David E. Reese

A Noninvasive Blood-based Combinatorial Proteomic Biomarker Assay to Detect Breast Cancer in Women Under the Age of 50 Years

Background: Despite significant advances in breast imaging, the ability to detect breast cancer (BC) remains a challenge. To address the unmet needs of the current BC detection paradigm, 2 prospective clinical trials were conducted to develop a blood‐based combinatorial proteomic biomarker assay (Videssa Breast) to accurately detect BC and reduce false positives (FPs) from suspicious imaging findings. Patients and Methods: Provista‐001 and Provista‐002 (cohort one) enrolled Breast Imaging Reporting and Data System 3 or 4 women aged under 50 years. Serum was evaluated for 11 serum protein biomarkers and 33 tumor‐associated autoantibodies. Individual biomarker expression, demographics, and clinical characteristics data from Provista‐001 were combined to develop a logistic regression model to detect BC. The performance was tested using Provista‐002 cohort one (validation set). Results: The training model had a sensitivity and specificity of 92.3% and 85.3% (BC prevalence, 7.7%), respectively. In the validation set (BC prevalence, 2.9%), the sensitivity and specificity were 66.7% and 81.5%, respectively. The negative predictive value was high in both sets (99.3% and 98.8%, respectively). Videssa Breast performance in the combined training and validation set was 99.1% negative predictive value, 87.5% sensitivity, 83.8% specificity, and 25.2% positive predictive value (BC prevalence, 5.87%). Overall, imaging resulted in 341 participants receiving follow‐up procedures to detect 30 cancers (90.6% FP rate). Videssa Breast would have recommended 111 participants for follow‐up, a 67% reduction in FPs (P < .00001). Conclusions: Videssa Breast can effectively detect BC when used in conjunction with imaging and can substantially reduce unnecessary medical procedures, as well as provide assurance to women that they likely do not have BC. Micro‐Abstract: To improve breast cancer diagnosis, 2 prospective clinical trials were conducted to test (n = 351) and validate (n = 210) Videssa Breast. If used in conjunction with imaging, Videssa Breast could have reduced unnecessary biopsies by up to 67%. These results support the joint use of breast imaging and Videssa Breast to better inform clinical decisions for women under age 50.

Integration of Serum Protein Biomarker and Tumor Associated Autoantibody Expression Data Increases the Ability of a Blood-Based Proteomic Assay to Identify Breast Cancer

Despite significant advances in breast imaging, the ability to accurately detect Breast Cancer (BC) remains a challenge. With the discovery of key biomarkers and protein signatures for BC, proteomic technologies are currently poised to serve as an ideal diagnostic adjunct to imaging. Research studies have shown that breast tumors are associated with systemic changes in levels of both serum protein biomarkers (SPB) and tumor associated autoantibodies (TAAb). However, the independent contribution of SPB and TAAb expression data for identifying BC relative to a combinatorial SPB and TAAb approach has not been fully investigated. This study evaluates these contributions using a retrospective cohort of pre-biopsy serum samples with known clinical outcomes collected from a single site, thus minimizing potential site-to-site variation and enabling direct assessment of SPB and TAAb contributions to identify BC. All serum samples (n = 210) were collected prior to biopsy. These specimens were obtained from 18 participants with no evidence of breast disease (ND), 92 participants diagnosed with Benign Breast Disease (BBD) and 100 participants diagnosed with BC, including DCIS. All BBD and BC diagnoses were based on pathology results from biopsy. Statistical models were developed to differentiate BC from non-BC (i.e., BBD and ND) using expression data from SPB alone, TAAb alone, and a combination of SPB and TAAb. When SPB data was independently used for modeling, clinical sensitivity and specificity for detection of BC were 74.7% and 77.0%, respectively. When TAAb data was independently used, clinical sensitivity and specificity for detection of BC were 72.2% and 70.8%, respectively. When modeling integrated data from both SPB and TAAb, the clinical sensitivity and specificity for detection of BC improved to 81.0% and 78.8%, respectively. These data demonstrate the benefit of the integration of SPB and TAAb data and strongly support the further development of combinatorial proteomic approaches for detecting BC.

dtectDx Breast: A serum biomarker test for breast cancer detection used in conjunction with traditional mammography screening.

18 Background: Provista Diagnostics has developed a test that analyzes serum concentrations of 5 protein biomarkers in order to detect breast cancer. The dtectDx Breast test utilizes a proprietary algorithm that has been described previously (Weber et al. 2010). In this study, it was noted that the algorithm performs best in women under age 50. The aim of this study was to evaluate the performance characteristics of dtectDx Breast in women under age 50 in a commercial setting and compare the results with data from the previous clinical study. METHODS The dtectDx Breast test measures the concentrations of IL-8, IL-12, VEGF, CEA, and HGF via ELISA. These data combined with select patient characteristics and Provistas proprietary algorithm result in a test value that is characterized as normal or elevated. dtectDx Breast test reports issued for women under age 50 were reviewed from a 3-year time period and prescribing physicians were interviewed regarding follow-up care and outcome measures (largely imaging studies, if warranted). RESULTS Of the 908 patients, 8 samples were rejected based on serum quality. Of the remaining 900 patients, 121 were reported as elevated (12.7%). In 4 cases, these elevated results were confirmed cases of breast cancer. Of these, 2 patients initially showed no screening evidence of cancer, but upon further evaluation (after receipt of dtectDx Breast results) were diagnosed with breast cancer. dtectDx correctly identified DCIS 66% of the time (n=2). CONCLUSIONS These results describe the use of dtectDx Breast in a clinical setting and confirm that the assay behaves similarly to previously published results (Weber et al 2010). While the false-positive rate is higher than predicted (12.7% vs 6.8%), the assay correctly identified 4 of 4 invasive cancers and 2 of 3 DCIS cases. Since two of the invasive cancer cases were originally not detected via standard screening procedures, the assay has demonstrated important clinical utility when used in conjunction with mammography/standard of care. Here we show that, in the commercial patient population, when combined with standard of care, dtectDx Breast improves the detection of breast cancer in women under 50.

Breast density does not impact the ability of Videssa® Breast to detect breast cancer in women under age 50

Breast density is associated with reduced imaging resolution in the detection of breast cancer. A biochemical approach that is not affected by density would provide an important tool to healthcare professionals who are managing women with dense breasts and suspicious imaging findings. Videssa® Breast is a combinatorial proteomic biomarker assay (CPBA), comprised of Serum Protein Biomarkers (SPB) and Tumor Associated Autoantibodies (TAAb) integrated with patient-specific clinical data to produce a diagnostic score that reliably detects breast cancer (BC) as an adjunctive tool to imaging. The performance of Videssa® Breast was evaluated in the dense (a and b) and non-dense (c and d) groups in a population of n = 545 women under age 50. The sensitivity and specificity in the dense breast group were calculated to be 88.9% and 81.2%, respectively, and 92.3% and 86.6%, respectively, for the non-dense group. No significant differences were observed in the sensitivity (p = 1.0) or specificity (p = 0.18) between these groups. The NPV was 99.3% and 99.1% in non-dense and dense groups, respectively. Unlike imaging, Videssa® Breast does not appear to be impacted by breast density; it can effectively detect breast cancer in women with dense and non-dense breasts alike. Thus, Videssa® Breast provides a powerful tool for healthcare providers when women with dense breasts present with challenging imaging findings. In addition, Videssa® Breast provides assurance to women with dense breasts that they do not have breast cancer, reducing further anxiety in this higher risk patient population.

Abstract P4-01-07: A liquid biopsy test for breast cancer detection provides consistent diagnostic results in patients over six months

Current methods of breast cancer detection are often confounded by imaging limitations, such as lesion size, benign breast tissue, and dense breasts. These limitations result in unnecessary biopsies due to false positive findings based on imaging. Despite the increased ability to detect early breast cancer, the over-use of biopsy remains an issue. There is a critical need for new approaches to breast cancer detection that improve diagnostic accuracy when clinical assessment is challenging. Provista Diagnostics has developed Videssa® Breast - a blood-based proteomic test that measures serum protein biomarkers (SPBs) and tumor-associated autoantibodies (TAAbs). Patient biochemical data is combined with clinical data to generate a diagnostic score that correlates with either the absence or presence of breast cancer (Grades I through III). The ability of Videssa® breast to detect cancer (Invasive Breast Cancer and Ductal Carcinoma in situ) was evaluated using prospective, multi-center clinical trials. The Provista-001 study enrolled 351 women ages 25-49 and included a follow-up visit at 6 months with an additional blood draw. Eligible patients included women assessed as ACR BIRADS® 3 or 4 on imaging with no history of breast cancer or prior breast biopsy. Serum samples from the initial visit and 6 month follow-up visit of Provista-001 were analyzed using Videssa® Breast to determine if diagnostic results for benign subjects were similar over the course of the study. Samples were analyzed for SPBs and TAAbs in order to determine whether analyte levels and diagnostic scores change over a 6-month period in patients diagnosed with a benign breast condition. Linear regression data for analytes shows overall high fidelity between the initial visit and follow-up. In addition, samples that were TAAb-positive for a given target at the initial visit tended to remain positive at follow-up. Sample background, deriving from unidentified immunological factors, can confound the analytical output when measuring TAAbs in serum. Interestingly, sample background was highly reproducible between both visits, suggesting that these values are related to inherent patient-specific factors. Overall, these data demonstrate high analytical reproducibility for in expression of independent Videssa® Breast biomarkers in patients diagnosed with a benign breast condition over the course of six months. Data for 236 women were compared between visits and demonstrated greater than 80% concordance in diagnostic status. The ability of Videssa® breast to provide consistent diagnostic results over 6 months further supports use of the test as an adjunct to imaging for the early detection of breast cancer and provides physicians with an additional tool that can be used to inform the decision to biopsy or increase vigilance through active monitoring. Citation Format: Benson KL, Henderson MC, Silver M, Gordon K, Borman S, Letsios E, Tran Q, Mulpuri R, Reese DE. A liquid biopsy test for breast cancer detection provides consistent diagnostic results in patients over six months [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-01-07.

Abstract P5-02-02: The final study report on the performance of Klarify™. Assessment of full data set from NCT01839045 a 6-month liquid biopsy panel run in women under the age of 50 that were initially assessed as a high risk population

The precise diagnosis of breast lesions represents a significant problem in women under the age of 50, especially given the high prevalence of confounding factors such as dense breast. No new approaches have been developed to augment standard of care in the more precise detection of breast cancer. The combination of breast imaging with a robust protein signature that would detect biochemical cues of breast cancer offers a potentially attractive approach to detection regardless of the quality of the radiographic evidence. We have recently tested a protein signature (KARIFY BREAST™) composed of immune-regulatory cytokines, growth factors and tumor-associated autoantibodies (TAAbs). Here, we confirm the hypothesis that this protein signature, combined with standard of care can increase the precision of the diagnosis of breast cancer in women under the age of 50. We have tested this method in a prospective study of 351 women at 8 centers across the US in a randomized and blinded manner. Presented is both data from the initial blood draw and results of the six-month follow up blood draw. The achievement of 93% sensitivity and greater than a 80 percent specificity was demonstrated. Methods: Provista-001 enrolled 351 patients from 9 sites across the US and will follow patients for 6 months prior to first blood draw under IRB approval. Upon enrollment, patients were randomized to either training or validation groups. Clinical truth was set at equal to or greater than 80% sensitivity/specificity. Serum protein biomarkers and autoantibodies identified in prior proteomic screens were measured prior to biopsy. Individual biomarker (25 serum protein biomarkers (SPB) and TAAbs) concentrations were measured , together with specific patient data were evaluated using various logistic regression models. Additionally, 200 patients were used as a training set to develop and refine new models, which were then validated in the remaining 151 subjects. Clinical findings were compared to biopsy (largely BIRADS 4) or were followed for 6 months and re-assessed (BIRADS 3). The novel algorithm utilizing patient data, SPBs and TAAb concentrations and regression models were able to distinguish benign from breast cancer lesions in a statistically significant manner. Importantly, the SPBs alone were unable to adequately distinguish benign lesions, consistent with prior work. However, the addition of TAAbs markedly increased both the sensitivity (93%) and specificity (80.3%) of the assay in this group of women. The use of the algorithm in conjunction with imaging detected more lesions than imaging alone. Our findings suggest that when used in combination, the protein signature developed here and breast imaging provides a more precise detection methodology than either alone. This is particularly important in women under the age of 50 where detection is difficult. The follow-up data at six months (BIRADS 3) have yielded additional data in this understudied group of women. Such as the apparent lack of effect of breast density on early detection when using the algorithm. Citation Format: Reese DE, Lourenco A, Mulpuri R, Borman S, Benson K, Alpers J, Silver M. The final study report on the performance of Klarify™. Assessment of full data set from NCT01839045 a 6-month liquid biopsy panel run in women under the age of 50 that were initially assessed as a high risk population. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-02-02.

Provista 002 prospective randomized trial utilizing Videssa in conjunction with imaging to detect breast cancer from patients with imaging findings age 25-75.

31 Background: An approach to detection that relies on biochemical markers of breast cancer would significantly contribute to more accurate detection in women with suspicious lesions. The combination of imaging, which identifies anatomical anomalies consistent with cancer with proteomic approaches promises to provide a powerful detection paradigm. A proteomic detection approach would provide a powerful tool for the detection of breast cancer in women with dense breast, a diagnosis that is difficult utilizing imaging alone. While protein signatures for the presence of breast cancer have remained elusive, we have developed a novel approach that combines serum protein biomarkers with tumor-associated autoantibodies. We utilized prospectively collected serum samples to develop novel algorithms for use in conjunction with imaging. We tested whether the assay was able to distinguish benign from invasive breast cancers in a prospective, randomized setting. METHODS Provista-002 enrolled 509 patients from multiple sites across the US and followed for 6 months after the first blood draw under IRB approval. Patients were consented after assessment of a BIRADS 3 or 4 and considered eligible if they were between 25 and 75 years of age, no history of cancer, no prior breast biopsy within the last six months, and were assessed as BIRADS 3 or 4 within 28 days. Upon enrollment, patients were randomized to either training or validation groups. Clinical truth was considered equal to or greater than 80% sensitivity and/or specificity. Serum protein biomarkers and tumor-associated autoantibodies identified in prior proteomic screens were measured prior to biopsy in a blinded and randomized fashion. Individual biomarker concentrations, together with specific patient data were evaluated using various logistic regression models developed from prior studies. RESULTS Provista-002 demonstrated a clear difference between women under the age of 50 from over the age of 50 in both markers required for early detection and the algorithm (models) used to distinguish benign from invasive breast cancer/DCIS. This is the first study that demonstrates clearly that modeling of proteomic patterns differs significantly in the BIRADS 3/4 setting and in the detection of early breast cancer lesions. As demonstrated in Provista - 001, we did not observe a statistical difference between early detection in women with dense breast and those with mostly fatty breast. The ability of the Videssa assay to distinguish between invasive breast cancer/DCIS from benign breast conditions was demonstrated as 85.7% sensitivity and 82.4% specificity for women under the age of 50 (although, unfortunately all lesions were pathologically confirmed to be CIS) and in women over the age of 50, the sensitivity was 86.4% and specificity was 83%. CONCLUSIONS As above, both age groups of women needed distinct marker sets and linear regressions to distinguish benign (non-clinically significant) lesions from those that needed further evaluation (DCIS and IBC). CLINICAL TRIAL INFORMATION NCT02078570.

Age-related variations: A retrospective analysis of 851 prospectively collected patient samples to determine the benefit of combining combinatorial protein biomarker assay for risk assessment in women with dense breast.

27 Background: Clinicians often experience difficulty in differentiating benign lesions from invasive breast cancers in patients designated as dense breast. A major limitation of radiological breast cancer screening methods involves a decrease in sensitivity and specificity in women with dense breast. Thus, we sought to test whether Klarify Breast, a combinatorial protein-based biomarker panel could improve early detection of significant breast lesions in a controlled fashion. Clearly, a diagnostic assay that would provide biochemical evidence in the patients clinical course is greatly needed. METHODS We have conducted two independent, multi-center, prospective clinical trials to establish the clinical validity of Klarify Breast - an assay that uses multiple Serum Protein Biomarkers (SPBs), Tumor-Associated Autoantibodies (TAAbs), patient specific clinical data and develop a score to differentiate patients with benign breast disease from those with invasive breast cancer. Independent panels of biomarkers and associated algorithms were developed using prospectively collected samples from women under age 50 (n = 351) and from women ages 25-75 (n = 500). Here, we present the benefit of integrating the results of Klarify Breast test with patient imaging to best assess risk in women with dense breast. RESULTS While performance of the assay was somewhat age dependent (women under the age of 50 demonstrated a higher sensitivity and specificity than women over the age of 50). Here we present data that both groups of women clearly benefit from the addition of a biomarker assay combined with standard of care imaging in identifying invasive breast lesions. CONCLUSIONS Clearly, in women with dense breast, where radiologic studies alone do not permit full assessment in women with a dense breast finding. The biomarker test here, comprised of TAAbs and SPBs, offers a clear advantage in terms of NPV, PPV, Sensitivity and specificity. The results argue strongly for the use of appropriate biomarkers to augment imaging based breast cancer screening in women with dense breast or those who are at high risk. CLINICAL TRIAL INFORMATION NCT01839045, NCT02078570.

Abstract P5-03-05: Provista-001 a multi-center prospective study of protein signature used in the differentiation of benign breast lesions from invasive breast cancer in women under the age of 50 with a BI-RADS 3 or 4

Background: Over-diagnosis of breast lesions represents a significant problem in detection and screening of breast cancer, especially in women under the age of 50. Despite this issue, few new approaches have been developed to augment standard of care in the more precise detection of breast cancer. The combination of breast imaging, which provides anatomical evidence, with a robust protein signature that would detect biochemical cues of breast cancer offers an attractive approach to this problem. We have recently identified a protein signature composed of immune-regulatory cytokines, growth factors and tumor derived autoantibodies. Here, we test the hypothesis that a protein signature, combined with standard of care can greatly increase the precision of breast cancer diagnosis in women under the age of 50 in a randomized and blinded study. Methods: Provista-001 enrolled 351 patients from 10 sites across the US and will follow patients for 6 months following the first blood draw under IRB approval. Patients were consented after first assessment of a BIRADS 3 or 4 and considered eligible if they were under the age of 50, no history of cancer, no prior breast biopsy, and were assessed as BIRADS 3 or 4 within 21 days of blood draw. Upon enrollment, patients were randomized to either training or validation groups. Clinical truth was set at equal to or greater than 80% sensitivity/specificity. Serum protein biomarkers and autoantibodies identified in prior proteomic screens were measured in serum samples collected prior to biopsy. Individual biomarker (22 serum protein and autoantibodies) concentrations, together with specific patient data were evaluated using various logistic regression models developed from prior retrospective studies. A training set, comprised of 200 patients, was used to develop and refine new models, which were then validated in the remaining 151 subjects. Clinical findings were compared to biopsy (largely BIRADS 4) or will be followed for 6 months and re-assessed (BIRADS 3). Results: The novel algorithm utilizing patient data, serum protein and autoantibody concentrations combined with regression models was able to distinguish benign from breast cancer lesions in a statistically significant manner. Importantly, the serum protein biomarkers alone were unable to adequately distinguish benign lesions, consistent with prior work. However, the addition of tumor autoantibodies markedly increased both the sensitivity and specificity of the assay in this group of women. The use of the algorithm in conjunction with imaging was more accurate than imaging alone in this population. Conclusions: Our current findings suggest that when used in combination, the protein signature developed here and breast imaging provides a more precise detection methodology than either alone. This is particularly important in women under the age of 50 where a low prevalence of disease makes detection difficult. The follow-up data at six months (BIRADS 3) will yield additional data in this understudied group of women. Additional studies utilizing the protein signature with women over the age of 50 are currently underway. Citation Format: David E Reese, Michael Silver, Susan Yeh, Sherri Borman, Henderson C Meredith. Provista-001 a multi-center prospective study of protein signature used in the differentiation of benign breast lesions from invasive breast cancer in women under the age of 50 with a BI-RADS 3 or 4 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-03-05.

Use of serum biomarkers to differentiate benign breast lesions from invasive breast cancer in women with a BI-RADS 3 or 4.

20 Background: The Provista Biomarker Assay (PBA) combines multiple serum biomarkers, measured at high sensitivity, in order to determine the likelihood of an invasive breast tumor at the time of testing. PBA, when paired with traditional breast imaging technologies, provides a biochemical tool that can help guide additional imaging or biopsy decisions. As part of our ongoing efforts to characterize clinical performance, we conducted a proof-of-concept study wherein PBA assay performance was tested in prospectively enrolled BI-RADS 3 and 4 patients. METHODS To maximize performance and sensitivity, we utilized the Meso Scale Discovery ELISA-based system to analyze a randomized cohort of 350 prebiopsy serum samples from women under the age of 50. Enrollment in the trial required a BI-RADS 3 or 4 radiologic assessment utilizing multiple imaging modalities. Serum proteins and autoantibodies were measured for each patient and applied to a proprietary statistical algorithm. RESULTS We were able to obtain statistically significant differentiation using the Provista Biomarker Assay in women under age 50 who were characterized as BI-RADS 3 or 4. The majority of patients in the study were diagnosed with a benign breast lesion, consistent with prior studies. However, the data indicate that PBA, when used in combination with imaging techniques, more accurately informs clinical decision-making. CONCLUSIONS The Provista Biomarker Assay provides a powerful tool for physicians to more accurately assess patients with a BI-RADS 3 or 4 classification. With more clinical evidence, the assay may translate into changes in clinical treatment decisions for women under the age of 50 with a BI-RADS 3 or 4 classification. CLINICAL TRIAL INFORMATION NCT01839045.