92d5be4a57814a3864a293116af0f7ac cancers-17-03848.pdf 7ba21199c59089c1858fbe1acffe090fc0cd9b8f cancers-17-03848.pdf 3b13598d90435815bf75b605a61c6f5773feb699e6ac59b9d436626591a775fa cancers-17-03848.pdf Title: Towards a New Standard: Prospective Validation of Ex Vivo Fusion Confocal Microscopy for Intraoperative Margin Assessment in Breast-Conserving Cancer Surgery Subject: Background/Objectives: Accurate intraoperative margin assessment is essential for ensuring complete tumour excision in breast-conserving surgery, minimising local recurrence, and avoiding reoperations. Ex vivo fusion confocal microscopy (EVFCM) provides real-time, high-resolution imaging of fresh, unfixed tissues that closely resembles conventional histological imaging. This study aimed to validate the diagnostic performance and clinical feasibility of EVFCM for real-time intraoperative margin assessment during breast cancer surgery. Methods: A prospective observational diagnostic validation study was conducted using 144 breast tissue specimens. The samples were stained with acridine orange and fast green and scanned using a VivaScope 2500M-G4 system. Two breast pathologists independently evaluated the EVFCM images, blinded to the conventional histology results, which served as the reference standard. The diagnostic accuracy, sensitivity, specificity, and interobserver agreement were calculated using Cohen’s . Results: Interobserver agreement was almost perfect for neoplasia detection (97.3%, = 0.942) and tumour type classification (93.8%, = 0.883). The EVFCM achieved 93.7% sensitivity and specificity, with 94.0% accuracy for tumour detection ( = 0.929, p < 0.001); 95.8% accuracy for tumour type classification ( = 0.925, p < 0.001); and 95.1% accuracy for invasive subtype identification ( = 0.907, p < 0.001). For margin assessment, EVFCM achieved 80% sensitivity, 100% specificity, and 99.3% accuracy ( = 0.857, p < 0.001), whereas margin distance evaluation (<2 mm vs. 2 mm) yielded 75% sensitivity, 100% specificity, and 98.6% accuracy ( = 0.854, p < 0.001). Conclusions: EVFCM enables rapid, high-resolution imaging of fresh breast tissue, facilitating real-time intraoperative margin evaluation with excellent diagnostic concordance and workflow efficiency. Its integration into surgical practice could reduce re-excisions, enhance oncological safety, and improve patient outcomes in breast-conserving surgeries. Keywords: breast-conserving surgery; intraoperative margin assessment; ex vivo fusion confocal microscopy; digital microscopy; breast cancer diagnosis Author: Daniel Humaran, Ana Castillo, Lidia Blay, Iciar Pascual, Karol Matute-Molina, Javiera Pérez-Anker, Susana Puig, Pedro L. Fernández and Joan F. Julián Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sun Nov 30 07:43:14 2025 CET ModDate: Sun Nov 30 07:49:18 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3930615 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- HUVLVJ+URWPalladioL-Ital Type 1 Custom yes yes yes 107 0 SSXPJJ+VnURWPalladioL Type 1 Custom yes yes yes 108 0 PZJEKD+URWPalladioL-Bold Type 1 Custom yes yes yes 109 0 AYXMDM+EURM10 Type 1 Builtin yes yes yes 110 0 FVUIVA+URWPalladioL-Roma Type 1 Custom yes yes yes 111 0 ZISOQP+CMSY10 Type 1 Builtin yes yes yes 165 0 KIDOMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 235 0 KIDOMK+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 236 0 KIDOML+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 237 0 KIDOMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 304 0 KIDOMK+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 305 0 KIDOML+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 306 0 RSPJNP+EURB10 Type 1 Builtin yes yes yes 520 0 KIDOMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 493 0 KIDOMK+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 494 0 KIDOML+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 495 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 546 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-12-19 05:11:16 CET RepresentationInformation: cancers-17-03848.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-12-18 19:10:24 CET Size: 3930615 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 692 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Study Design and Setting Item: Title: Surgical Procedure Item: Title: Staining Protocol, Image Acquisition, and Histopathological Processing Item: Title: Image Evaluation Item: Title: Statistical Analysis Item: Title: Ethical Considerations Item: Title: Results Children: Item: Title: Study Population and Case Flow Item: Title: Inter-Observer Agreement Between Pathologists Item: Title: Diagnostic Performance of EVFCM Compared with Paraffin Histology Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Towards a New Standard: Prospective Validation of Ex Vivo Fusion Confocal Microscopy for Intraoperative Margin Assessment in Breast-Conserving Cancer Surgery Author: Daniel Humaran, Ana Castillo, Lidia Blay, Iciar Pascual, Karol Matute-Molina, Javiera Pérez-Anker, Susana Puig, Pedro L. Fernández and Joan F. Julián Subject: Background/Objectives: Accurate intraoperative margin assessment is essential for ensuring complete tumour excision in breast-conserving surgery, minimising local recurrence, and avoiding reoperations. Ex vivo fusion confocal microscopy (EVFCM) provides real-time, high-resolution imaging of fresh, unfixed tissues that closely resembles conventional histological imaging. This study aimed to validate the diagnostic performance and clinical feasibility of EVFCM for real-time intraoperative margin assessment during breast cancer surgery. Methods: A prospective observational diagnostic validation study was conducted using 144 breast tissue specimens. The samples were stained with acridine orange and fast green and scanned using a VivaScope 2500M-G4 system. Two breast pathologists independently evaluated the EVFCM images, blinded to the conventional histology results, which served as the reference standard. The diagnostic accuracy, sensitivity, specificity, and interobserver agreement were calculated using Cohen’s . Results: Interobserver agreement was almost perfect for neoplasia detection (97.3%, = 0.942) and tumour type classification (93.8%, = 0.883). The EVFCM achieved 93.7% sensitivity and specificity, with 94.0% accuracy for tumour detection ( = 0.929, p < 0.001); 95.8% accuracy for tumour type classification ( = 0.925, p < 0.001); and 95.1% accuracy for invasive subtype identification ( = 0.907, p < 0.001). For margin assessment, EVFCM achieved 80% sensitivity, 100% specificity, and 99.3% accuracy ( = 0.857, p < 0.001), whereas margin distance evaluation (<2 mm vs. 2 mm) yielded 75% sensitivity, 100% specificity, and 98.6% accuracy ( = 0.854, p < 0.001). Conclusions: EVFCM enables rapid, high-resolution imaging of fresh breast tissue, facilitating real-time intraoperative margin evaluation with excellent diagnostic concordance and workflow efficiency. Its integration into surgical practice could reduce re-excisions, enhance oncological safety, and improve patient outcomes in breast-conserving surgeries. Keywords: breast-conserving surgery; intraoperative margin assessment; ex vivo fusion confocal microscopy; digital microscopy; breast cancer diagnosis Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Sun Nov 30 07:43:14 CET 2025 ModDate: Sun Nov 30 07:49:18 CET 2025 ID: 0xfd7df6e286fc39a7e8e823fdc7c37f0d, 0x2414d332364962ec94cc11388c45744c Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: WBVKDJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 112 LastChar: 112 FontDescriptor: FontName: WBVKDJ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ZISOQP+CMSY10 FontSubset: true FirstChar: 2 LastChar: 21 FontDescriptor: FontName: ZISOQP+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: RSPJNP+EURB10 FontSubset: true FirstChar: 20 LastChar: 20 FontDescriptor: FontName: RSPJNP+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true ToUnicode: true Font: BaseFont: HUVLVJ+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: HUVLVJ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: SSXPJJ+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 225 FontDescriptor: FontName: SSXPJJ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: PZJEKD+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 233 FontDescriptor: FontName: PZJEKD+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AYXMDM+EURM10 FontSubset: true FirstChar: 20 LastChar: 22 FontDescriptor: FontName: AYXMDM+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: FVUIVA+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: FVUIVA+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 105, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 165, 140, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 130, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 131, 643, 142, 656 Annotation: Subtype: Link Rect: 209, 643, 220, 656 Annotation: Subtype: Link Rect: 283, 643, 294, 656 Annotation: Subtype: Link Rect: 132, 629, 143, 642 Annotation: Subtype: Link Rect: 315, 629, 326, 642 Annotation: Subtype: Link Rect: 409, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 356, 87, 375 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 465, 496, 472, 505 ActionDest: 75 Annotation: Subtype: Link Rect: 273, 454, 280, 463 ActionDest: 75 Annotation: Subtype: Link Rect: 493, 454, 500, 463 ActionDest: 75 Annotation: Subtype: Link Rect: 500, 454, 507, 463 ActionDest: 75 Annotation: Subtype: Link Rect: 217, 412, 224, 421 ActionDest: 75 Annotation: Subtype: Link Rect: 227, 412, 234, 421 ActionDest: 75 Annotation: Subtype: Link Rect: 316, 384, 323, 393 ActionDest: 75 Annotation: Subtype: Link Rect: 326, 384, 338, 393 ActionDest: 75 Annotation: Subtype: Link Rect: 475, 315, 487, 324 ActionDest: 75 Annotation: Subtype: Link Rect: 491, 315, 502, 324 ActionDest: 181 Annotation: Subtype: Link Rect: 431, 287, 443, 296 ActionDest: 75 Annotation: Subtype: Link Rect: 444, 287, 456, 296 ActionDest: 75 Annotation: Subtype: Link Rect: 456, 287, 468, 296 ActionDest: 181 Annotation: Subtype: Link Rect: 358, 245, 370, 254 ActionDest: 181 Annotation: Subtype: Link Rect: 371, 245, 383, 254 ActionDest: 181 Annotation: Subtype: Link Rect: 386, 245, 398, 254 ActionDest: 181 Annotation: Subtype: Link Rect: 494, 217, 506, 226 ActionDest: 181 Annotation: Subtype: Link Rect: 321, 175, 333, 184 ActionDest: 181 Annotation: Subtype: Link Rect: 334, 175, 346, 184 ActionDest: 181 Annotation: Subtype: Link Rect: 349, 175, 361, 184 ActionDest: 181 Annotation: Subtype: Link Rect: 486, 147, 498, 157 ActionDest: 181 Annotation: Subtype: Link Rect: 501, 147, 513, 157 ActionDest: 181 Annotation: Subtype: Link Rect: 205, 92, 217, 101 ActionDest: 75 Annotation: Subtype: Link Rect: 218, 92, 230, 101 ActionDest: 181 Annotation: Subtype: Link Rect: 230, 92, 242, 101 ActionDest: 181 Annotation: Subtype: Link Rect: 245, 92, 257, 101 ActionDest: 181 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 439, 744, 451, 754 ActionDest: 75 Annotation: Subtype: Link Rect: 206, 717, 218, 726 ActionDest: 181 Annotation: Subtype: Link Rect: 444, 661, 456, 670 ActionDest: 75 Annotation: Subtype: Link Rect: 231, 591, 243, 600 ActionDest: 75 Annotation: Subtype: Link Rect: 244, 591, 256, 600 ActionDest: 181 Annotation: Subtype: Link Rect: 256, 591, 268, 600 ActionDest: 181 Annotation: Subtype: Link Rect: 269, 591, 281, 600 ActionDest: 181 Annotation: Subtype: Link Rect: 411, 536, 423, 545 ActionDest: 181 Annotation: Subtype: Link Rect: 367, 410, 379, 419 ActionDest: 181 Annotation: Subtype: Link Rect: 379, 410, 391, 419 ActionDest: 210 Page: Sequence: 4 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 404, 593, 416, 602 ActionDest: 210 Annotation: Subtype: Link Rect: 294, 523, 306, 532 ActionDest: 75 Annotation: Subtype: Link Rect: 371, 495, 383, 504 ActionDest: 210 Annotation: Subtype: Link Rect: 485, 439, 497, 448 ActionDest: 210 Page: Sequence: 6 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 524, 337, 531, 349 ActionDest: 295 Annotation: Subtype: Link Rect: 551, 337, 558, 349 ActionDest: 296 Annotation: Subtype: Link Rect: 362, 295, 369, 307 ActionDest: 61 Annotation: Subtype: Link Rect: 552, 241, 559, 253 ActionDest: 61 Page: Sequence: 8 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 394, 118, 401, 130 ActionDest: 65 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 215, 157, 227, 166 ActionDest: 210 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 459, 577, 471, 586 ActionDest: 181 Annotation: Subtype: Link Rect: 318, 536, 330, 545 ActionDest: 75 Annotation: Subtype: Link Rect: 461, 522, 473, 531 ActionDest: 181 Annotation: Subtype: Link Rect: 552, 296, 559, 308 ActionDest: 61 Annotation: Subtype: Link Rect: 544, 173, 556, 182 ActionDest: 75 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 358, 563, 369, 572 ActionDest: 181 Annotation: Subtype: Link Rect: 270, 159, 282, 169 ActionDest: 75 Annotation: Subtype: Link Rect: 283, 159, 295, 169 ActionDest: 181 Annotation: Subtype: Link Rect: 295, 159, 307, 169 ActionDest: 181 Annotation: Subtype: Link Rect: 308, 160, 320, 169 ActionDest: 210 Annotation: Subtype: Link Rect: 192, 132, 204, 141 ActionDest: 210 Annotation: Subtype: Link Rect: 351, 76, 363, 85 ActionDest: 181 Annotation: Subtype: Link Rect: 519, 76, 531, 85 ActionDest: 210 Page: Sequence: 14 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 388, 410, 400, 419 ActionDest: 210 Page: Sequence: 16 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 520, 584, 557, 595 Annotation: Subtype: Link Rect: 398, 558, 436, 569 Annotation: Subtype: Link Rect: 122, 521, 160, 531 Annotation: Subtype: Link Rect: 237, 482, 275, 492 Annotation: Subtype: Link Rect: 135, 444, 173, 454 Annotation: Subtype: Link Rect: 59, 419, 96, 429 Annotation: Subtype: Link Rect: 102, 419, 138, 429 Annotation: Subtype: Link Rect: 95, 380, 133, 390 Annotation: Subtype: Link Rect: 139, 380, 175, 390 Annotation: Subtype: Link Rect: 131, 329, 168, 339 Annotation: Subtype: Link Rect: 175, 329, 211, 339 Annotation: Subtype: Link Rect: 104, 291, 141, 301 Annotation: Subtype: Link Rect: 59, 253, 96, 263 Annotation: Subtype: Link Rect: 232, 213, 269, 224 Annotation: Subtype: Link Rect: 185, 175, 223, 186 Annotation: Subtype: Link Rect: 229, 175, 265, 186 Annotation: Subtype: Link Rect: 158, 150, 195, 161 Annotation: Subtype: Link Rect: 201, 150, 237, 161 Annotation: Subtype: Link Rect: 95, 125, 132, 135 Annotation: Subtype: Link Rect: 450, 98, 488, 109 Annotation: Subtype: Link Rect: 494, 98, 530, 109 Annotation: Subtype: Link Rect: 263, 73, 301, 84 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 297, 743, 334, 754 Annotation: Subtype: Link Rect: 424, 705, 461, 716 Annotation: Subtype: Link Rect: 164, 668, 201, 678 Annotation: Subtype: Link Rect: 104, 629, 142, 639 Annotation: Subtype: Link Rect: 59, 591, 96, 601 Annotation: Subtype: Link Rect: 102, 591, 138, 601 Annotation: Subtype: Link Rect: 149, 552, 187, 563 Annotation: Subtype: Link Rect: 193, 552, 229, 563 Annotation: Subtype: Link Rect: 242, 526, 279, 537 Annotation: Subtype: Link Rect: 423, 501, 461, 512 Annotation: Subtype: Link Rect: 358, 475, 396, 486 Annotation: Subtype: Link Rect: 281, 437, 318, 448 Annotation: Subtype: Link Rect: 324, 437, 360, 448 Annotation: Subtype: Link Rect: 389, 399, 427, 410 Annotation: Subtype: Link Rect: 135, 361, 173, 371 Annotation: Subtype: Link Rect: 270, 322, 308, 333 Annotation: Subtype: Link Rect: 456, 296, 493, 307 Annotation: Subtype: Link Rect: 59, 259, 96, 269 Annotation: Subtype: Link Rect: 59, 221, 96, 231 Annotation: Subtype: Link Rect: 194, 182, 231, 193 Annotation: Subtype: Link Rect: 435, 156, 472, 167 Annotation: Subtype: Link Rect: 231, 118, 268, 129 Annotation: Subtype: Link Rect: 460, 79, 497, 90 Annotation: Subtype: Link Rect: 504, 79, 540, 90 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 118, 731, 155, 742 Annotation: Subtype: Link Rect: 161, 731, 197, 742 Annotation: Subtype: Link Rect: 204, 692, 241, 703 Annotation: Subtype: Link Rect: 247, 692, 283, 703 Annotation: Subtype: Link Rect: 339, 667, 377, 678 Annotation: Subtype: Link Rect: 383, 667, 419, 678 Annotation: Subtype: Link Rect: 56, 641, 496, 652 Annotation: Subtype: Link Rect: 81, 591, 119, 601 Annotation: Subtype: Link Rect: 337, 565, 375, 576 Annotation: Subtype: Link Rect: 381, 565, 417, 576 Checksum: 508d8dc5 Type: CRC32 Checksum: 92d5be4a57814a3864a293116af0f7ac Type: MD5 Checksum: 7ba21199c59089c1858fbe1acffe090fc0cd9b8f Type: SHA-1 Checksum: 3b13598d90435815bf75b605a61c6f5773feb699e6ac59b9d436626591a775fa Type: SHA-256