Preliminary reports suggest that it may be possible to diagnose breast cancer from a blood test, but an expert approached for comment says there are questions over clinical utility. A next-generation systems biology approach using a library of single-stranded oligodeoxynucleotides (ssODNs) may be used to interrogate exosomes from a blood sample to determine whether a woman has breast cancer or not. Using the Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) platform developed by Caris Life Sciences, the study authors were able to generate "aptamer" profiles (ssODNs from the library) specific for breast cancer using blood samples. The study was published online February 20 in Scientific Reports. "The key feature of the ADAPT platform is its ability to understand perturbations in biological systems and highlight these features in their entirety," David Spetzler, PhD, president and chief scientific officer, Caris Life Sciences, and co-corresponding author of the study, told Medscape Medical News. "The ADAPT platform is a systems biology approach and does not look for specific aberrations associated with cancer," he added. "This is an interesting preliminary report suggesting that this assay may distinguish patients with established cancers from normal individuals," Daniel Hayes, MD, professor of breast cancer research at the University of Michigan Comprehensive Cancer Center, Ann Arbor, told Medscape Medical News. "However, substantial research needs to be done to introduce such an assay into routine clinical practice. It appears that the assay has analytical validity, although it is modestly difficult to determine from this report," he added. Dr Hayes is also president of the American Society for Clinical Oncology. He noted that the views he was providing are his own and that he is not speaking in any official capacity. Plasma Exosome Profiling The study authors indicate that "ADAPT relies on sample fractionation to identify and characterize specific subpopulations of macromolecules and complexes in blood plasma, including those residing on the surface of exosomes." In practice, exosomes are extracellular vesicles, which cells secrete into the circulation. By capturing information present in exosomes, it is possible to get snapshots of what health looks like (using blood from control persons) and what disease looks like (using blood from patients). Exosomes can be generated from plasma samples using either polyethylene glycol precipitation or ultracentrifugation and visualized with transmission electron microscopy. In developing ADAPT, the investigators started with a random ssODNs library of 35-nucleotide variable regions with 1011 sequences (L0). L0 was then used with exosomes from pooled blood plasma of 59 biopsy-positive breast cancer patients to generate the L1 library of ssODNs. Similarly, L2 was prepared using exosomes from a pooled blood plasma of 60 women. Dr Spetzler indicated that the biopsy-positive samples were likely from women diagnosed with early-stage breast cancer. L1 and L2 were characterized using next-generation sequencing (NGS). They were amplified and mixed together to generate the profiling library L3 (~106 ssODNs). NGS quantified recovery of each sequence in L3. The study authors showed that the ssODNs in L2, when used with controls, had aptamer-like properties: they could bind with high affinity to specific molecules in exosomes of controls. In a pull-down experiment using aptamers from L2, the authors showed that these were encoded by tumor suppressors (eg, HIST1H2BK, MORC, AHNAK, and TRIM29). Dr Spetzler indicated that identifying specific proteins in blood from cancer patients was not possible, because the procedure for identifying proteins requires a large volume of blood. However, by incubating plasma samples of each cancer patient with L3, it was possible to profile exosomes/proteins from individual cancer patients to generate patient-specific ssODNs profiles (or aptamer profiles). The efficiency of the L3 library was further enhanced by generating L2000 — a selection of 2000 aptamers from L3 based on four factors, which included a fold-change in binding between tumor and control samples, effect size (using a small cohort of patients), and statistical methods to tell the difference between cancer and noncancer. "L2000 provides the ability to mitigate the risk of false-positive discovery," Dr Spetzler said. The study investigators used the L2000 library to interrogate exosomes from 500 individuals (206 cancer patients; 177 breast biopsy-negative patients; and 117 self-declared healthy persons). Information from these aptamer profiles confirmed that L2000 provides adequate information to differentiate between the three populations. "This suggests that L2000 is measuring components related to abnormalities within the breast," the study authors note. "Our study suggests that with refinement of the diagnostic performance, we can have a valuable tool that will aid in the diagnosis of breast cancer when mammography is uninformative, as in women with dense breasts," Dr Spetzler told Medscape Medical News. Although this study provides a glimpse into how ADAPT can be used to diagnose breast cancer, it may have wide applicability in other diseases, the study authors suggest. "While ADAPT in its current form still requires further refinement to bring it to a clinical standard, we envision...[that it] will provide a useful tool for the massively parallel analysis of complex interactome networks of biomolecules in their native state in healthy and diseased conditions," they write. Clinical Utility Is Questioned A big issue is with regard to the clinical utility of ADAPT, Dr Hayes commented in an interview with Medscape Medical News. That requires high levels of evidence (preferably from prospectively conducted studies) that for a given use, a patient's outcome would be better through use of the test than it would have been had the test not been used, he explained. In addition, Dr Hayes noted that the development of ADAPT raises several questions: "What is its intended use? Risk recategorization? True screening? Differential diagnosis?" he asked. As president of the company preparing ADAPT for commercialization, Medscape Medical News asked Dr Spetzler to address these questions. Dr Spetzler said that as " a diagnostic tool able to detect breast cancer, it may be ready in 2 years. However, 2 years will not be adequate to indicate whether detecting breast cancer early with ADAPT will have an impact on survival," he said. "That may take a decade," he added. What about its use as a screening test, which may obviate the need for mammography? Dr Spetzler answered that rather than a method of screening, this technology may be better utilized to identify a subset of patients who may respond to HER2-targeted therapies, such as trastuzumab (Herceptin, Genentech/Roche). Currently, trastuzumab is either overprescribed in HER2-positive patients or underprescribed in HER2-negative patients, he explained. Dr Spetzler also indicated that because breast cancer is a heterogeneous disease with different molecular subtypes, the ssODN library should be able to distinguish across the heterogeneous disease landscape with unique aptamer signatures. "This proof-of-concept study of our ADAPT Biotargeting platform in the diagnostics space provides evidence of the platform's potential for other applications such as drug target discovery and development, biomarker identification and therapeutics," Dr Spetzler commented in the company press release. "Diagnosing and informing treatment are two different components [of patient management]," he said. With respect to diagnosis, Dr Spetzler indicated that as a matter of social responsibility, ADAPT needs to do better than the prostate-specific antigen (PSA) test does for diagnosing men with prostate cancer. "The performance of this test has to be better. We need sensitivity and specificity in the high 90s," he said. "We propose doing a double-blinded, prospective study to validate this test," Dr Spetzler said. "The test is independent of breast density and may be favored when mammography and/or magnetic resonance imaging is uninformative," he added. However, Dr Hayes noted several issues that need to be resolved. He is not certain that ADAPT would replace mammographic screening. To serve as a true screening test would require that if a test result were negative, nothing more would need to be done; but if it were positive, then the patient would proceed to diagnostic breast imaging. An aspect of current clinical management is differential diagnosis of breast abnormality by physical examination or mammographic imaging, Dr Hayes pointed out. If negative, no further workup is required, thus avoiding unnecessary biopsy. If positive, the patient proceeds with biopsy. Dr Hayes noted that the intended use of the test is very important. In each case, sensitivity and specificity, and therefore negative predictive value and positive predictive value, need to be carefully determined, he explained. Distinguishing women with established cancer from those without is an important first step in clinical validity, but that alone is not sufficient, he pointed out. A study must include patients with benign diseases of the breast and of other organ sites, as well as premalignant or even malignant conditions elsewhere, Dr Hayes explained. "If it is positive and yet the breast imaging is negative, what then?" he asked. "Since it [ADAPT] is unlikely to be a perfect test in any of these situations, one must decide how willing one is to accept either a false negative [if one is going to use it to replace the accepted standard of care] or a false positive [if one is going to provide some form of care that would not have been performed in the absence of the test] in the context of the current paradigm for that use," Dr Hayes said. "These are critical questions, and they place a high bar in the pathway towards introduction of such a test in the clinic," Dr Hayes told Medscape Medical News. "Past experience, such as with the Ovacheck test [for ovarian cancer], which was introduced quite prematurely, and even PSA, which was widely introduced before prospective randomized trials gave us any insight into proper use, provide us with a need to be cautious," he added. Source
