Methods for assaying enriched nucleic acids
Inventors
Perou, Charles M. • Parker, Joel S. • Marron, James Stephen • Nobel, Andrew • Bernard, Philip S. • Ellis, Matthew J. • Mardis, Elaine • Nielsen, Torsten O. • Cheang, Maggie Chon U.
Assignees
British Columbia Cancer Agency BCCA • University of North Carolina at Chapel Hill • University of Utah • Washington University in St Louis WUSTL • University of Utah Research Foundation Inc
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Publication Number
US-12595518-B1
Publication Date
2026-04-07
Expiration Date
Abstract
In an aspect, a method comprises providing a biological sample of a subject, and assaying nucleic acid molecules derived from RNA expression products of the biological sample to determine an RNA expression level of a plurality of genes, wherein the assaying comprises use of probes having sequences complementary to the nucleic acid molecules derived from the RNA expression products, to enrich the nucleic acid molecules for the plurality of genes.
Core Innovation
A biological sample of a subject is provided, and nucleic acid molecules derived from RNA expression products of the biological sample are assayed to determine RNA expression levels corresponding to genes of the subject. The assaying uses capture probes having sequences complementary to the nucleic acid molecules derived from the RNA expression products. A gene expression profile is generated comprising RNA expression levels for a plurality of genes consisting of 40 to 50 genes selected from a specified gene set and a set of housekeeping genes.
The generated gene expression profile is compared to each of a set of centroids, where the set of centroids corresponds to a plurality of breast cancer intrinsic subtypes. A centroid distance based on the gene expression profile is used to assign the biological sample to a breast cancer intrinsic subtype among the plurality of breast cancer intrinsic subtypes corresponding to the centroid. The assignment is based at least upon the distance of the gene expression profile to the centroid.
Centroids are constructed using training breast cancer samples labeled by intrinsic subtype and compared to reference gene expression data using a nearest centroid algorithm. The RNA expression levels are normalized prior to comparing in the centroid comparison, including normalization using a set of housekeeping genes. Subtype assignment supports predicting whether a subject will respond or not respond to neoadjuvant therapy based on the assigned intrinsic subtype.
Claims Coverage
The independent claim is directed to assaying RNA expression products using capture probes, generating a defined gene expression profile with housekeeping genes, comparing the profile to breast cancer intrinsic subtype centroids, and assigning a subtype based on centroid distance. Additional features cover centroid construction using training-labeled samples and reference expression data, normalization, and prediction of neoadjuvant therapy response.
Capture probe-based nucleic acid assay for RNA expression products
Assaying nucleic acid molecules derived from RNA expression products of the biological sample to determine RNA expression levels, wherein the assaying comprises use of capture probes having sequences complementary to the nucleic acid molecules.
Defined multi-gene expression profile with housekeeping genes
Generating a gene expression profile comprising RNA expression levels for a plurality of genes consisting of 40 to 50 genes selected from a specified gene set and a set of housekeeping genes.
Centroid comparison for breast cancer intrinsic subtypes
Comparing the gene expression profile to each of a set of centroids, wherein the set of centroids correspond to a plurality of breast cancer intrinsic subtypes.
Intrinsic subtype assignment by gene expression profile centroid distance
Assigning the biological sample to a breast cancer intrinsic subtype corresponding to a centroid among the plurality of breast cancer intrinsic subtypes based at least upon a distance of the gene expression profile to the centroid.
Centroid construction using training subtypes and reference expression data
Detecting RNA expression levels of genes in training breast cancer samples labeled by intrinsic subtype, generating a training gene expression profile, and constructing subtype centroids by comparing the training profile to reference gene expression data using a nearest centroid algorithm.
Normalization of RNA expression levels prior to centroid comparison
Normalizing RNA expression levels of a plurality of genes before comparing the normalized gene expression levels in the comparing step, including normalization using a set of housekeeping genes.
Predicting neoadjuvant response from assigned intrinsic subtype
Predicting whether a subject will respond or not respond to neoadjuvant therapy based on the breast cancer intrinsic subtype assigned.
The claim set centers on capture-probe RNA expression assays, a defined intrinsic gene expression profile including housekeeping genes, comparison to subtype centroids, and assignment based on centroid distance. Dependent refinements include centroid construction from training breast cancer samples, normalization prior to comparison, and use of the assigned subtype to predict response or non-response to neoadjuvant therapy.
Stated Advantages
PAM50-based subtype/risk is reported to predict neoadjuvant endocrine therapy failure and prognosis after adjuvant endocrine treatment.
One-month PAM50 subtype and one-month high-risk proliferation scores are reported to correlate with poorer response, less down-staging, worse end-of-treatment Ki67, and higher relapse.
Documented Applications
Predicting whether a subject will respond or not respond to neoadjuvant therapy based on the assigned breast cancer intrinsic subtype.
Assessing neoadjuvant endocrine therapy outcomes including RECIST response, pathological down-staging (T size), log-normalized Ki67 at end of treatment, and relapse, using PAM50 intrinsic subtype and proliferation-weighted ROR risk scores at baseline versus one month.
Evaluating prognosis after adjuvant endocrine treatment, including reported relationships between PAM50 subtype and ROR-C in ER+ breast cancer cohorts and disease-specific survival and relapse-free survival, with additional stratification reported by axillary lymph node status.
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