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A highly accurate breast cancer diagnostic test for effective personalized treatment and assessment of therapy response.

Periodic Reporting for period 1 - Multiplex8+ (A highly accurate breast cancer diagnostic test for effective personalized treatment and assessment of therapy response.)

Reporting period: 2020-05-01 to 2022-01-31

Breast cancer (BCa) is a heterogeneous disease, a spectrum of many subtypes with distinct biological features that lead to differences in treatment response and clinical outcomes. The diagnosis of BCa still suffers from imprecise diagnostic tests often relying on a single technology, and consequently failing to deliver accurate data for selecting the most effective treatment.
Diagnosis of BCa typically involves the pathological examination of a patient specimens (e.g. biopsy, resected tumor) using histological and immunohistochemical analyses. Although this provides critical data about cellular morphology, the information is largely qualitative, and the number of biomarkers is minimal. On the other hand, multigene assays provide crucial quantitative data on numerous genes involved in the progression of cancer, but they lack insights into cellular morphology and the spatial relationships about gene expression. The limitations of these approaches can lead to inadequate or inappropriate treatment causing significant burden to patients, families, and society.
We are developing a revolutionary multiplexed BCa diagnostic test called Multiplex8+, which combines morphological visualization and sequencing technologies in one single test with a high level of specificity/sensitivity (see Figure 1). The test consists of using RNA fluorescent in situ hybridization (RNA-FISH) to visualize a panel of biomarkers. Then, based on the expression of these biomarkers, we dissect out regions of interest using laser capture microdissection. With this tumor-enriched sample, we conduct total RNA-sequencing to survey gene expression in a spatially resolved manner, effectively mitigating biases that arise from tumor heterogeneity and capturing valuable information about spatial relationships and single cell gene expression.
By leveraging the latest advances in artificial intelligence and machine learning, we can combine the visualization and sequencing data with the clinical and pathological patient data to identify unique gene signatures that provide actionable insights for potential treatment options, risk for recurrence, and survival. The information from Multiplex8+ is then used to generate a score that precisely identifies the patient-specific BCa profile: a so-called BCa barcode that will help oncologists design a personalized and effective treatment plan.
Our main objectives are both technical and strategic. From the technical standpoint, we aim to improve assay performance and accuracy by optimizing protocols for tissue preparation, RNA-FISH, and RNA-sequencing, scale up the number of biomarkers from 8 to 25, and automate aspects of the workflow. Strategically, we strive to complete a large-scale retrospective validation of Multiplex8+, engage with KOLs and other stakeholders to facilitate fundraising and validation projects (e.g. companion diagnostic trial), revamp our commercialization and branding strategy, and establish an ISO 15189-accredited lab to offer Multiplex8+ as a laboratory service.
During the first reporting period, we 1) optimized our in-house RNA-FISH protocol as well as a commercial RNA-FISH protocol from Advanced Cell Diagnostics so that we can visualize a panel of 8 breast cancer biomarkers; 2) optimized a laser capture microdissection RNA sequencing protocol for low input, degraded tumor specimens; 3) acquired 144 m2 of additional lab space and invested over 500 000 Euro in laboratory equipment all dedicated to the Multiplex8+ workflow, setting us up with the necessary infrastructure for ISO 15189 accreditation and commercialization; 4) automated key parts of the workflow including fluorescent microscopy, image analysis using machine learning, RNA extraction, and bioinformatics; 5) initiated our retrospective clinical validation after receiving the first tranche of over 300 breast cancer specimens from one of our collaborating biobanks; and 6) expanded our PCT patent application covering Mulitplex8+ to six national phase patent applications.
Our Multiplex8+ test combines histological assessment, biomarker visualization using RNA-FISH, and multigene surveillance using RNA-sequencing into a single test, alleviating the individual limitations of each approach, and capturing the best of both worlds – tissue morphology and spatially-resolved multigene expression. This merging of technologies in a streamlined, cost-effective test creates synergies that go beyond the state of the art. The results from RNA-FISH and RNA-sequencing can cross-validate one another leading to more accurate results, mitigating misdiagnosis, and consequently inappropriate (e.g. unnecessary chemotherapy) or inadequate treatments. Utilizing laser capture microdissection allows us to profile gene expression in tumor-enriched specimens, thus providing new insights into tumor heterogeneity, which is essentially lost in current multigene assays. By providing Multiplex8+ as a laboratory service, we can provide comprehensive testing solutions to hospitals and oncologists that lack the infrastructure and expertise, thus enabling personalized and precision oncology for the entire world.
We are MultiplexDX. A picture of our team at the Comenius University Science Park.
An overview of the Multiplex8+ test.
We attended the online Bio Digital conference giving us the to present our company in the form of a