WO2024191598A1

WO2024191598A1 - Labeled amplifier oligonucleotides for detecting low or ultra low expression of cancer markers and uses in managing cancer treatments

Info

Publication number: WO2024191598A1
Application number: PCT/US2024/017535
Authority: WO
Inventors: Xiaoxian LI; Ji-Hoon Lee
Original assignee: Emory University; Georgia Tech Research Institute; Georgia Tech Research Corp
Current assignee: Emory University; Georgia Tech Research Institute; Georgia Tech Research Corp
Priority date: 2023-03-10
Filing date: 2024-02-27
Publication date: 2024-09-19
Anticipated expiration: 2025-09-10

Abstract

Disclosed herein are methods of managing and treating a cancer patient. In certain embodiments, methods comprise using low levels of quantified signals from cancer associated nucleic acid detection to determine whether to treat patient with an anticancer agent. In certain embodiments, the cancer associated nucleic acid is HER2 and/or TROP2 mRNA expression. In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer or lung cancer.

Description

LABELED AMPLIFIER OLIGONUCLEOTIDES FOR DETECTING LOW OR ULTRA LOW EXPRESSION OF CANCER MARKERS AND USES IN MANAGING CANCER TREATMENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/451,426 filed March 10, 2023 and U.S. Provisional Application No. 63/605,770 filed December 4, 2023. The entirety of each of these applications is hereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 22210PCT.xml. The XML file is 6 KB, was created on Feb 26, 2024, and is being submitted electronically via the USPTO patent electronic filing system.

BACKGROUND

ENHERTU® (fam-trastuzumab deruxtecan-nxki) is a HER2-directed antibody and topoisomerase inhibitor conjugate indicated for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer. Human epidermal growth factor receptor 2 (HER2 also referred to as ERBB2) expression in breast cancer are typically evaluated by immunohistochemistry (IHC) assays. Based on IHC scores, breast cancer can be categorized as HER2-negative (0 to 1+), HER2-equivocal (2+), or HER2-positive (3+). Tumors with a HER2 IHC score of 0 can be further classified as either 0 (no membranous staining) or ultralow (incomplete and faint membranous staining in greater than 0 but less than or equal to 10% of tumor cells).

Treating cancer at an early stage is generally considered to provide an optimal chance of survival. Current commercial HER2 immunohistochemistry (IHC) assays are inconsistent and suboptimal for early detection and treatment. The HER2 IHC 0 and 1+ cases can be difficult to evaluate and separate because the semi quantitative scoring of IHC relies on subjective evaluations from pathologists. Patients diagnosed with HER2 0 using IHC may ultimately develop HER2 expressing breast cancer. Thus, there is a need to develop methods for more accurately identifying abnormal cancerous HER2 express to instigate beneficial targeted anticancer therapy.

Fernandez et al. report studies on the examination of low ERBB2 (HER2) protein expression in breast cancer tissue and report there is poor inter-observer agreement with only 26% concordance between HER2 IHC 0 and 1+ evaluations. JAMA Oncol 8: 1-4, 2022.

Vinatzer et al. report expression of HER2 in human breast cancer and quantitative realtime reverse transcription-PCR as a diagnostic alternative to immunohistochemistry and fluorescence in situ hybridization. Clin Cancer Res, 2005,11:8348-57.

Baehner et al. repot HER2 assessments comparing fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories. J Clin Oncol, 2010, 28:4300-6.

Wang et al. report an in-situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn, 2012, 14(l):22-9. See also US Pat. No. 8,658,361.

Wang et al. report automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma. J Mol Diagn, 2013,15(2):210-9.

Vassilakopoulou et al. report in situ quantitative measurement of HER2 mRNA predicts benefit from trastuzumab-containing chemotherapy in a cohort of metastatic breast cancer patients. PLoS One, 2014, 9(6):e99131.

Schulz et al. report simultaneous multiplexed imaging of mRNA and proteins with subcellular resolution in breast cancer tissue samples by mass cytometry. Cell Syst, 2018, 6:25- 36.

Choi et al. report in situ hybridization chain reaction. The Company of Biologists Ltd, Development (2018) 145, devl65753. See also US Pat. No. 11,713,485.

Moutafi et al. report quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer. Lab Invest, 2022, 102(10): 1101-1108.

Mosele et al. report trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression. Nat Med 29:2110-2120, 2023

References cited herein are not an admission of prior art. SUMMARY

In certain embodiments, methods for managing and treating a cancer patient include the steps of exposing a tissue sample with target probes that hybridize with a cancer associated nucleic acid, amplifier oligonucleotides, and labeled oligonucleotides, and detecting the labeled oligonucleotide in the tissue sample, and quantifying the labeled oligonucleotide signals as an indication of the presence of the cancer in the tissue sample. In certain embodiments, the method further comprises using the quantified signals to determine whether to treat patient with an anticancer agent. In certain embodiments, the cancer associated nucleic acid is HER2 and/or TROP2 mRNA expression.

In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer or lung cancer. In certain embodiments, this disclosure relates to methods of detecting and quantifying a low or an ultra-low quantity of Human epidermal growth factor 2 (HER2) mRNA expression comprising: providing a group of immobilized cells or tissue sample from a patient comprising a target HER2 mRNA sequence, wherein the target HER2 mRNA comprises a first target sequence and a second target sequence; contacting the sample with a first target probe and a second target probe, wherein the first target probe comprises a first probe segment that hybridizes with the first target sequence and a first probe second segment, wherein the second target probe comprises a second probe segment that hybridizes with the second target sequence and second probe second segment; further contacting the sample with a preamplifier oligonucleotide, wherein the preamplifier oligonucleotide comprises a first preamplifier segment that hybridizes with the first probe second segment and a second preamplifier segment that hybridizes the second probe second segment, wherein the preamplifier oligonucleotide further comprises preamplifier target segments; further contacting the sample with amplifier oligonucleotides, wherein the amplifier oligonucleotides comprise a first segment that hybridizes with the preamplifier target segments and second amplifier segments; and further contacting the sample with labeled oligonucleotides, wherein the labeled oligonucleotides hybridize with the second amplifier segments; and detecting and/or quantifying the labeled oligonucleotide signals, wherein quantifying the labeled oligonucleotide signals indicate quantified expression of the target RNA in an amount associated with a risk of developing life threating cancer that is a low or an ultra-low quantity.

In certain embodiments, a sample from the patient is first determined by HERZ immunohistochemistry (IHC) assay to be a score of 0. In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of score ultra-low (UL). In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of low or 1+. In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of 2+ or 3+.

In certain embodiments, the methods further comprise recoding the detected and quantified labeled oligonucleotide signals on a non-transitory computer readable medium. In certain embodiments, the methods further comprise comparing the detected and quantified labeled oligonucleotide signals to a normal, control, or reference value. In certain embodiments, the methods further comprise diagnosing the subject as having breast cancer, when compared to a normal, control, or reference value. In certain embodiments, the methods further comprise recoding the diagnosis or comparison on a non-transitory computer readable medium. In certain embodiments, the methods further comprise communicating the diagnosis, comparison, detection, quantification, or implications thereof to a medical profession and/or the patient.

In certain embodiments, this disclosure relates to methods of diagnosing and treating breast cancer comprising; quantifying target RNA which is HERZ in a breast tissue sample of a subject using the methods reported herein, wherein if the subject is diagnosed with a low or ultra-low expression of the HER2 mRNA, administering to the subject an effective amount of an anti-cancer agent, performing radiation therapy, and/or removing the tissue by a surgical intervention. In certain embodiments, the anti-cancer agent is anti-HERZ antibody therapy, e.g., trastuzumab deruxtecan. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 illustrates the detection of HER mRNA in a tissue sample using target probes, amplifier oligonucleotides, and fluorescent labels. A pair of oligonucleotide target probes have first segments that hybridized to HER mRNA. First segments of preamplifier oligonucleotides hybridize with second segments of the target probes, first segments of amplifier oligonucleotides hybridize with second segments of the preamplifier oligonucleotides, and fluorescent labels are linked to oligonucleotide sequences that hybridize to the second segments of the amplifier oligonucleotides.

Figures 2A-2D show scatter plots of HER2 RNAscope™ results vs. protein levels and IHC H-scores.

Figure 2A shows data from scatter plots of HER2 protein levels vs. RNAscope™ results (dots/cell) from tissue cores that had protein levels above the limit of detection (less than or equal to 3 amol/mm²), including the commercial and patient tissue cores.

Figure 2B shows data from scatter plots of HER2 protein levels vs. RNAscope™ results from the patient tissue cores that had protein levels above the limit of detection (less than or equal to 3 amol/mm²).

Figure 2C shows data from scatter plots of RNAscope™ results vs. IHC H-scores from PATHWAY™ assay.

Figure 2D shows data from scatter plots of RNAscope™ results vs. IHC H-scores from HercepTest™ assay.

Figures 3 A-D show data from quantified protein levels and RNAscope™ results according to IHC scores.

Figure 3A shows data on HER2 protein levels according to IHC scores using the PATHWAY™ assay.

Figure 3B shows data on HER2 protein levels according to IHC scores using the HercepTest™ assay.

Figure 3C shows data on HER2 RNAscope™ results according to IHC scores using the PATHWAY™ assay. Inset: HER2 RNA levels of IHC 0, UL, and 1+ cases; UL: ultralow.

Figure 3D shows data on HER2 RNAscope™ results according to IHC scores using the HercepTest™ assay. Inset: HER2 RNA levels of IHC 0, UL, and 1+ cases; UL: ultralow. DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. An “embodiment” refers to an example of the invention, and it is not intended that this disclosure necessarily be limited to such example. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as presented or amended during prosecution.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

"Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to methods and compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an oligonucleotide, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled nucleotide or the covalent attachment of biotinyl moieties to an oligonucleotide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling oligonucleotides are known in the art and may be used. Examples of labels include, but are not limited to, the following: fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), radioisotopes or radionucleotides (such as ¹⁸F, ³⁵S, or ¹³¹I), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary fluorescent antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. "Subject" refers to any animal, preferably a human patient, livestock, horse, cow, pig, chicken, turkey, mouse, rodent, monkey, dog, cat, or other domestic pet. The subject may be a female or male subject.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.

The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

"Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5- fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC). In certain embodiments, methods disclosed herein include measurements that are compared to a normal or reference value. In some embodiments, the reference value refers to a control e.g., threshold value, indicative of a HERZ immunohistochemistry (IHC) assay to be a score of 1+ , low, ultra-low (UL), or 0; the course of disease without treatment; a healthy subject or an average of healthy subjects (normal); a subject at a different time intervals, e.g., prior to, during, or after a treatment.

As used herein, a “reference value” can be an absolute value; a relative value; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample or a large number of samples, such as from patients or normal individuals.

A “normalized measured” value refers to a measurement taken and adjusted to take background into consideration. Background subtraction to obtain total fluorescence is considered a normalized measurement. The background subtraction allows for the correction of background fluorescence that is inherent in the optical system and assay buffers.

Unless stated otherwise as apparent from the following discussion, it will be appreciated that terms such as “detecting,” “receiving,” “quantifying,” “mapping,” “generating,” “registering,” “determining,” “obtaining,” “processing,” “computing,” “deriving,” “estimating,” “calculating,” “inferring” or the like may refer to the actions and processes of a computer system, or similar electronic computing device, that manipulates and transforms data represented as physical (e.g., electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage, transmission or display devices. Embodiments of the methods described herein may be implemented using computer software. If written in a programming language conforming to a recognized standard, sequences of instructions designed to implement the methods may be compiled for execution on a variety of hardware platforms and for interface to a variety of operating systems. In addition, embodiments are not described with reference to any particular programming language. It will be appreciated that a variety of programming languages may be used to implement embodiments of the disclosure.

In some embodiments, the disclosed methods may be implemented using software applications that are stored in a memory and executed by a processor (e.g., CPU) provided on the system. In some embodiments, the disclosed methods may be implanted using software applications that are stored in memories and executed by CPUs distributed across the system. As such, the modules of the system may be a general purpose computer system that becomes a specific purpose computer system when executing the routine of the disclosure. The modules of the system may also include an operating system and micro instruction code. The various processes and functions described herein may either be part of the micro instruction code or part of the application program or routine (or combination thereof) that is executed via the operating system.

It is to be understood that the embodiments of the disclosure may be implemented in various forms of hardware, software, firmware, special purpose processes, or a combination thereof. In one embodiment, the disclosure may be implemented in software as an application program tangible embodied on a computer readable program storage device. The application program may be uploaded to, and executed by, a machine comprising any suitable architecture. The system and/or method of the disclosure may be implemented in the form of a software application running on a computer system, for example, a mainframe, personal computer (PC), handheld computer, server, cloud, etc. The software application may be stored on a recording media locally accessible by the computer system and accessible via a hard wired or wireless connection to a network, for example, a local area network, or the Internet.

It is to be further understood that because some of the constituent system components and method steps depicted in the accompanying figures may be implemented in software, the actual connections between the systems components (or the process steps) may differ depending upon the manner in which the disclosure is programmed. Given the teachings of the disclosure provided herein, one of ordinary skill in the related art will be able to contemplate these and similar implementations or configurations of the disclosure.

Methods of managing and treating a cancer

This disclosure relates to methods of managing cancer by detecting, measuring, quantifying cancer markers, diagnosing, and/or treating a cancer patient including the steps of exposing a sample, e.g., tissue, with target probes that hybridize with a cancer associated nucleic acid, amplifier oligonucleotides, and labeled oligonucleotides, and detecting the labeled oligonucleotide in the tissue sample, and quantifying the labeled oligonucleotide signals as an indication of the presence of cancerous cells in the tissue sample. In certain embodiments, methods further comprise using the quantified signals to determine whether to treat patient with an anti-cancer agent and administering to the patient an effective amount of an anticancer agent(s) the patient in need thereof. In certain embodiments, the cancer associated nucleic acid is HER2 and/or TR0P2 mRNA expression. In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer or lung cancer.

In certain embodiments, this disclosure relates to methods of detecting and quantifying a low or an ultra-low quantity of Human epidermal growth factor 2 (HER2) mRNA expression comprising: providing a group of immobilized cells or tissue sample from a patient comprising a target HER2 mRNA sequence, wherein the target HER2 mRNA comprises a first target sequence and a second target sequence; contacting the immobilized tissue sample with a first target probe and a second target probe, wherein the first target probe comprises a first probe segment that hybridizes with the first target sequence and a first probe second segment, wherein the second target probe comprises a second probe segment that hybridizes with the second target sequence and second probe second segment; further contacting the immobilized sample with a preamplifier oligonucleotide, wherein the preamplifier oligonucleotide comprises a first preamplifier segment that hybridizes with the first probe second segment and a second preamplifier segment that hybridizes the second probe second segment, wherein the preamplifier oligonucleotide further comprises preamplifier target segments; further contacting the sample, e.g., immobilized tissue, with amplifier oligonucleotides, wherein the amplifier oligonucleotides comprise a first segment that hybridizes with the preamplifier target segments and second amplifier segments; and further contacting the sample with labeled oligonucleotides, wherein the labeled oligonucleotides hybridize with the second amplifier segments; and detecting and quantifying the labeled oligonucleotide signals, wherein quantifying the labeled oligonucleotide signals indicate quantified expression of the target RNA in an amount associated with a risk of developing life threating cancer that is a low or an ultra-low quantity.

In certain embodiments, between one or more or all of the contacting steps a washing or purifying step may be used to remove excess oligonucleotide that failed to hybridize to the designed targets.

In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of 0. In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of score ultra-low (UL). In certain embodiments, a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of low or 1+.

In certain embodiments, the sample is from patient with breast cancer or lung cancer. In certain embodiments, the sample is from patient that received a prior anti-HER2-based drug regimen. In certain embodiments, the subject is diagnosed with metastatic cancer. In certain embodiments, the sample is from patient that has developed disease recurrence during or within six months of completing therapy. In certain embodiments, the sample is from patient with unresectable or metastatic that developed disease recurrence. In certain embodiments, the sample is from a subject during or within 6 months of completing adjuvant chemotherapy, or a patient with unresectable or metastatic non-small cell lung cancer (NSCLC). In certain embodiments, the sample is from a patient with tumors having activating HER2 (ERBB2) mutations and have received a prior systemic therapy.

In certain embodiments, the methods further comprise recoding the detected and quantified labeled oligonucleotide signals on a non-transitory computer readable medium. In certain embodiments, the methods further comprise comparing the detected and quantified labeled oligonucleotide signals to a normal, control, and/or reference value. In certain embodiments, the methods further comprise diagnosing the subject as having breast cancer or lung cancer, when compared to a normal, control, and/or reference value. In certain embodiments, the methods further comprise recoding the diagnosis and/or comparison on a non-transitory computer readable medium. In certain embodiments, the methods further comprise communicating the diagnosis, comparison, detection, quantification, and/or implications thereof to a medical profession or the subject/patient from which the sample was obtained. In certain embodiments, the communication is verbally, by electronic mail, paper mail, or storage of the information on a computer database accessible by the patient.

In certain embodiments, this disclosure relates to methods of diagnosing and treating breast cancer comprising; quantifying target RNA which is HER2 in a breast tissue sample of a subject using the methods reported herein, wherein if the subject is diagnosed with a low or ultra-low expression of the HER2 mRNA administering to the subject an effective amount of an anti-breast cancer agent, performing radiation therapy, and/or removing the tissue by a surgical intervention. In certain embodiments, the anti-cancer agent is anti-HER2 antibody therapy, e.g., trastuzumab deruxtecan. In certain embodiments, the methods further comprise detecting, measuring, quantifying, diagnosing, and/or treating a cancer patient including the steps of exposing a tissue sample from the patient with target probes that hybridize with a cancer associated RNA sequence providing target bound probes to the cancer associated RNA, and exposing the target bound probes to amplifier oligonucleotides and labeled oligonucleotides that bind the amplifier oligonucleotides, and detecting the labeled oligonucleotide in the tissue sample, and quantifying the labeled oligonucleotides as an indication of the presence of the cancer associated with the patient.

In certain embodiments, the methods further comprise treating the patient by administering to the patient an anti-cancer agent if the patient is diagnosed with cancer due to having the cancer associated RNA at or above a low or ultra-low level threshold level.

In certain embodiments, the methods of detecting HER2 and/or TR0P2 expression comprise providing a group of immobilized cells or tissue sample comprising a target RNA sequence, wherein the target RNA comprises a first target sequence and a second target sequence, contacting the immobilized tissue sample with a first target probe and a second target probe, wherein the first target probe comprises a first probe segment that hybridizes with the first target sequence and a first probe second segment, wherein the second target probe comprises a second probe segment that hybridizes with the second target sequence and second probe second segment, further contacting the immobilized tissue sample with a preamplifier oligonucleotide, wherein the preamplifier oligonucleotide comprises a first preamplifier segment that hybridizes with the first probe second segment and a second preamplifier segment that hybridizes the second probe second segment, wherein the preamplifier oligonucleotide further comprises preamplifier target segments, further contacting the immobilized tissue sample with amplifier oligonucleotides; wherein the amplifier oligonucleotides comprise a first segment that hybridizes with the preamplifier target segments and second amplifier segments; further contacting the immobilized tissue sample with labeled oligonucleotides, wherein the labeled oligonucleotides hybridize with the second amplifier segments.

In certain embodiments, the detection of HER2 and/or TR0P2 mRNA in a tissue sample using target probes, amplifier oligonucleotides, and fluorescent labels, wherein a pair of oligonucleotide target probes have first segments that hybridized to HER2 and/or TR0P2 mRNA in special proximity, wherein first segments of preamplifier oligonucleotides hybridize with second segments of the target probes, amplifier oligonucleotides hybridize with second segments preamplifier oligonucleotides, and fluorescent labels are linked oligonucleotide sequences that hybridize to the second segments of the amplifier oligonucleotides.

In certain embodiments, the fluorescent labels optionally can be designed to bind directly to second segments of the preamplifier probes and/or optionally the amplifier oligonucleotides which are fluorescently labeled hairpin molecular beacons capable of polymerized self-binding by in situ hybridization chain reaction.

In certain embodiments, the target RNA is Human epidermal growth factor 2 (HER2) mRNA. In certain embodiments, the target mRNA is (SEQ ID NO: l).In certain embodiments, the target RNA is trophoblast cell-surface antigen 2 (TR0P2) mRNA. In certain embodiments, the target mRNA is (SEQ ID NO: 2). In certain embodiments, the labeled oligonucleotides are fluorescently or diaminobenzidine (DAB) labeled.

In certain embodiments, methods further comprise detecting, measuring, or quantifying the labeled oligonucleotide signal. In certain embodiments, methods further comprise comparing the detected, measured of quantified labeled oligonucleotide signal to a normal or reference value. In certain embodiments, methods further comprise recoding the detected, measured, or quantified labeled oligonucleotide signal on a non-transitory computer readable medium. In certain embodiments, methods further comprise diagnosing the subject of having a disease of condition associated with elevated or suppressed expression of the RNA, e.g., breast cancer, when compared to a normal, diseased, or other reference value. In certain embodiments, methods further comprise recoding the diagnosis or comparison on a non-transitory computer readable medium. In certain embodiments, methods further comprise communicating the diagnosis, comparison, detection, measurement, quantification, for implications thereof to a medical profession or a patient from which the sample was obtained, e.g., communicating to the patient that the patient is in need of treatment.

In certain embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue. In certain embodiments, methods further comprise visualizing, detecting, measuring, or quantifying mRNA using labels detected by a bright-field microscope (with chromogenic labels) or an epifluorescence microscope (with fluorescent labels).

In certain embodiments, the sample is breast tissue and further comprising quantifying the labeled oligonucleotides as associated with having low or ultra-low expression of target RNA such as HER2 and/or TROP2. In certain embodiments, methods of diagnosing and treating cancer comprising; quantifying target RNA which is HER2 and/or TROP2 in a sample of a subject using any of the methods of the presented claims, wherein if the subject is diagnosed with a quantified expression of the target RNA in an amount associated with a risk of developing life threating cancer, administering to the subject an effective amount of an anti-cancer agent, performing radiation therapy, and/or removing the tissue by a surgical intervention.

In certain embodiments, methods further comprise measuring the quantified expression of the target RNA in an amount associated with a risk of developing life threating cancer is a low or an ultra-low quantity, i.e., tissue cores had greater than 0% but less than or equal to 10% incomplete membranous staining.

In certain embodiments, methods diagnosing and treating breast cancer comprising; quantifying target RNA which is HER2 and/or TROP2 in a breast tissue sample of a subject using any of the methods disclosed herein, wherein if the subject is diagnosed with a low or ultra-low expression of the target RNA, administering to the subject an effective amount of an anti -breast cancer agent, performing radiation therapy, and/or removing the tissue by a surgical intervention. In certain embodiments, the anti-cancer agent is trastuzumab deruxtecan.

In certain embodiments, methods of diagnosing and treating breast cancer comprise; quantifying target RNA which is HER2 in a breast tissue sample of a subject using any of the methods disclosed herein, wherein if the subject is diagnosed with a no expression of the target RNA, administering to the subject an effective amount of TROP2-directed antibody and topoisomerase inhibitor conjugate, e.g., sacituzumab govitecan.

In certain embodiments, methods of diagnosing and treating breast cancer comprise; quantifying target RNA which is TROP2 in a breast tissue sample of a subject using any of the methods disclosed herein, wherein if the subject is diagnosed with a no expression of the target RNA, administering to the subject an effective amount of HER2-directed antibody and topoisomerase inhibitor conjugate, e.g., trastuzumab deruxtecan.

Quantifying HER2 expression in breast cancer

Experiments were performed to determine whether HER2 protein levels correlate with quantified mRNA levels. Trastuzumab deruxtecan (T-DXd) is clinically approved to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemistry (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd.

HER2 expression in breast cancer tissue microarray (TMA) cores was measured using the PATHWAY™ and HercepTest™ IHC assays, and the corresponding RNA levels were evaluated by RNAscope™. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in the cores. RNAscope™ was also performed in metastatic biopsies from patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope™ strongly correlated with HER2 protein levels and with HER2 IHC H-scores from the PATHWAY™ and HercepTest™ assays. However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY™ and HercepTest™. The RNA levels were significantly higher in responders than in non-responders to T-DXd; the cut-offs for RNA level with 10% and 30% response rates were 0.98 and 8.1 dots/cell, respectively by RNAscope™ in breast cancer. RNAscope™ is an assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, i.e., identify patients who would benefit from T-DXd.

Human epidermal growth factor receptor 2 (HER2/ERBB2) expression and gene amplification in breast cancer are currently evaluated by immunohistochemistry (IHC) and in situ hybridization (ISH). Based on IHC scores, breast cancer can be categorized as HER2-negative (0 to 1 +), HER2-equivocal (2+), or HER2-positive (3+). Tumors with a HER2 IHC score of 0 can be further classified as either 0 (no membranous staining) or ultralow (incomplete and faint membranous staining in >0 but <10% of tumor cells). Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of trastuzumab, which recognizes the HER2 protein on the tumor cell surface, and a cytotoxic topoisomerase I inhibitor (payload), through an enzyme- cleavable linker.

T-DXd was clinically approved to treat patients with metastatic HER2-low breast cancer based on results from a clinical trial. In the trial, HER2-low breast cancer was defined as tumors that were IHC 1+ or 2+ but ISH negative. Patients with HER2-low breast cancer can be treated with T-DXd; however, it is unclear which of these patients would benefit. The HER2 IHC 0 and 1+ cases can be difficult to evaluate and separate because the semiquantitative scoring of IHC heavily relies on subjective evaluation from pathologists. This issue is particularly problematic in sub-categorizing breast cancers with HER2-low expression levels (0, ultralow, or 1+) as it includes patients with HER2 -ultralow breast cancers. It has been reported that there is poor inter-observer agreement with only 26% concordance between HER2 IHC 0 and 1+ evaluations among 18 pathologists. See Fernandez et al: Examination of Low ERBB2 Protein Expression in Breast Cancer Tissue. JAMA Oncol 8: 1-4, 2022. These distinctions are important because inaccurate evaluation of HER2 IHC may lead to suboptimal treatment.

Patients with metastatic HER2-low breast cancer may not be able to receive T-DXd if HER2 is erroneously assessed as 0 instead of 1+ by IHC. On the other hand, if one can effectively identify patients who are unlikely to respond to T-DXd, these patients may be able to receive other more suitable treatment regimens such as sacituzumab govitecan, TROP2 antibody based therapy. Therefore, there is a need to develop HER2 and TROP2 evaluations that can be objectively quantified.

RNAscope™ is an RNA in situ hybridization (ISH) assay that allows evaluation of RNA levels using typical unstained formalin-fixed paraffin-embedded (FFPE) tissue slide. Experiments reported herein indicate that HER2 RNA levels measured by RNAscope™ can be objectively quantified and strongly correlate with HER2 protein levels and responses of patients to T-DXd treatment.

Experiments reported herein indicate that HER2 RNA levels from RNAscope™ were strongly correlated with HER2 protein levels and responses of patients to T-DXd. Current IHC assays are suboptimal in evaluating HER2-low breast cancers and therefore cannot identify which patients would benefit from T-DXd. RNAscope™ is a promising alternative to IHC assays to quantify HER2 expression in tumors.

Studies of patients with HER2 DNA gene-amplified breast cancers showed these patients had worse prognosis, but their disease responded to HER2 -targeted therapy. Other studies showed correlation between HER2 RT-PCR gene amplification and HER2 RNA and protein levels. The RT-PCR method may generate false-positive or false-negative results when in situ carcinoma or normal epithelial or stromal tissues are included. As a result, currently, IHC assays are recommended as the first-line assay to evaluate HER2 expression in breast cancer. The HER2 protein levels show dichotomous separation between HER2 gene-amplified and non-amplified breast cancers, and inter-observer agreement among pathologists in evaluating HER2-positive tumors is high. IHC results correlated better with tumor response to neoadjuvant HER2 -targeted therapy and chemotherapy than did ISH results in HER2-positive breast cancers. However, IHC assays are suboptimal in evaluating HER2-low breast cancers. The IHC assays were not designed to differentiate among tumors with continuous low HER2 protein expression levels, and there is poor interobserver agreement among pathologists in evaluating HER2-low expression levels.

Experiments reported herein indicate that both HER2 protein and HER2 RNA levels were similar between IHC 0, UL, and 1+ cases based on both the PATHWAY™ and HercepTest™ assays. In addition, experiments reported herein indicate no correlation between HER2 RNA levels and H-scores in IHC 0, UL, and 1+ cases. These results indicate that current clinically approved IHC assays are suboptimal in evaluating HER2-low tumors. A simple assay that can be objectively quantified is urgently needed.

Experiments reported herein indicate that the RNA levels measured by RNAscope™ were strongly associated with protein levels measured by the AQUA™ method and the H-scores in IHC 1+, 2+, and 3+ cases, indicating RNAscope™ is a robust assay in evaluating RNA levels. The RNAscope™ results were significantly higher in the metastatic biopsies obtained right before T- DXd treatment in responders compared to those in non-responders. The response rate of patients to T-DXd was better classified by the RNAscope™ scores than by the IHC scores. Cut-offs of RNAscope™ results to predict 10% and 30% response rates to T-DXd treatment were identified. Experiments also indicate that high HER2 RNA levels were associated with negative lymph node metastasis. Some tissue cores with high protein levels had relatively low RNA levels. The range of HER2 RNA levels in the responders to T-DXd was wide (6.4±8.2 dots/cell), which suggests other factors besides HER2 level may play a role in tumor response.

Experiments disclosed herein indicate RNAscope™ may be used as a quantitative and reliable assay to evaluate HER2 expression in all breast cancers. The results of the RNAscope™ assay can be objectively quantified. RNAscope™ results were strongly associated with HER2 protein levels and responses of patients to T-DXd treatment. It is contemplated to be an alternative to IHC assays in evaluating HER2 expression in breast cancers, or in conjunctions with IHC assays, when results provide a score of 1+, UL, or 0. Robust HER2 RNA quantification in tumor cells by RNAscope™

Tumor IHC stains were obtained by the PATHWAY™ assay. Subcellular RNA dot signals were used to quantify HER2 RNA levels via RNAscope™. The annotation and selection of tumor cells was done using QuPath™, whose cell detection algorithm effectively selected only tumor cells. The algorithm was applied to the 526 tissue microarray (TMA) cores and the 32 metastatic biopsy tissues from the patients who were treated with T-DXd.

HER2 RNA levels by RNAscope™ were strongly correlated with HER2 protein levels and IHC H-scores except when H-scores were low

HER2 protein levels were quantified by AQUA™ in 287 TMA cores (100 commercial and 187 patient tissue cores). Among these, 48 cores (31 commercial cores and 16 clinical cores) had HER2 protein levels above the LOD (greater than or equal to 3 amol/mm²). The remaining cores had protein levels below the LOD and therefore were excluded from further analyses.

In all 48 TMA cores with detectable protein levels, RNAscope™ HER2 RNA levels, and protein levels were moderately correlated (Fig 2A). Because the pre-analytical and storage conditions of the commercial cores were not clear, the correlation in the clinical cores were further analyzed. In these cores, RNA and protein levels were strongly correlated (Fig 2B).

The correlation between the RNAscope™ RNA levels and the IHC H-scores were examined. The RNA levels were strongly correlated with the IHC H-scores from both the PATHWAY™ assay (Fig 2C) and the HercepTest™ assay (Fig 2D). However, closer examination showed that the strong correlation between RNA levels and IHC H-scores was mainly driven by IHC 1+ to 3+ cases; the correlation disappeared in IHC 0 and 1+ cases.

No significant difference in HER2 protein or RNA levels between IHC 0, ultralow, and 1+ cases

To evaluate whether the IHC assays can efficiently evaluate HER2-low breast tumors, the quantified HER2 protein levels by IHC scores from the PATHWAY™ and HercepTest™ assays were examined using the 48 tissue cores with protein levels above the LOD. Protein levels significantly differed between IHC 1+, 2+ and 3+ cases based on both the PATHWAY™ (Fig 3A) and HercepTest™ assays (Fig 3B). However, quantified protein levels did not significantly differ between the 0, UL, and 1+ cases based on either the PATHWAY™ (Fig 3A) or HercepTest™ assay (Fig 3B). Additionally, when 0 and UL cases were combined, there was no significant difference between the combined 0 and UL cases and the 1+ cases.

The correlation between the HERZ RNA levels by RNAscope™ and the IHC scores (0, UL, 1+, 2+, and 3+) from the PATHWAY™ and HercepTest™ assays using results from the 526 TMA cores were examined. Similar to the results from the protein level analyses, although RNA levels significantly differed between IHC 1+, 2+ and 3+ cases based on both the PATHWAY™ (Fig 3C) and HercepTest™ assays (Fig 3D), RNA levels did not significantly differ between IHC 0, UL, and 1+ cases by either the PATHWAY™ assay (Fig 3C) or the HercepTest™ assay (Fig 3D). These data strongly indicate that the IHC assays are suboptimal in differentiating different protein levels and different RNA levels in HER2-low breast cancers.

HER2 RNA levels were significantly correlated with the response rate of patients to T-DXd treatment

The correlation between HER2 RNAscope™ results and response rates of patients were evaluated using the 32 metastatic biopsy samples from 23 patients who were treated with T-DXd. The HERZ RNA levels were significantly higher in responders (6.4 ± 8.2 dots/cell, n=12) than in non-responders (2.6 ± 2.2 dots/cell, n=20) (Fig. 4A). A cut-off value was identified by which the RNAscope™ results predicted rate of response to T-DXd treatment. The cut-offs for RNA level with 10% and 30% response rates were 0.98 and 8.1 dots/cell, respectively. The response rates in these patients by IHC results (Fig. 4B) were 60% in 1+ (n=5), 32% in 2+ (n=22), and 50% in 3+ (n=8).

Logistic regression was performed to correlate HERZ RNA levels with clinicopathologic variables from the 243 patients with TMA cores. RNAscope™ results were averaged for duplicate cores from the same patient. Increased RNA levels were significantly associated with decreased likelihood of lymph node metastasis. No association of HER2 RNA level was observed with age, race, ER or PR status, Nottingham histologic grade, T or M category, or overall survival.

Tissue Microarrays

Tissue microarray (TMA) cores with a diameter of 1.0 mm were used for RNAscope™ and IHC evaluations. The TMA cores included 100 commercially available breast cancer cores and cores from patients with clinicopathologic information including race, sex, Nottingham histologic grade, staging information, estrogen receptor (ER) and progesterone receptor (PR) status, survival, and treatment information, including chemotherapy, hormonal therapy, and radiation therapy information.

HERZ IHC Assays and Scoring Methods

The Ventana PATHWAY™ assay (Roche) and the HercepTest™ assay (Dako) are commonly used and clinically approved HER2 IHC assays. These tests were performed on TMA cores. Two scoring methods were used to evaluate the IHC staining results. First, using the current ASCO/CAP recommendations, overall IHC scores of 0, ultralow (UL), 1+, 2+, or 3+ were assigned. The UL score was given when the tissue cores had greater than 0 % but less than 10% incomplete membranous staining. In the second method, an H score was calculated as a weighted percentage multiplied by staining intensity: incomplete membranous staining (score 1), weak to moderate complete membranous staining (score 2), or strong complete membranous staining (score 3), resulting in H-scores ranging from 0 to 300.

Patients Treated With T-DXd

Metastatic biopsies were initially identified that were obtained before T-DXd treatment from patients. Of these biopsies, some were bone metastases with decalcified tissues and were excluded from analyses. Furthermore, other additional samples were excluded owing to insufficient tumor cells. In total, RNAscope™ results were evaluated from 32 metastatic biopsies from 23 patients who had HER2-low or HER2-positive metastatic breast carcinoma. Clinicopathologic information was collected including race, sex, site of metastasis, ER, PR, and HER2 status and detailed treatment information including targeted therapy, chemotherapy, hormonal therapy, and radiation therapy. Response was evaluated based on imaging and clinical evaluations. Responders were defined as patients who had stable or decreased disease burden after T-DXd treatment. Non-responders were defined as patients whose disease progressed on T-DXd treatment based on imaging and/or clinical evaluations.

RNAscope™ Assay and Quantification of HERZ RNA Levels

The RNAscope™ kit was purchased from a commercial supplier. One FFPE tissue section (5 mm) was subjected to the RNAscope™ FFPE assay kit. Tissue sections were treated with heat and protease and then hybridized with probes to the HER2/ERBB2 RNA molecules using an auto- stainer. RNAscope™ whole-slide images were analyzed to quantify subcellular ISH dots per cell. The whole-slide images generated by the scanner were loaded into QuPath™. The color vectors for hematoxylin staining and RNAscope™ ISH dots were computed using region-of-interest annotations. Subcellular RNAscope™ dots in the images were quantification. A slide rectangular window of region-of-interest annotations was created to cover the arrays, followed by a QuPath™ algorithm for tumor cell detection and stromal cell deselection; then, subcellular detection of RNAscope™ dots based on intensity, shape, and cluster sizes were analyzed. The resulting RNA levels were the average of number of subcellular dots among the tumor cells annotated by QuPath™.

Quantification of HER2 Protein

HER2 protein levels were quantified in the commercial TMA cores and TMA cores from patients who had breast cancers using the AQUA™ method of automated quantitative immunofluorescence (QIF) which calculates expression on a continuous scale by quantifying immunofluorescence pixel intensity per unit area, standardized against a cell line array with known HER2 protein levels determined by mass spectrometry and the intensities of fluorescence signals were converted to attomoles (amol) per square millimeter, using the pixel area expressing cytokeratin to determine the denominator. The limit of detection (LOD) for the protein level was equal or greater than 3 amol/mm².

Claims

1. A method of detecting and quantifying a low or an ultra-low quantity of Human epidermal growth factor 2 (HERZ) mRNA expression comprising: providing a group of immobilized cells or tissue sample from a patient comprising a target HER.2 mRNA sequence, wherein the target HER2 mRNA comprises a first target sequence and a second target sequence; contacting the immobilized tissue sample with a first target probe and a second target probe, wherein the first target probe comprises a first probe segment that hybridizes with the first target sequence and a first probe second segment, wherein the second target probe comprises a second probe segment that hybridizes with the second target sequence and a second probe second segment; further contacting the immobilized tissue sample with a preamplifier oligonucleotide, wherein the preamplifier oligonucleotide comprises a first preamplifier segment that hybridizes with the first probe second segment and a second preamplifier segment that hybridizes the second probe second segment, wherein the preamplifier oligonucleotide further comprises preamplifier target segments; further contacting the immobilized tissue sample with amplifier oligonucleotides, wherein the amplifier oligonucleotides comprise a first segment that hybridizes with the preamplifier target segments and second amplifier segments; and further contacting the immobilized tissue sample with labeled oligonucleotides, wherein the labeled oligonucleotides hybridize with the second amplifier segments; and detecting and quantifying the labeled oligonucleotide signals, wherein quantifying the labeled oligonucleotide signals indicate quantified expression of the target RNA in an amount associated with a risk of developing life threating cancer that is a low or an ultra-low quantity.

2. The method of claim 1 wherein a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of 0.

3. The method of claim 1 , wherein a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of score ultra-low (UL).

4. The method of claim 1 wherein a sample from the patient is first determined by HER2 immunohistochemistry (IHC) assay to be a score of low or 1+.

5. The method of claim 1 further comprising recoding the detected and quantified labeled oligonucleotide signals or implications thereof on a non-transitory computer readable medium.

6. The method of claim 1 further comprising comparing the detected and quantified labeled oligonucleotide signals to a normal, control, or reference value.

7. The method of claim 6 further comprising diagnosing the subject as having breast cancer and of need of treatment.

8. The method of claim 7 further comprising recoding the diagnosis on a non-transitory computer readable medium.

9. The method of claim 7 further comprising communicating the diagnosis, comparison, detection, quantification, or implications thereof to a medical profession or a patient from which the sample was obtained.

10. A method of diagnosing and treating breast cancer comprising; quantifying target RNA which is HER2 in a breast issue sample of a subject using the method of claim 1; wherein if the subject is diagnosed with a low or ultra-low expression of the HER2 mRNA administering to the subject an effective amount of an anti -breast cancer agent, performing radiation therapy, and/or removing the tissue by a surgical intervention.

11. The method of claim 9, wherein the anti-cancer agent is trastuzumab deruxtecan.