Digital Biopsy with Fluorescence Confocal Microscopy for the Real-Time Diagnosis of Prostate Cancer - Beyond the Abstract

The ability to generate digital microscopical images from fresh tissue could be a game changer for the achievement of a real-time diagnosis. Fluorescence confocal microscopy is an optical technique that allows immediate acquisition of digital images from ex vivo unprocessed tissues. Digital images display a hematoxylin-eosin (H&E) appearance and are immediately available for remote consultation. Moreover, the analysis with confocal microscopy has been proven to preserve the integrity of the tissue for further conventional and/or immunohistochemical analysis.1

We aimed to evaluate the diagnostic accuracy of confocal microscopy in the clinical setting of prostate biopsy.

A total of 427 biopsy cores were retrieved from 54 men undergoing prostate biopsy for cancer suspicion. All cores have been immediately analyzed with confocal microscopy to obtain digital images, uploaded, and stored into a web platform. Then, the same cores underwent the conventional pathological analysis with digitalization and web storage of hematoxylin-eosin images. A panel of four independent pathologists was randomly shown and blindly interpreted all images – those from confocal microscopy and their corresponding H&E.

The diagnostic performance of confocal microscopy was evaluated in terms of agreement with the conventional pathology for the detection of prostate cancer: overall, we found 95.1% of correct diagnosis (k=0.84) with confocal microscopy, with an AUC of 0.92. As a secondary endpoint, we tested the concordance between pathologists for the detection of prostate cancer, and again, a high level of agreement was found (K=0.86) for digital biopsies. We concluded that confocal microscopy is able to detect the presence of cancer in a real-time fashion with almost perfect accuracy.

Some key findings need to be noted for further implementation of the technology.

First, fluorescence confocal microscopy does not require specific training nor seems prone to a learning curve effect. Specimens could be prepared by the urological staff while biopsy is going on and digital images are uploaded. From the pathologist’s viewpoint, the analysis with confocal microscopy seems not to require a steep learning curve:1 even if non-specifically addressed, the almost perfect agreement with H&E from pathologists naïve to the novel technology confirms the easy interpretation regardless of their expertise and background.

Second, as grade attribution is concerned, the variability between pathologists is still a matter of concern for both conventional pathology and confocal microscopy. Gleason grade takes into account the heterogeneity of prostate cancer;2 nevertheless, it requires both the classification of morphological patterns and the estimation of their extent, assessed visually and translated into an ordinal scale. Indeed, grade attribution is prone to inter-observer variability.2

The current study confirmed just a modest level of agreement between pathologists for the discrimination between ISUP 1 and ISUP>1, for both conventional pathology and confocal microscopy (K=0.47 and K=0.49, respectively). Consistently, a recent study on digital biopsy with the confocal microscope confirmed the difficult discrimination between Gleason 3+4 and 3+3 scores, as appears with H&E; opposite, larger areas of Gleason 4 patterns are reliably recognizable.3 Further studies are required to clarify the issue.

Beyond grade attribution, the ability to diagnose prostate cancer still under biopsy may definitely change the landscape of prostate disease management.4,5 Merging biopsy and BPH treatment in patients with abnormal PSA, checking the accuracy of focal treatment,4 the real-time control of surgical margins5 are current feasible opportunities of fluorescence confocal microscopy.

Written by: Maria Chiara Sighinolfi & Bernardo Rocco, ASST Santi Paolo e Carlo, Milan, Italy, University of Milan, Italy


  1. Bertoni L, Puliatti S, Reggiani Bonetti L, Maiorana A, Eissa A, Azzoni P, Bevilacqua L, Spandri V, Kaleci S, Zoeir A, Sighinolfi MC, Micali S, Bianchi G, Pellacani G, Rocco B, Montironi R. Ex vivo fluorescence confocal microscopy: prostatic and periprostatic tissues atlas and evaluation of the learning curve. Virchows Arch. 2020 Apr;476(4):511-520. doi: 10.1007/s00428-019-02738-y. Epub 2020 Jan 6
  2. Egevad L, Swanberg D, Delahunt B, Ström P, Kartasalo K, Olsson H, Berney DM, Bostwick DG, Evans AJ, Humphrey PA, Iczkowski KA, Kench JG, Kristiansen G, Leite KRM, McKenney JK, Oxley J, Pan CC, Samaratunga H, Srigley JR, Takahashi H, Tsuzuki T, van der Kwast T, Varma M, Zhou M, Clements M, Eklund M. Identification of areas of grading difficulties in prostate cancer and comparison with artificial intelligence assisted grading. Virchows Arch. 2020 Dec;477(6):777-786. doi: 10.1007/s00428-020-02858-w. Epub 2020 Jun 15.PMID: 32542445
  3. Titze U, Hansen T, Brochhausen C, Titze B, Schulz B, Gunnemann A, Rocco B, Sievert KD. Diagnostic Performance of Ex Vivo Fluorescence Confocal Microscopy in the Assessment of Diagnostic Biopsies of the Prostate. Cancers (Basel). 2021 Nov 13;13(22):5685. doi: 10.3390/cancers13225685
  4. Selvaggio O, Falagario UG, Bruno SM, Recchia M, Sighinolfi MC, Sanguedolce F, Milillo P, Macarini L, Rastinehad AR, Sanchez-Salas R, Barret E, Lugnani F, Rocco B, Cormio L, Carrieri G. Intraoperative Digital Analysis of Ablation Margins (DAAM) by Fluorescent Confocal Microscopy to Improve Partial Prostate Gland Cryoablation Outcomes. Cancers (Basel). 2021 Aug 30;13(17):4382. doi: 10.3390/cancers13174382
  5. Rocco B, Sarchi L, Assumma S, Cimadamore A, Montironi R, Reggiani Bonetti L, Turri F, De Carne C, Puliatti S, Maiorana A, Pellacani G, Micali S, Bianchi G, Sighinolfi MC. Digital Frozen Sections with Fluorescence Confocal Microscopy During Robot-assisted Radical Prostatectomy: Surgical Technique.Eur Urol. 2021 Dec;80(6):724-729. doi: 10.1016/j.eururo.2021.03.021. Epub 2021 May 6

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