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

Project Information

Multiplex8+

Grant agreement ID: 946693

DOI

10.3030/946693

Project closed

EC signature date 30 March 2020

Start date 1 May 2020

End date 28 February 2023

Funded under

• INDUSTRIAL LEADERSHIP - Innovation In SMEs

Total cost

€ 3 499 325,00

EU contribution

€ 2 449 527,50

Coordinated by

MULTIPLEXDX S.R.O.
Slovakia

Periodic Reporting for period 2 - Multiplex8+ (A highly accurate breast cancer diagnostic test for effective personalized treatment and assessment of therapy response.)

Reporting period: 2022-02-01 to 2023-02-28

Summary of the context and overall objectives of the project

Breast cancer (BCa) is a heterogeneous disease, a spectrum of subtypes with distinct biological features that lead to differences in treatment response and clinical outcomes. The diagnosis of BCa still suffers from imprecise diagnostics often relying on a single technology, and consequently failing to deliver accurate data for selecting the most effective treatment.

Diagnosis of BCa typically involves pathological examination of a patient specimen (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 cancer, but they lack insights into cellular morphology and the spatial relationships of gene expression. These limitations can lead to inadequate or inappropriate treatment causing significant burden to patients, families, and society.

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 -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 diagnostics), revamp our commercialization and branding strategy, and establish an ISO 15189-accredited lab to offer Multiplex8+ as a laboratory service.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We are developing a revolutionary multiplexed BCa diagnostic test called Multiplex8+, which combines visualization and sequencing technologies in one single test with high 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 advances in AI and machine learning, we combine the visualization and sequencing data with clinical and pathological patient data to identify unique gene signatures that provide actionable insights for potential treatment options, risk for recurrence, and survival. The info from Multiplex8+ is then used to generate a score that precisely identifies the patient-specific BCa profile: a so-called BCa barcode that helps oncologists design a personalized treatment plan.

We have applied this technology to a cohort of 1080 FFPE breast tumors each containing detailed clinicopathological data. This retrospective validation has demonstrated the analytical validity of Multiplex8+ by showing high concordance with biobank immunohistochemistry results for the four main breast cancer biomarkers. Gene expression profiling has also shown Multiplex8+ as an effective tool for determining the intrinsic molecular subtype of breast cancer and providing superior stratification of subtypes based on tumor biology and survival. We have also identified gene signatures that can identify patients that are more likely to respond to therapies such as immunotherapies and new classes of antibody-drug conjugates.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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, and making strides to resolve equivocal cases of low biomarker expression (e.g. Her2-low). 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.

Presenting Multiplex8+ at the Cancer and Metabolic Disorders Conference.

MultiplexDX celebrating the International Day of Women and Girls in Science

Presenting Multiplex8+ to Eduard Heger, the Prime Minister of Slovakia.

Our team hosted students from the Faculty of Natural Sciences of Comenius University and showed them

The Multiplex8+ test includes multiplexed RNA-FISH followed by laser capture microdissection and RNA

MultiplexDX at Arab Health 2023 in Dubai.

Our Multiplex8+ image won the 2023 Advanced Cell Diagnostics calendar contest