Current assays fail to address breast cancer's complex biology and accurately predict treatment response. On a retrospective cohort of 1082 female breast tissues, we develop and validate mFISHseq, which integrates multiplexed RNA fluorescent in situ hybridization with RNA-sequencing, guided by laser capture microdissection. This technique ensures tumor purity, unbiased whole transcriptome profiling, and explicitly quantifies intratumoral heterogeneity. Here we show mFISHseq has 93% accuracy compared to immunohistochemistry. Our consensus subtyping and risk groups mitigate single sample discordance, provide early and late prognostic information, and identify high risk patients with enriched immune signatures, which predict response to neoadjuvant immunotherapy in the multicenter, phase II, prospective I-SPY2 trial. We identify putative antibody-drug conjugate (ADC)-responsive patients, as evidenced by a 19-feature T-DM1 classifier, validated on I-SPY2. Deploying mFISHseq as a research-use only test on 48 patients demonstrates clinical feasibility, revealing insights into the efficacy of targeted therapies, like CDK4/6 inhibitors, immunotherapies, and ADCs.

• MeSH
• dospělí MeSH
• hybridizace in situ fluorescenční * metody   MeSH
• imunohistochemie MeSH
• imunokonjugáty terapeutické užití   MeSH
• imunoterapie metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery * genetika   metabolismus   MeSH
• nádory prsu * genetika   farmakoterapie   metabolismus   MeSH
• neoadjuvantní terapie MeSH
• prognóza MeSH
• prospektivní studie MeSH
• retrospektivní studie MeSH
• sekvenční analýza RNA metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze II MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• imunokonjugáty MeSH
• nádorové biomarkery * MeSH

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.

• Klíčová slova
• RNA-FISH, RNA-sequencing, antibody drug-conjugates, breast cancer, laser capture microdissection, spatial biology,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH