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Background The crucial point in making treatment decisions for breast cancer patients is the assessment of tumour aggressiveness. The established prognostic markers may be insufficient to stratify cancer patients into treatment relevant risk groups. Emerging evidence indicates that mechanical properties of cancer cells and their microenvironment that occur on a nanometre scale play a critical role in cancer invasion and metastases. Therefore, detecting these nanomechanical changes could serve as biomarker of cancer aggressiveness. Trial design We conduct a prospective, blinded study in a routine clinical setting. Using minimal invasive breast biopsies we measure the nanomechanical (stiffness) properties of human breast tissue with our atomic force microscope (AFM) based method known as ARTIDIS (Automated and Reliable Tissue Diagnostics). These properties can only be measured using fresh (non-fixed) tissue under physiological conditions (Custodiol transplant buffer). This novel method is based on the use of a micro-fabricated 20nm-sharp tip that indents several thousand individual locations across tissue specimens within 60-180 minutes. Each indentation effectively measures the stiffness of local structures (e.g. cancer cells, extracellular matrix) located under the tip. Thus we obtain a quantitative, biopsy-wide, nanomechanical profile. Post-AFM the same biopsy is used for routine histopathological diagnosis, the current diagnostic gold standard to which the nanomechanical profile is then correlated. Eligibility criteria All women undergoing a minimal invasive breast biopsy (core needle or vacuum assisted biopsy) at the breast centre of the University of Basel. Exclusion criteria: age younger than 18years, necrotic/disintegrated biopsy, and technical limitations Specific aims Our primary endpoint is to differentiate benign from cancerous breast lesions based on their nanomechanical properties. Our secondary endpoint is to subclassify biopsies with cancerous lesions into the current four main breast cancer subgroups (Luminal A, Luminal B, HER2+ and basal-like). Statistical analysis The full dataset will include all patients with valid AFM measurements. Primary analysis: the proportion of true positive results divided by the total number of patients with malignant tumour (sensitivity) will be estimated and presented together with its 95% confidence interval. The histological diagnosis of the same biopsy as analysed by AFM will serve as gold standard. Present accrual and target accrual Present accrual as of June 12, 2017: 200 breast tissue biopsies. Target accrual is 508 biopsies. This will allow for a power of 0.8 and a sensitivity of 90%. Contact information: Rosemarie.Burian@usb.ch Citation Format: Burian R, Appenzeller T, Oertle P, Raez C, Lim R, Forte S, Dellas S, Münst S, Obermann E, Plodinec M. Atomic force microscopy (AFM) - a novel nanotool for cancer diagnostics: A prospective, blinded study of nanomechanical profiling of human breast tissue as a potential biomarker for stratifying low- and high-risk breast cancer subtypes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-06-05.

Clinical treatment of breast cancer is successful. What remains a challenge for individualized therapy is the prediction of which cancer responds to which treatment. Emerging evidence indicates that mechanical alterations on a (sub-) cellular level are markers of cancer aggressiveness. Detecting these could serve as biomarker and help optimize cancer diagnosis, therapy choice, and patient follow-up. We developed a diagnostic platform called ARTIDIS (Automated and Reliable Tissue Diagnostics) based on atomic force microscopy (AFM)-type nanomechanical testing. ARTIDIS uses a 20-nanometer-sharp tip measuring 10'000 miniscule indentation across the biopsy's surface and quantifies stiffness on a molecular level. The relative size and distribution of stiffness values is integrated into a data analytics tool that systematically analyses tissue biomechanics in correlation to clinical parameters, histological prognostic markers, and treatment yielding prognostic and predictive value. To demonstrate ARTIDIS' clinical utility for diagnosis and development of prognostic and predictive biomarkers we conducted a prospective blinded clinical study on 545 patients. All patients undergoing a core needle or vacuum biopsy of the breast qualified. Within the routine clinical workflow, ARTIDIS analyzed fresh biopsies in a physiological solution preserving the biopsy's viability for histological and genetic analysis. Per patient, one representative biopsy was measured, marked and reunited with the patient's other biopsies for standard histological procedure. In total, 583 biopsies from N=545 patients were analyzed: 62.1% were B1-B4 (normal, benign, of uncertain malignant potential, or suspicious); a third (36.9%) malignant (B5a-B5d), and six specimens (1%) not assignable. Half of all B5b lesions were Luminal B, followed by Luminal A, Luminal B-like, HER2-positive or triple negative. The analysis of the primary endpoint (N=520 patients qualified, N=3 were excluded due to missing information) demonstrated ARTIDIS' ability to detect cancer in all samples including lesions with We could separate more aggressive cases within the Luminal B subtype (CI 95%; sensitivity 83%, specificity 82%, AUC = 0.86). Secondary analysis also indicated a similarity between the majority of B3 (uncertain) and B2 (benign) lesions and distinct differences from all B5a and B5b, which could help reduce overtreatment of B3 lesions. This large prospective single center study demonstrated ARTIDIS' ability to differentiate benign from malignant lesions and to further subclassify specific breast cancer subtypes. In addition, Luminal B cancers exhibited nanomechanical profiles associated with better or worse prognosis and treatment outcome. The final analysis results provide cutoffs that will be assessed in the upcoming large multicenter prospective trial. Finally, this study helped to determine clinical utility of nanomechanical characterization of breast and other solid cancers for prediction and treatment optimization. Citation Format: Rosemarie Burian, Ahmed Jizawi, Flora Scott, Sabine Schädelin, Christian Raez, Simone Muenst-Soysal, Tatjana Vlaijnic, Ellen Obermann, Sophie Dellas, Serafino Forte, Zlatko Marušić, Tobias Appenzeller, Philipp Oertle, Nam Seung-Zln, Gregory Zaugg, Milan Liepelt, Gabriel Zihlman, Roderick Lim, Marko Loparic, Marija Plodinec. First clinical validation of the physical biomarker based on the nanomechanical tissue profiling for rapid breast cancer diagnosis, prognosis and predication of treatment outcome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-273.

Mechanical properties and physical interactions of cells and their microenvironment that occur at nanometre scale play a critical role in cancer progression and metastatic dissemination (https://physics.cancer.gov). Nanomechanical profiling of breast tissue enables fast and precise cancer diagnosis. Detecting extreme viscoelastic (soft) cancer cells in biopsies provides a direct biomarker of cancer aggressiveness (Plodinec et. al., 2012). In an ongoing single centre clinical trial conducted in a routine clinical setting we distinguish benign from cancerous breast lesions within 2.5 hours using an atomic force microscope (AFM)-based method known as ARTIDIS. ARTIDIS employs a micro-fabricated 20nm-sharp tip which indents 10,000 locations/sample to measure the stiffness of cellular and matrix structures. In a comprehensive web-based platform we collect, verify and analyze these 2.54 millions of AFM indentations in relation to over 100 clinical parameters /patient using artificial intelligence. In our interim analysis of core needle and vacuum biopsies (N=254), 34 samples were classified as B1 (normal); 127 as B2 (benign); 12 as B3 (uncertain malignant potential); 0 as B4 (suspicious); 18 as B5a (ductal carcinoma in situ), and 58 as B5b (malignant). The diagnostic AFM ROC curve of B1 and B2 lesions vs. all B5 lesions (CI 95%; 100% sensitivity, 90% specificity, AUC = 0.98) demonstrated ARTIDIS outstanding ability to detect cancer in samples with >80% neoplastic tissue. An analysis including lesions with Citation Format: Rosemarie Burian, Ahmed Jizawi, Gabriel Zihlmann, Tobias Appenzeller, Philipp Oertle, Christian Raez, Roderick Y. Lim, Serafino Forte, Sophie Dellas, Simone Muenst, Tatjana Vlajnic, Ellen Obermann, Maria Ricci, Kevin Grimm, Ferdinand Niedermann, Marko Loparic, Marija Plodinec. A prospective, double-blinded clinical study using atomic force microscopy for fast diagnosis and subtyping of low and high-risk breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3140.