The current gold standard for the diagnosis of bladder cancer is cystoscopy, which is invasive and painful for patients. Therefore, noninvasive urine cytology is usually used in the clinic as an adjunct to cystoscopy; however, it suffers from low sensitivity. Here, a novel noninvasive, label-free approach with high sensitivity for use with urine is presented. Coherent anti-Stokes Raman scattering imaging of urine sediments was used in the first step for fast preselection of urothelial cells, where high-grade urothelial cancer cells are characterized by a large nucleus-to-cytoplasm ratio. In the second step, Raman spectral imaging of urothelial cells was performed. A supervised classifier was implemented to automatically differentiate normal and cancerous urothelial cells with 100% accuracy. In addition, the Raman spectra not only indicated the morphological changes that are identified by cytology with hematoxylin and eosin staining but also provided molecular resolution through the use of specific marker bands. The respective Raman marker bands directly show a decrease in the level of glycogen and an increase in the levels of fatty acids in cancer cells as compared to controls. These results pave the way for "spectral" cytology of urine using Raman microspectroscopy.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatographyetandem mass spectrometryebased proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive highgrade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS. (Am J Pathol 2019, 189: 619e631; https://doi.org/10. 1016/j.ajpath.2018.11.018) Bladder cancer is the second most common urogenital malignancy, with approximately 430,000 new cases diagnosed worldwide in 2012. 1,2 Approximately 70% to 75% of patients are newly diagnosed with nonmuscle invasive, mostly low-grade bladder cancer, whereas 25% to 30% of de novo diagnoses are high-grade bladder cancer at the stage of infiltration of the muscularis, with metastases observed in 10% of cases. 3e5 A significant percentage (50% to 70%) of patients diagnosed with nonmuscle invasive cancer will have multiple recurrences, and 10% to 15% will even progress to invasion. 3,6,7 A special case of bladder cancer is carcinoma in situ (CIS), which is a flat, noninvasive tumor with a rate of progression to an invasive state of approximately 54%. 8