Breast tissue imaging atlas using ultra-fast confocal microscopy to identify cancer lesions.

New generation ultra-fast fluorescence confocal microscopy (UFCM) allows to image histological architecture of fresh breast tissue and may be used for ex vivo intraoperative analysis for margin status. The criteria to identify breast tumoral and non-tumoral tissues in UFCM images are still objects of investigation. The objective of the study was to create an atlas of ex vivo UFCM images of breast tissues and breast carcinomas based on the first extensive collection of large field-of-view UFCM breast images. One hundred sixty patients who underwent conserving surgery for breast cancer were included. Their fresh surgical specimens were sliced, stained with acridine orange, and imaged at high resolution with large-field-of-view UFCM. The resulting images were digitally false colored to resemble frozen sections. Each UFCM image was correlated with the corresponding definitive histology. Representative images of normal tissue, inflammation, benign lesions, invasive carcinoma (IC), and ductal carcinoma in situ (DCIS) were collected. A total of 320 large-field images were recorded from 58 IC of no special type, 44 invasive lobular carcinomas, 1 invasive mucinous carcinoma, 47 DCIS, 2 lobular carcinomas in situ, and 8 specimens without cancer. Representative images of the main components of the normal breast and the main types of ICs and DCIS were annotated to establish an UFCM atlas. UFCM enables the imaging of the fresh breast tissue sections. Main morphological criteria defined in traditional histopathology such as tissue architecture and cell features can be applied to describe UFCM images content. The generated atlas of the main normal or tumoral tissue features will support the adoption of this optical technology for the intraoperative examination of breast specimens in clinical practice as it can be used to train physicians on UFCM images and develop artificial intelligence algorithms. Further studies are needed to document rare breast lesions.

Real-World Diagnostic Accuracy of the On-Site Cytopathology Advance Report (OSCAR) Procedure Performed in a Multidisciplinary One-Stop Breast Clinic.

Fine-needle aspiration (FNA) cytology has been widely used for the diagnosis of breast cancer lesions with the objective of differentiating benign from malignant masses. However, the occurrence of unsatisfactory samples and false-negative rates remains a matter of concern. Major improvements have been made thanks to the implementation of rapid on-site evaluation (ROSE) in multidisciplinary and integrated medical settings such as one-stop clinics (OSCs). In these settings, clinical and radiological examinations are combined with a morphological study performed by interventional pathologists. The aim of our study was to assess the diagnostic accuracy of the on-site cytopathology advance report (OSCAR) procedure on breast FNA cytologic samples in our breast OSC during the first three years (April 2004 till March 2007) of its implementation. To this goal, we retrospectively analyzed a series of 1820 breast masses (1740 patients) radiologically classified according to the American College of Radiology (ACR) BI-RADS lexicon (67.6% being either BI-RADS 4 or 5), sampled by FNA and immediately diagnosed by cytomorphology. The clinicoradiological, cytomorphological, and histological characteristics of all consecutive patients were retrieved from the hospital computerized medical records prospectively registered in the central information system. Histopathological analysis and ultrasound (US) follow-up (FU) were the reference diagnostic tests of the study design. In brief, we carried out either a histopathological verification or an 18-month US evaluation when a benign cytology was concordant with the components of the triple test. Overall, histology was available for 1138 masses, whereas 491 masses were analyzed at the 18-month US-FU. FNA specimens were morphologically nondiagnostic in 3.1%, false negatives were observed in 1.5%, and there was only one false positive (0.06%). The breast cancer prevalence was 62%. Diagnostic accuracy measures of the OSCAR procedure with their 95% confidence intervals (95% CI) were the following: sensitivity (Se) = 97.4% (96.19-98.31); specificity (Sp) = 94.98% (92.94-96.56); positive predictive value (PPV) = 96.80% (95.48-97.81); negative predictive value (NPV) = 95.91% (94.02-97.33); positive likelihood ratio (LR+) = 19.39 (13.75-27.32); negative predictive ratio (LR-) = 0.03 (0.02-0.04), and; accuracy = 96.45% (95.42-97.31). The respective positive likelihood ratio (LR+) for each of the four categories of cytopathological diagnoses (with their 95% CI) which are malignant, suspicious, benign, and nondiagnostic were 540 (76-3827); 2.69 (1.8-3.96); 0.03 (0.02-0.04); and 0.37 (0.2-0.66), respectively. In conclusion, our study demonstrates that the OSCAR procedure is a highly reliable diagnostic approach and a perfect test to select patients requiring core-needle biopsy (CNB) when performed by interventional cytopathologists in a multidisciplinary and integrated OSC setting. Besides drastically limiting the rate of nondiagnostic specimens and diagnostic turn-around time, OSCAR is an efficient and powerful first-line diagnostic approach for patient-centered care.

Automatic diagnosis and classification of breast surgical samples with dynamic full-field OCT and machine learning.

Purpose: The adoption of emerging imaging technologies in the medical community is often hampered when they provide a new unfamiliar contrast that requires experience to be interpreted. Dynamic full-field optical coherence tomography (D-FF-OCT) microscopy is such an emerging technique. It provides fast, high-resolution images of excised tissues with a contrast comparable to H&E histology but without any tissue preparation and alteration. Approach: We designed and compared two machine learning approaches to support interpretation of D-FF-OCT images of breast surgical specimens and thus provide tools to facilitate medical adoption. We conducted a pilot study on 51 breast lumpectomy and mastectomy surgical specimens and more than 1000 individual 1.3×1.3 mm2 images and compared with standard H&E histology diagnosis. Results: Using our automatic diagnosis algorithms, we obtained an accuracy above 88% at the image level (1.3×1.3 mm2) and above 96% at the specimen level (above cm2). Conclusions: Altogether, these results demonstrate the high potential of D-FF-OCT coupled to machine learning to provide a rapid, automatic, and accurate histopathology diagnosis with minimal sample alteration.

Breast carcinoma detection in ex vivo fresh human breast surgical specimens using a fast slide-free confocal microscopy scanner: HIBISCUSS project.

BACKGROUND: New generation ultra-fast fluorescence confocal microscopy allows the ex vivo intraoperative analysis of fresh tissue. The High resolution Imaging for Breast carcInoma detection in ex vivo Specimens after breast Conserving sUrgery by hiStolog Scanner (HIBISCUSS) project aimed to develop an online learning program to recognize the main breast tissue features on ultra-fast fluorescence confocal microscopy images and to evaluate the performance of surgeons and pathologists in diagnosing cancerous and non-cancerous breast tissue in ultra-fast fluorescence confocal microscopy images. METHODS: Patients who underwent conservative surgery or mastectomy for breast carcinoma (invasive or in situ lesions) were included. The fresh specimens were stained with a fluorescent dye and imaged using a large field-of-view (20 cm2) ultra-fast fluorescence confocal microscope. RESULTS: One hundred and eighty-one patients were included. The images from 55 patients were annotated to generate learning sheets and images from 126 patients were blindly interpreted by seven surgeons and two pathologists. The time for tissue processing and ultra-fast fluorescence confocal microscopy imaging was between 8 and 10 min. The training program was composed of 110 images divided into nine learning sessions. The final database for blind performance assessment comprised 300 images. The mean duration for one training session and one performance round was 17 and 27 min respectively. The performance of pathologists was almost perfect with 99.6 per cent (standard deviation (s.d.) 5.4 per cent) accuracy. Surgeons' accuracy significantly increased (P = 0.001) from 83 per cent (s.d. 8.4 per cent) in round 1 to 98 per cent (s.d. 4.1 per cent) in round 7 as well as the sensitivity (P = 0.004). Specificity increased without significance from 84 per cent (s.d. 16.7 per cent) in round 1 to 87 per cent (s.d. 16.4 per cent) in round 7 (P = 0.060). CONCLUSION: Pathologists and surgeons showed a short learning curve in differentiating breast cancer from non-cancerous tissue in ultra-fast fluorescence confocal microscopy images. Performance assessment for both specialties supports ultra-fast fluorescence confocal microscopy evaluation for intraoperative management. REGISTRATION NUMBER: NCT04976556 (http://www.clinicaltrials.gov).

Diagnostic Challenges and Pitfalls of Mammary Pleomorphic Adenoma Illustrated by a Case Report.

Mammary pleomorphic adenoma is a biphasic tumor, characterized by epithelial-myoepithelial components with myxochondroid stroma, resembling the prototypic pleomorphic adenoma of the salivary glands. We report the multiple diagnostic pitfalls raised by a mammary pleomorphic adenoma, initially diagnosed as mucinous carcinoma on fine-needle aspiration (FNA) cytology and invasive carcinoma on needle core biopsy. The final diagnosis was made on the surgical specimen. As the term "pleomorphic" suggests, this tumor can present various phenotypes, some of which might be misleading on both FNA cytology or needle core biopsy. Rearrangements in PLAG1 and HMGA2 genes have not been detected in our patient. Mammary pleomorphic adenoma is considered a benign tumor, despite rare local recurrence and malignant behavior. Its correct identification, despite the difficulties, is essential to avoid unnecessary aggressive treatment.

Axillary reverse mapping using near-infrared fluorescence imaging in invasive breast cancer (ARMONIC study).

BACKGROUND: Axillary lymph node dissection (ALND) in patients with breast cancer has potential side effects, including upper-limb lymphedema. Axillary reverse mapping (ARM) is a technique that enables discrimination of the lymphatic drainage of the upper limb in the axillary lymph node basin from that of the breast. We aimed to evaluate ARM node identification by near-infrared (NIR) fluorescence imaging during total mastectomy with ALND and then to analyze potential predictive factors of ARM node involvement. METHODS: The study enrolled 119 patients diagnosed with invasive breast cancer with an indication for ALND. NIR imaging using indocyanine green dye was performed in 109 patients during standard ALND to identify ARM nodes and their corresponding lymphatic ducts. RESULTS: 94.5% of patients had ARM nodes identified (95%CI = [88.4-98.0]). The ARM nodes were localized in zone D in 63.4% of cases. Metastatic axillary lymph nodes were found in 55% in the whole cohort, and 19.4% also had metastasis in ARM nodes. Two patients had metastatic ARM nodes but not in the remaining axillary lymph nodes. No serious adverse events were observed. Only the amount of mitosis was significantly associated with ARM node metastasis. CONCLUSIONS: ARM by NIR fluorescence imaging could be a reliable technique to identify ARM nodes in real-time when ALND is performed. The clinical data compared with ARM node histological diagnosis showed only the amount of mitosis in the diagnostic biopsy is a potential predictive factor of ARM node involvement. CLINICAL TRIAL REGISTRATION: NCT02994225.

Near-Infrared Fluorescence Axillary Reverse Mapping (ARM) Procedure in Invasive Breast Cancer: Relationship between Fluorescence Signal in ARM Lymph Nodes and Clinical Outcomes.

The near-infrared (NIR) fluorescence axillary reverse mapping (ARM) procedure is a promising tool to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND). The ARMONIC clinical trial was conducted to validate the technique on a large cohort of patients and to analyze the predictive clinical factors for ARM lymph node metastasis. For the first time, the fluorescence signal intensity from the ARM lymph nodes was measured and correlated with clinical findings. A total of 109 patients with invasive breast cancer and indications of mastectomy and ALND underwent the NIR fluorescence ARM procedure. Indocyanine green was administered by intradermal injection followed by intraoperative identification and resection of the ARM lymph nodes with NIR fluorescence camera guidance. The fluorescence signal intensity and signal distribution were then measured ex vivo and compared with clinical outcomes. ARM lymph nodes were successfully identified by fluorescence in 94.5% of cases. The mean normalized fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes (p = 0.3728). At the microscopic level, the fluorescence signal distribution was focally intense in lymphoid tissue areas. Only the preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with a higher ARM node fluorescence signal intensity (p = 0.0253), though it was not significantly associated with the pathological nodal (pN) status (p = 0.8081). Based on an optimal cut-off fluorescence value, the final sensitivity and specificity of the NIR fluorescence ARM procedure for ARM lymph node metastatic involvement were 64.7% and 47.3%, respectively. Although our preliminary results did not show that fluorescence signal intensity is a reliable diagnostic tool, the NIR fluorescence ARM procedure may be useful for ARM lymph node identification. Clinical trial registration: NCT02994225.

[2021 update of the GEFPICS' recommendations for HER2 status assessment in invasive breast cancer in France]. / Mise à jour 2021 des recommandations du GEFPICS pour l'évaluation du statut HER2 dans les cancers infiltrants du sein en France.

The last international guidelines on HER2 determination in breast cancer have been updated in 2018 by the American Society of Clinical Oncology and College of American Pathologists, on the basis of a twenty-year practice and results of numerous clinical trials. Moreover, the emerging HER2-low concept for 1+ and 2+ non amplified breast cancers lead to refine French practices for HER2 status assessment. The GEFPICS group, composed of expert pathologists, herein presents the latest French recommendations for HER2 status evaluation in breast cancer, taking into account the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, HER2 status assessment remains one of the most important biomarkers in breast cancer and its quality guaranties the optimal patients' care. French pathologists' commitment in theranostic biomarker quality is more than ever required to provide the most efficient cares in oncology.

Adipose Tissue Properties in Tumor-Bearing Breasts.

The tissue stroma plays a major role in tumors' natural history. Most programs for tumor progression are not activated as cell-autonomous processes but under the conditions of cross-talks between tumor and stroma. Adipose tissue is a major component of breast stroma. This study compares adipose tissues in tumor-bearing breasts to those in tumor-free breasts with the intention of defining a signature that could translate into markers of cancer risk. In tumor-bearing breasts, we sampled adipose tissues adjacent to, or distant from the tumor. Parameters studied included: adipocytes size and density, immune cell infiltration, vascularization, secretome and gene expression. Adipose tissues from tumor-bearing breasts, whether adjacent to or distant from the tumor, do not differ from each other by any of these parameters. By contrast, adipose tissues from tumor-bearing breasts have the capacity to secrete twice as much interleukin 8 (IL-8) than those from tumor-free breasts and differentially express a set of 137 genes of which a significant fraction belongs to inflammation, integrin and wnt signaling pathways. These observations show that adipose tissues from tumor-bearing breasts have a distinct physiological status from those from tumor-free breasts. We propose that this constitutive status contributes as a non-cell autonomous process to determine permissiveness for tumor growth.

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group.

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

[GEFPICS' guidelines for management of breast cancer tissue samples in the neoadjuvant setting]. / Recommandations du GEFPICS pour la prise en charge des prélèvements dans le cadre du traitement néoadjuvant du cancer du sein.

Neoadjuvant therapy is an increasing treatment option in the management of breast cancer. The tumor response to neoadjuvant therapy, especially the pathological complete response, is a validated endpoint frequently used in clinical trials. However, there is still a lack of standardization for the surgical specimen management in the neoadjuvant setting. This leads to heterogeneity in the specimen handling and might lead to significant bias for the prognostic assessment of patients or in clinical trials. The GEFPICS group, composed of expert breast cancer pathologists, herein presents guidelines for the management of breast and axillary specimen before treatment (management of biopsy, items of the pathological report) and after neoadjuvant therapy (specimen handling, histological assessment of response, items of the pathological report and response grading systems).

Preoperative Breast Magnetic Resonance Imaging in Women With Local Ductal Carcinoma in Situ to Optimize Surgical Outcomes: Results From the Randomized Phase III Trial IRCIS.

PURPOSE: We evaluated the addition of breast magnetic resonance imaging (MRI) to standard radiologic evaluation on the re-intervention rate in women with ductal carcinoma in situ (DCIS) undergoing breast-conserving surgery. PATIENTS AND METHODS: Women with biopsy-proven DCIS corresponding to a unifocal microcalcification cluster or a mass less than 30 mm were randomly assigned to undergo MRI or standard evaluation. The primary end point was the re-intervention rate for positive or close margins (< 2 mm) in the 6 months after randomization ( ClinicalTrials.gov identifier: NCT01112254). RESULTS: A total of 360 patients from 10 hospitals in France were included in the study. Of the 352 analyzable patients, 178 were randomly assigned to the MRI arm, and 174 were assigned to the control arm. In the intent-to-treat analysis, 82 of 345 patients with the assessable end point were reoperated for positive or close margins within 6 months, resulting in a re-intervention rate of 20% (35 of 173) in the MRI arm and 27% (47 of 172) in the control arm. The absolute difference of 7% (95% CI, -2% to 16%) corresponded to a relative reduction of 26% (stratified odds ratio, 0.68; 95% CI, 0.41 to 1.1; P = .13). When considering only the per-protocol population with an assessable end point, the difference was 9% (stratified odds ratio, 0.59; 95% CI, 0.35 to 1.0; P = .05). Total mastectomy rates were 18% (31 of 176) in the MRI arm and 17% (30 of 173) in the control arm (stratified P = .93). For 100 lesions seen on MRI, nonmass-like enhancement was more predominant (82%) than mass enhancement (20%). Nevertheless, no specific morphologic and kinetic parameters for DCIS were identified. CONCLUSION: The study did not show sufficient surgical improvement with the use of preoperative MRI to be clinically relevant in DCIS staging. However, this could be reconsidered with the improvement of new MRI sequences and new modalities in magnetic resonance techniques.

Clinical Response to Induction Chemotherapy Predicts Outcome after Combined-Modality Therapy in Inflammatory Breast Cancer.

PURPOSE: To assess predictors of outcome in a cohort of Inflammatory Breast Cancer (IBC) patients receiving induction chemotherapy followed by local treatment. METHODS: We retrospectively reviewed 95 non-metastatic IBC patient files. RESULTS: Complete clinical response (cCR) was obtained in 15 (16%) patients. Median follow up was 13.4 years (IC95%: 10.4-14.6). Loco-regional control (LC), disease-free survival (DFS), and overall survival (OS) at 5 years were 85%, 41%, and 55%, respectively; cCR was associated with better DFS and OS in multivariate analyses adjusted for age (p = 0.02). CONCLUSIONS: Clinical response to upfront chemotherapy predicts the outcome of patients affected by IBC.

Low risk of invasive lobular carcinoma of the breast in carriers of BRCA1 (hereditary breast and ovarian cancer) and TP53 (Li-Fraumeni syndrome) germline mutations.

BACKGROUND: Invasive lobular carcinoma (ILC) of the breast has epidemiological, molecular and clinical specificities, and should likely be considered a unique entity. As for genetic susceptibility, CDH1 germline mutations predispose exclusively to ILC. Data are however scarce regarding ILC in women with BRCA1/2 (Hereditary Breast and Ovarian Cancer) and TP53 (Li-Fraumeni syndrome) germline mutations. METHODS: We included all breast cancers from female patients tested at our institute between 1992 and 2016 (n = 3469) for which pathology data were available. ILC proportion comparison according to mutational status was performed by a chi-squared test. The impact of susceptibility genes on ILC proportion was investigated by univariate logistic regression with wild-type patients as reference. RESULTS AND DISCUSSION: There were 265 (7.64%) ILC: 2/342 (0.58%) in BRCA1 patients, 24/238 (10%) in BRCA2 patients, 1/57 (1.75%) in TP53 patients and 238/2832 (8.4%) in non-carriers. The majority of breast cancers in all groups were invasive ductal and ductal in situ carcinomas. The difference in ILC proportion was highly significant (P < 0.001). Compared to wild-type patients, BRCA1 was associated with a lower ILC proportion (OR 0.064 [95% CI 0.016;0.259], P < 0.0001). BRCA2 OR was 1.222 [95%CI 0.785;1.902] (P = 0.374), TP53 OR was 0.195 [95%CI 0.027;1.412] (P = 0.105). ILC are therefore underrepresented in BRCA1 and TP53 mutation carriers. Formal significance (P = 0.05) was not reached for TP53, but statistical power was only 38%. Based on ILC incidence in the general population, we make the hypothesis that BRCA1 and TP53 do not predispose to ILC, as the few occurrences of ILC in mutation carriers could be attributed to chance and not to germline mutations. Our observations will be useful to clinical cancer geneticists managing patients with ILC, as a BRCA1 or TP53 mutation in these patients would be unlikely. Genetic counseling should be adapted accordingly.

Immunohistochemistry for Diagnosis of Metastatic Carcinomas of Unknown Primary Site.

Immunohistochemistry has become an essential ancillary examination for the identification and classification of carcinomas of unknown primary site (CUPs). Over the last decade, the diagnostic accuracy of organ- or tumour-specific immunomarkers and the clinical validation of effective immunohistochemical panels has improved significantly. When dealing with small sample sizes, diagnostic accuracy is crucial, particularly in the current era of targeted molecular and immune-based therapies. Effective systematic use of appropriate immunohistochemical panels enables accurate classification of most of the undifferentiated carcinomas as well as careful preservation of tissues for potential molecular or other ancillary tests. This review discusses the algorithmic approach to the diagnosis of CUPs using CK7 and CK20 staining patterns. It outlines the most frequently used tissue-specific antibodies, provides some pitfalls essential in avoiding potential diagnostic errors and discusses the complementary tools, such as molecular tumour profiling and mutation-specific antibodies, for the improvement of diagnosis and prediction of the treatment response.

COX2/PTGS2 Expression Is Predictive of Response to Neoadjuvant Celecoxib in HER2-negative Breast Cancer Patients.

BACKGROUND: The prognostic and predictive role of cyclo-oxygenase-2 (COX2) in breast cancer is still debated, and in particular, its role as a target of COX2 inhibitor (celecoxib) in neoadjuvant setting. MATERIALS AND METHODS: We analyzed a series of 156 breast cancer samples from patients of the COX2 inhibitor-treated arm included in the REMAGUS-02 randomized phase II trial. COX2 gene expression was assessed by reverse transcription and quantitative polymerase chain reaction using ribonucleic acid from frozen biopsies. Pathological complete response (pCR) was the surrogate end-point. RESULTS: Significantly higher rates of grade 3, and estrogen and progesterone receptor negativity were observed in tumors with the highest expression of COX2. pCR rates were significantly higher in COX2-overexpressing tumors in patients receiving celecoxib. The test for interaction between COX2 gene expression and the celecoxib effect was statistically significant (p<0.01), but was not retained in the multivariate analysis. CONCLUSION: COX2 overexpression is predictive of pCR in patients with celecoxib-treated tumors. The efficacy of celecoxib in breast cancer might be improved by quantification of COX2 gene expression.

Daily Biopsy Diagnosis in Surgical Pathology: Concordance Between Light Microscopy and Whole-Slide Imaging in Real-Life Conditions.

OBJECTIVES: The current challenge for the various digital whole-slide imaging (WSI) systems is to be definitively validated for diagnostic purposes. We designed a concordance study between glass slide and digital slide diagnosis in real-life conditions, coupled with an ergonomic study. METHODS: Three senior pathologists evaluated, first in glass slides and then in digital slides, 119 biopsy cases, including 749 slides, with 332 H&E saffron stains and 417 additional techniques, mainly immunohistochemistry. RESULTS: All digital slides, including specially stained slides, were interpretable. Concordance between glass slides and digital slides was observed in 87.4% of cases. Minor discordances were observed in 12 (10.1%) cases and major discordances, with therapeutic impact, in three (2.5%), including one related to WSI. The satisfaction of participants was high and increased with time. CONCLUSIONS: Our study confirms the feasibility and accuracy of WSI diagnosis, even for cases having multiple samples and requiring special staining techniques, such as immunohistochemistry and in situ hybridization.

Preclinical ex vivo evaluation of the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses.

OBJECTIVES: To assess the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses in freshly removed surgical specimens, in preparation for its clinical development. METHODS: Sixty-four breast masses from consenting patients who had undergone either a lumpectomy or a mastectomy were included. Label-free fluorescence spectral acquisitions were obtained with a 25G fibre-containing needle inserted into the mass. Data from benign and malignant masses were compared to establish the most discriminating thresholds and measurement algorithms. Accuracy was verified using the bootstrap method. RESULTS: The final histological examination revealed 44 invasive carcinomas and 20 benign lesions. The maximum intensity of fluorescence signal was discriminant between benign and malignant masses (p < .0001) whatever their sizes. Statistical analysis indicated that choosing five random measurements per mass was the best compromise to obtain high sensitivity and high negative predictive value with the fewest measurements. Thus, malignant tumours were identified with a mean sensitivity, specificity, negative and positive predictive value of 98.8%, 85.4%, 97.2% and 93.5%, respectively. CONCLUSION: This new in situ tissue autofluorescence evaluation device allows accurate discrimination between benign and malignant breast masses and deserves clinical development. KEY POINTS: • A new device allows in situ label-free fluorescence analysis of ex vivo breast masses • Maximum fluorescence intensity discriminates benign from malignant masses (p < .0001) • Five random measurements allow a high negative predictive value (97.2%).