Breast cancer is notoriously complex, making it challenging to predict how patients will respond to treatments. Traditional tests often fall short in capturing this complexity, leading to inconsistent results and less effective treatment strategies. Researchers at the Comenius University Science Park have developed a new tool called mFISHseq that offers a more accurate and detailed view of breast cancer biology.

What Is mFISHseq?

mFISHseq combines advanced technologies like RNA fluorescent in situ hybridization (RNA-FISH) with RNA sequencing, guided by laser capture microdissection. This means researchers can focus on specific tumor cells, ensuring pure samples and detailed whole-transcriptome profiling. Unlike other methods, mFISHseq excels at quantifying intratumoral heterogeneity—essentially, the variations within a single tumor that can affect treatment outcomes.

Study design, workflow, and analytical validity

a A retrospective cohort of 1,082 formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing. Annotation of the tumor on a hematoxylin and eosin (H&E) section and the biomarker expression derived from multiplexed RNA-FISH were used to select regions of interest (ROIs) for LCM from cresyl violet sections. These tumor-enriched samples were then sequenced to characterize gene expression signatures to provide diagnostic, prognostic, and predictive inferences from the cohort clinical data. DEGs, Differential expressed genes; IHC, immunohistochemistry. Created with BioRender.com. b Analytical validity of mFISHseq compared to immunohistochemistry assessed by receiver operating characteristic (ROC) curves in 1013 breast tumors stratified into 70:30 training (n = 701) and test (n = 312) datasets. AUC, area under the curve. The table shows biomarker thresholds defined in the training set by maximizing concordance (Cohen’s κ) between RNA-SEQ transcripts per million (TPM) expression values and immunohistochemistry results for each biomarker. These thresholds were then applied to the test set. Source data are provided as a Source Data file.

Why is This Important?

The study demonstrated that mFISHseq achieves an impressive 93% accuracy compared to traditional immunohistochemistry, a standard testing method. Beyond accuracy, it helps define breast cancer subtypes and risk groups more reliably, reducing the chance of inconsistent results.

By analyzing data from a large clinical trial (I-SPY2), the researchers found that mFISHseq identifies high-risk patients with unique immune signatures, making it possible to predict who will benefit from immunotherapy. It also pinpoints patients likely to respond to targeted treatments like antibody-drug conjugates (ADCs) and CDK4/6 inhibitors.

Clinical Potential

mFISHseq isn’t just a research tool—it’s already proving its clinical value. When tested on a group of 48 patients, it provided actionable insights into treatment strategies. This approach promises to guide the use of cutting-edge therapies, ensuring patients receive the treatments most likely to work for their specific cancer type.

The Future of Breast Cancer Care

By integrating cutting-edge technologies, mFISHseq offers a path toward more personalized and effective breast cancer treatments. Its ability to unravel the complexities of tumors and predict treatment responses marks a significant step forward in the fight against breast cancer.