On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

E.D.P., B.H., N.Val., N.B., D.G., S.G., H.I., T.O., S.B., J.B., K.B., D.D., Z.K., M.K., D.L., V.Mam., V.Man., N.Voj., and P.Č. are current, or former employees of MultiplexDX, a biotechnology company that is developing a lab developed diagnostic test called Multiplex8+ (https://www.multiplexdx.com/products/multiplex-eight-plus), which is based on the research presented in the manuscript. P.Č. and E.D.P. are inventors and MultiplexDX, s.r.o is the assignee on patent applications that were filed in relation to the technology and research outlined in the manuscript. T.T. and F.P. are members of the Scientific Advisory board at MultiplexDX. All other authors declare no competing interests.

This project was supported by the European Union's Horizon 2020 research and innovation programme under an EIC Accelerator grant (agreement No 946693) awarded to MultiplexDX s.r.o. (Pavol Cekan as PI). FP is funded in part by an NIH/NCI P50 CA24779 01 grant. The funding sources had no role in the design and conduct of the study, interpretation of the data, writing of the manuscript, and decision to submit the manuscript for publication.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Ethics Committee of the Bratislava Self-Governing Region gave ethical approval for this work (Ref. No. 05320/2020/HF). In addition, the Ethics Commission of the Medical University of Graz on behalf of Biobank Graz gave ethical approval for this work (No. 34-354 ex 21/21, 1158-2022).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The data that support the findings of this study are available within the paper and its supplementary information files. Reports from the 26 patients that underwent a research use only version of the diagnostic test (called Multiplex8+) can be found at https://www.multiplex8.com/medical-professional. The underlying data, custom code and scripts used for bioinformatic analyses, and access to the full dataset for research and/or commercial use may also be obtained upon a reasonable request from the lead corresponding author (P.&Ccaron.).

https://www.multiplex8.com/medical-professional