVANT BASELINE

NEOADJUVANT FOLLOW UP

METASTATIC

HEALTHY

ACCEPTED MANUSCRIPT Figure 3: CTC counts in Neoadjuvant group by pathologic stage at time of cystectomy. The relative change in CTC counts between baseline and follow-up (1month) is shown for two subsets of patients: (a) patients that were pStage T0 and N0 at time of cystectomy, and (b) patients that were pStage T1 or N1 or greater at time of cystectomy. (a)

pStage = T0 and N0

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1000

100

Neo4

Neo10 CTC COUNT

10

Neo12

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Neo14

1

0 TIME POINT 1

(b)

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TIME POINT 2

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pStage = T+ and N+

0

TIME POINT 1

TIME POINT 2

Neo1 Neo3 Neo5 Neo6 Neo7 Neo8 Neo9 Neo11 Neo16 Neo17 Neo4 Neo10 Neo12 Neo14 Neo15

ACCEPTED MANUSCRIPT Figure 4: Assay repeatability. For a subset of patients, duplicate blood tubes were collected at the same time point for repeatability analysis. Normalized CTC counts are shown on log scale, with reference to the Low/Medium/High classification scheme. Concordance amongst these matched samples using this scheme was 100% (15/15).

92

77

23

1111

13

01

2017

56

01

5 2 281

01

31

132

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M AN U

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00

AC C

100 90 80 70 60 CTC 50 COUNT 40 30 20 10 0

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CTC Counts in Duplicate Blood Tubes

62

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Tube A Tube B

ACCEPTED MANUSCRIPT Figure 5: Comparison to CellSearch. The first 5 patients had matched samples sent for analysis on the IsoFlux System and the CellSearch CTC Test at both baseline and following 1 cycle of chemotherapy. For the five patients tested, no cells were detected using the CellSearch system.

CTC Count Comparison to CellSearch 70

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ISOFLUX - BASELINE

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CTC COUNT 30

ISOFLUX - FOLLOWUP CELLSEARCH - BASELINE

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10 0 0

8

7 0 0

0 0

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CELLSEARCH - FOLLOWUP

0 0

0 Neo3

Neo4

Neo5

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Neo2

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ACCEPTED MANUSCRIPT Figure 6: Analytical validation of NGS assay. A model tumor cell line (MDA-MB-231) was spiked into healthy donor blood, resulting in final CTC concentrations ranging from 0-8 CTC/mL blood. The cell line has 3 known somatic variants in the BRAF, KRAS, and TP53 genes. The NGS workflow was able to detect each of these variants for 17/18 true positives with only 1 false positive call made in the test group. The figure presents the correlation between allelic frequencies from runs at two different sites.

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equivalence

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KRAS

6%

TP53

4% 2%

4%

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Allelic Ratio Site 2

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BRAF

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Allelic Ratio Site 1

12%

14%

ACCEPTED MANUSCRIPT Figure 7: Clinical testing of NGS assay. Results from NGS analysis of clinical samples are presented for 8 patients in the neoadjuvant and metastatic settings, along with positive (spiked) and negative (healthy) controls. The same variant filtering criteria applied to the analytical samples was used for clinical sample analysis to produce high confidence variant calls. All 3 expected variants in the 2 positive controls were detected, and no false positives were detected in the negative controls. For patient samples, 4/8 (50%) had at least one somatic variant detected. Parallel NGS analysis was performed at two independent labs for a subset of samples (denoted with *) that showed concordant calls. CANCER STAGE Healthy Healthy Healthy Healthy Neoadjuvant Neoadjuvant Neoadjuvant Neoadjuvant Metastatic Metastatic Metastatic Metastatic

CTC COUNT 0 0 0 0 40 100 20 21 154 34 11 8

CTC PURITY VARIANTS IN GENE SEQUENCE 0% None 0% None 0% None* 0% None 10% FGFR2 19% PDGFRA* 12% None 8% EGFR 25% JAK2* 4% None* 15% None N/D None

MDA Cell Spike

Positive Control

N/D

N/D

MDA Cell Spike

Positive Control

60

8%

chr7:55221802 G>A p.(=) chr9:5073770 T>G p.Val617Phe

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KRAS, BRAF, TP53*

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LOCATION AND EFFECTS N/A N/A N/A N/A chr10:123279681 G>A p.Arg251Ter chr4:55144562 A>G p.Tyr679Cys

RI PT

PATIENT IDENTIFIER Healthy Healthy Healthy Healthy N17 - 1mo follow up N18 - 1mo follow up N22* N22 - 1 mo follow up* M9 M10 M12* M13*

KRAS, BRAF, TP53

chr7:140481417 C>A p.Gly464Val; chr12:25398281 C>T p.Gly13Asp; chr17:7577099 G>T p.Gly207Trp chr7:140481417 C>A p.Gly464Val; chr12:25398281 C>T p.Gly13Asp; chr17:7577099 G>T p.Gly207Trp

ACCEPTED MANUSCRIPT

List of commonly used abbreviations

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CTC – circulating tumor cell FNA – fine needle aspiration GC – combination chemotherapy, cisplatinum and gemcitabine gDNA – genomic deoxyribonucleic acid MVAC – combination chemotherapy for bladder cancer consisting of methotrexate, vinblastine, doxorubicin and cisplatin NGS – next generation sequencing

Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer.

We explored the diagnostic use of circulating tumor cells in patients with neoadjuvant bladder cancer using enumeration and next generation sequencing...
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