Patent Description:
The aberrant functioning of androgen receptor signaling is a central driving force behind prostatic tumorigenesis and its transition (or progression) into metastatic castration resistant disease (Feldman, <NUM>).

Methods for detecting expression of androgen receptor variants have been described previously. By way of example, <NPL>) describes an assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants Arv7 and Arv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. <NPL>)) describes a screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs). <NPL>) describes an assay in which ARv567 expression confers docetaxel sensitivity in vivo, while expression of the microtubule-independent ARv7 variant confers resistance. <CIT> describes a method for detecting and/or treating a subset of prostate cancer patients who may benefit from taxane treatment, wherein the method comprises testing whether an androgen receptor (AR) splice variant is present in a test sample obtained from the patient, wherein the androgen receptor variant can be ARv5,<NUM>,<NUM> or ARv7, or a combination thereof. <CIT> describes an in vitro method for determining if a tumor is resistant to androgen deprivation therapy, the method comprising determining the presence or absence of an androgen receptor splice variant selected from the group consisting of AR-V5 and AR- V10 in a tumor sample.

Hence, androgen deprivation therapy (ADT) is usually the first line of treatment for prostate cancer patients (Harris (<NUM>); Sridhar (<NUM>)). In addition to ADT, next-generation androgen receptor (AR) signaling inhibitors such as abiraterone, an inhibitor of androgen bio-synthesis, and enzalutamide, an antagonist of AR-ligand binding, are used in the treatment of prostate cancer.

However, many patients become resistant to ADT therapy as well to the next generation AR signaling inhibitors (enzalutamide and abiraterone). When prostate cancer patients progress following androgen receptor targeted therapies they are treated with taxane chemotherapy, the only class of cancer chemotherapy that improves survival of castration-resistant prostate cancer (mCRPC) patients. However, the effectiveness of taxanes can also be impaired by the development of drug resistance. Drug resistance is the number one cause of cancer death in such patients.

Methods of detecting drug resistance allow new therapeutic regimens to be utilized before the prostate cancer has progressed too far.

Prostate cancer drug resistance is due to the expression of androgen receptor (AR) splice variants (AR-Vs), which lack the ligand-binding domain and are constitutively active in the nucleus (Antonarakis, <NUM>). For example, expression of the androgen receptor splice variants AR-v7 or AR-V567 in circulating tumor cells (CTCs) isolated from the blood of prostate cancer patients has been correlated with resistance to enzalutamide and abiraterone. There is also evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes. Hence, assessment of the presence and/or amounts of different AR variants has clinical utility.

The inventors have shown that the androgen receptor (AR) binds microtubules (MTs), the primary target of taxanes, via its hinge domain and utilizes microtubules as tracks for its nuclear translocation and transcriptional activation of target genes. To assess the chemotherapy effect on targeting the microtubule-androgen receptor (MT-AR) axis and the nuclear accumulation of AR, the mechanism in CTCs derived from chemotherapy-naive mCRPC patients receiving taxane chemotherapy (docetaxel or cabazitaxel) was investigated in a multi institutional clinical trial. The results showed that clinical response to treatment was significantly associated with lower nuclear AR expression in CTCs and such reduced AR expression was predictive of the biochemical response to treatment for the individual patient (Antonarakis, <NUM>).

Sensitive assay methods are described herein that involve (a) capturing circulating cancer cells (e.g., by any method); (b) extracting mRNA and (c) detecting and quantifying each of AR-FL, AR-V7, AR-V567 in parallel digital droplet PCR assays. Such methods can detect androgen receptor and androgen receptor variants from prostate cancer subjects who may be in need thereof treatment.

The assay methods described herein are sensitive when using mRNA from as little as a half of a cell (<NUM> cell). These assay methods are also highly specific for each AR-V transcript and avoid co-detection of other similar variants, especially AR-V9, which is structurally similar to AR-V7. Currently available methods do not employ the highly sensitive methods described herein. For example, currently available methods do not adequately detect AR-V567, and do not have the specificity to distinguish which type of AR-variant, as well as the full-length AR (AR-FL), is being expressed and how much of each AR-variant (including AR-FL) is expressed. Currently available methods sometimes employ pre-amplification of transcripts, which is not needed in the methods described herein. No reference gene or standard curve is needed for the assay methods described herein. Instead of currently available multiplex assay procedures, separate assay mixtures can be used in the methods described herein, which helps to improve the assay sensitivity.

The claimed invention is defined in the appended claims.

The invention provides a method comprising:.

In certain embodiments, the method comprises one or more digital droplet PCR assays for detecting and quantifying AR-FL, each digital droplet PCR assay for detecting and quantifying AR-FL performed in a separate droplet comprising a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>; wherein the droplet can optionally further comprise a probe according to the sequence as set forth in SEQ ID NO: <NUM>.

In certain embodiments, the method comprises one or more digital droplet PCR assays for detecting and quantifying for AR-V7, each digital droplet PCR assay for detecting and quantifying for AR-V7 performed in a separate droplet comprising a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>; wherein the droplet can optionally further comprise a probe according to the sequence as set forth in SEQ ID NO: <NUM>.

In certain embodiments, the method comprises one or more digital droplet PCR assays for detecting and quantifying AR-v567es, each digital droplet PCR assay for detecting and quantifying AR-v567es performed in a separate droplet comprising a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>; wherein the droplet optionally further comprises a probe according to the sequence as set forth in SEQ ID NO: <NUM>.

In certain embodiments, the primers or probes are covalently or non-covalently bonded to one or two labels fluorescent, chemiluminescent, or radioactive labels.

In certain embodiments, capturing circulating cancer cells from the sample comprises isolating cells that express transferrin receptor, optionally further comprising depletion of CD45+ cells from the sample before isolating cells that express transferrin receptor.

In certain embodiments, the test subject is healthy.

In certain embodiments, the test subject is suspected of having cancer, or metastatic cancer, or prostate cancer.

In certain embodiments, the method comprises informing the test subject or medical personnel providing medical care for the test subject of detection, quantities, and/or quantification of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), or androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) levels detected or quantified in the parallel digital droplet PCR assays.

The invention provides a taxane for use in a method of treating prostate cancer in a subject, wherein the method comprises:.

The invention provides a composition comprising two or more types of primers or probes with at least <NUM> nucleotides of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, wherein one or more type of primer or probe is covalently or non-covalently bound to a label, wherein the composition comprises:
a primer having the sequence as set forth in SEQ ID NO: <NUM> and a primer comprising at least <NUM> nucleotides according to the sequence as set forth in SEQ ID NO: <NUM>, wherein the composition can optionally further comprise a probe comprising at least <NUM> nucleotides according to the sequence as set forth in SEQ ID NO: <NUM>.

In certain embodiments, the composition comprises a primer comprising at least <NUM> nucleotides according to the sequence as set forth in SEQ ID NO: <NUM> and a primer comprising at least <NUM> nucleotides according to the sequence as set forth in SEQ ID NO: <NUM>, wherein the composition can optionally further comprise a probe comprising at least <NUM> nucleotides according to the sequence as set forth in SEQ ID NO: <NUM>.

In certain embodiments, the composition comprises one or more primers or probes are covalently or non-covalently bonded to one or two fluorescent, chemiluminescent, or radioactive labels.

In certain embodiments, the method comprises capturing circulating cancer cells from the sample, comprising:.

Methods are described herein that involve (a) capturing circulating cancer cells (e.g., by any method); (b) extracting mRNA; and (c) detecting and quantifying each of AR-FL, AR-V7, and AR-V567 RNAs in parallel digital droplet PCR assays.

Any disclosures provided herein relating to methods for treatment of the human or animal body by surgery or therapy, or diagnostic methods practiced on the human or animal body, should be regarded as the disclosure of substances or compositions for use in such methods.

Androgen receptor variants ARv5,<NUM>,<NUM> and ARv7 (also known as AR3) appear to be the two most clinically prevalent splice variants. The ARv5,<NUM>,<NUM> variant is present in <NUM>% of tumor specimens from castration-resistant prostate cancer patients, and its expression arises in response to androgen deprivation therapy or abiraterone treatment (<NPL>); <NPL>)). The ARv7 variant is present in both benign and malignant prostate tissues but is generally enriched in metastatic disease (<NPL>); <NPL>)). Thus, the presence of androgen receptor splice variants is common in castration-resistant prostate cancer patients and is associated with resistance to current androgen deprivation therapies.

Sequences for various androgen receptors are available, for example, from the National Center for Biotechnology Information (see website at ncbi.

For example, a full length human androgen receptor (AR-FL) sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi. gov), which has accession number P10275. <NUM> (GI:<NUM>) and is shown below as SEQ ID NO:<NUM>.

The sequence of the androgen receptor can vary somewhat from one patient to another. For example, the number of the repetitive glutamine residues in androgen receptors (amino acids <NUM>-<NUM> of SEQ ID NO:<NUM>) can increase or decrease by any number between about <NUM>-<NUM> amino acids. Similarly, the number of repetitive glycine residues in androgen receptors (amino acids <NUM>-<NUM> of SEQ ID NO:<NUM>) can increase or decrease by any number between about <NUM>-<NUM> amino acids. Thus, the androgen receptor detected by the methods, reagents and devices described herein can have at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity to SEQ ID NO:<NUM>.

Sequence identity can be evaluated using sequence analysis software (e.g., via the NCBI tools, or the Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. <NUM> University Avenue. Madison, WI <NUM>). Such software matches similar sequences by assigning degrees of sequence identity to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Nucleotide sequences for the full-length human androgen receptor are also available from the NCBI database. For example, a cDNA sequence for the full length human androgen receptor is available as accession number M20132. <NUM> (GI:<NUM>), shown below as SEQ ID NO:<NUM>. <IMG>
<IMG>
<IMG>
<IMG>.

An mRNA encoding an androgen receptor can have at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity to an mRNA with or complementary to SEQ ID NO:<NUM>.

A sequence for androgen receptor variant <NUM>,<NUM>,7es [Homo sapiens] is also available from the NCBI database National Center for Biotechnology Information (see website at ncbi. This androgen receptor variant lacks exons <NUM>,<NUM>, and7. The NCBI database provides a sequence for a human androgen receptor variant <NUM>,<NUM>,7es with accession number ACZ81436. <NUM> (GI:<NUM>) (SEQ ID NO:<NUM>).

The sequence of the androgen receptor splice variant v5,<NUM>,<NUM> can vary somewhat from one patient to another. For example, the androgen receptor splice variant v5,<NUM>,<NUM> detected by the methods, reagents and devices described herein can have at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity to SEQ ID NO:<NUM>.

Nucleotide sequences for the human androgen receptor variant v5,<NUM>,<NUM> are also available from the NCBI database. For example, a cDNA sequence for the SEQ ID NO:<NUM> human androgen receptor variant v5,<NUM>,<NUM> is available as accession number GU208210. <NUM> (GI:<NUM>), shown below as SEQ ID NO:<NUM>. <IMG>
<IMG>
<IMG>.

An mRNA encoding an androgen receptor variant can have at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity to an mRNA with or complementary to SEQ ID NO:<NUM>.

The androgen receptor variant <NUM> (AR-V7) is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. A sequence for human androgen receptor variant <NUM> is available from the NCBI database with accession number ACN39559. <NUM> (GI:<NUM>) (SEQ ID NO:<NUM>).

The sequence of the androgen receptor splice variant v7 can vary somewhat from one patient to another. For example, the androgen receptor splice-variant v7 detected by the methods, reagents and devices described herein can have at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity, or at least <NUM>% sequence identity to SEQ ID NO:<NUM>.

Nucleotide sequences for the human androgen receptor variant v7 are also available from the NCBI database. For example, a cDNA sequence for the SEQ ID NO:<NUM> human androgen receptor variant v7 is available as accession number FJ235916. <NUM> (GI:<NUM>), shown below as SEQ ID NO:<NUM>. <IMG>
<IMG>
<IMG>.

Patients who are in need of evaluation for prostate cancer, or for progression of prostate cancer, or who can benefit from modified therapy for prostate cancer can provide samples for evaluation in the methods described herein. For example, patients who may be suffering from prostate cancer and/or patients who may be resistant or non-respondent to various drugs such as enzalutamide, abiraterone, taxanes, or a combination thereof can provide samples for evaluation in the methods described herein.

Taxanes refer to a class of compounds having a core ring system of three rings, A, B and C, as shown below. <CHM>
Examples of taxanes include paclitaxel, docetaxel, abraxane, and taxotere.

The sample can, for example, be circulating tumor cells, or prostate tissue sample. The development of metastases in patients with solid tumor malignancies can result from tumor cells entering the circulatory system and migrating to distant organs, where they extravasate and multiply. Circulating tumor cells (CTCs) are rare-as few as one cell per <NUM> million blood cells.

The samples can be obtained directly from a patient or indirectly from the patient. In other words, a sample can be obtained by one person and then tested or evaluated as described herein by a second person.

The sample can also, for example, be a fresh or frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue sample, routinely prepared and preserved in everyday clinical practice. The sample can be a biological fluid, such as, without limitation, whole blood, peripheral blood, ascites fluid, or a combination thereof.

For example, the samples used in the methods described herein can be peripheral blood samples or the circulating tumor cells (CTCs) obtained from peripheral blood samples. Peripheral blood samples can be collected from mCRPC patients, for example, in EDTA tubes. The collected blood samples ideally are processed within <NUM> hours of the time of withdrawing the blood.

A variety of technologies has been developed to improve the detection and capture of circulating tumor cells from the peripheral blood. These include density gradient centrifugation, immunomagnetic bead separation using monoclonal antibodies targeting epithelial cell-surface antigens, cell sorting using flow cytometry, filtration-based size separation and microfluidic devices. Although advances in circulating tumor cell capture have been made, the low frequency of circulating tumor cells in cancer patients, their heterogeneity, the lack of organ-specific capture approaches, and the plasticity of the circulating tumor cell population has limited the ability to capture and track all circulating tumor cells. Currently, the epithelial cell-adhesion molecule (EpCAM), represents an antigen of choice for the majority of microfluidic devices that have been developed to capture circulating tumor cells. However, as illustrated herein, capture of CTCs using epithelial cell-adhesion molecule (EpCAM) may not be an optimal method for obtaining CTCs that are useful for evaluating androgen receptor expression levels. Instead, selection of CTCs that express transferrin receptor (TfR) are a better pool for evaluation of androgen receptor expression levels.

In some cases, the sample used for extraction of RNA contains circulating tumor cells (CTCs). Such circulating tumor cells can be obtained from whole blood samples by isolation and/or enrichment of the circulating tumor cells by various methods. For example, in a first method circulating tumor cells can be isolated and enriched from peripheral blood samples by using a CD45 negative depletion Rosette Sep kit. according to the manufacturer's instructions (STEMCELL Technologies Inc. In another example, a second method for isolating and enriching circulating tumor cells from peripheral blood samples can involve using the prostate-specific membrane antigen (PSMA)-based geometrically enhanced immunocapture (GEDI) as described by Kirby (<NUM>), Galletti (<NUM>).

Total RNA can be extracted from the enriched circulating tumor cells pool using the RNAeasy Plus Micro kit (Qiagen) as per manufacturer's instructions. Aliquots of the RNA can be arrayed in separate test vessels. For example, the RNA can be arrayed in plates that have multiple wells, such as <NUM>-well plates.

Droplet Digital PCR (ddPCR) can be used to quantify the mRNA levels. In many cases, Droplet Digital PCR (ddPCR) is an improved method for distinguishing and quantifying the full-length AR and the various AR variants.

RNA aliquots can be prepared for detection and/or quantitation by mixing the RNA aliquots with nucleotides and one or more RNA/DNA polymerases. For example, the RNA aliquots can be mixed with available multiplexed master mixes of PCR enzyme/buffer from the One-Step RT ddPCR Advanced Kit for Probes (Bio-Rad), amplicons for a GUSB control DNA region, and primers that specifically bind to, detect, and can amplify the full-length androgen receptor (AR-FL) and AR-variants.

Specific primers are used that provide improved sensitivity and specificity for the full-length androgen receptor (AR-FL) and AR-variants. Such primer sequences are shown below.

In some cases, the primers and/or probes are labeled with one or more detectable labels. For example, the primers and/or probes can be labeled with <NUM>-carboxyfluorescein (<NUM>-FAM), but other labels can be used. Labels such as <NUM>-carboxyfluorescein (<NUM>-FAM), NED™ (Applera Corporation), HEX™ or VIC™ (Applied Biosystems); TAMRA™ labels (Applied Biosystems, CA, USA); chemiluminescent markers, Ruthenium probes; or radioactive labels (for example, tritium in the form of tritiated thymidine, <NUM>Sulfur, or <NUM>Pphosphorus) may also be used.

The primers and probes can have the sequences shown in Table <NUM>. However, they can also have at least one nucleotide difference, or at least two nucleotide differences, or at least three nucleotide differences, or at least four nucleotide differences, or at least five nucleotide differences from the sequences shown in Table <NUM>. In some cases, the primers and probes can be at least one nucleotide, or at least two nucleotides, or at least three nucleotides, or at least four nucleotides, or at least five nucleotides, or at least six nucleotides, or at least seven nucleotides longer or shorter than the sequences shown in Table <NUM>. Typically, the primers are shorter than about <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides, or <NUM> nucleotides. Primers are typically at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length. Probes can be longer than primers. For example, probes can generally be as long as <NUM> nucleotides in length. However, probes are conveniently less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length, or less than <NUM> nucleotides in length. Probes generally are at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length, or at least <NUM> nucleotides in length.

In some disclosures provided herein, a set of primers can be used that can include one or more of the following pairs of primers:.

In some cases, probes can be used that include one or more of the following sequences:.

Hence, for example, primers can have between <NUM> to <NUM> nucleotides, for example any range between <NUM> and <NUM> nucleotides, such as between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, or between <NUM> and <NUM> nucleotides etc..

For example, a "probe" can be an oligonucleotide that forms a hybrid structure with a target sequence contained in a molecule in a sample undergoing analysis, due to the complementarity of at least one sequence in the probe with the target sequence. Probes can include oligonucleotide sequences, for example, that are between <NUM> to <NUM> nucleotides, for example any range between <NUM> and <NUM> nucleotides, such as between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, between <NUM> and <NUM> nucleotides, or between <NUM> and <NUM> nucleotides etc..

Assay methods for detecting and/or quantifying androgen receptors can include methods such as real-time PCR, end-point PCR; end-point PCR with fluorescence detection, quantitative PCR, digital PCR, open- array PCR, digital drop PCR, quantitative digital PCR, quantitative real-time PCR, PCR suitable for high-throughput, and microarray detection / quantification methods.

The term "PCR" relates to polymerase chain reaction which is a procedure involving target amplification. The term "target amplification" relates to an enzymemediated procedure which is capable of producing billions of copies of nucleic acid target sequences. Procedures for PCR as a target amplification method are available to those of ordinary skill in the art. In general, conducting PCR involves mixing a sample of DNA, cDNA or RNA in a solution with at least two oligonucleotide primers that are prepared to be complementary to each strand of a DNA duplex, cDNA duplex, or an RNA:cDNA hybrid template. Nucleotide triphosphates (e.g., dNTPs) and a DNA polymerase, such as Taq polymerase, are used to catalyze the formation of DNA from the oligonucleotide primers and the dNTPs. At least one of the primers is a so called forward primer binding in <NUM>' to <NUM>' direction to the <NUM>' end of the first strand of the DNA; the so called reverse primer is binding in <NUM>' to <NUM>' direction to the <NUM>' end of the second strand of the DNA. The general principle of the PCR procedure foresees that the solution is heated to denature the double- stranded DNA to single-stranded DNA. After cooling down of the solution to the so called annealing temperature, the primers are able to bind to the separated DNA strands and the DNA polymerase catalyzes the generation of a new strand by joining the dNTPs to the primers. This process is repeated in several cycles resulting in a respective amount of amplified PCR products. The term "real-time PCR" relates to the detection of PCR products via fluorescence signals which are generated by cleavage of a dual labeled probe during hybridization of the PCR product. A dual labeled probe has a fluorescence dye and a quencher moiety. Examples of commonly used probes are TAQMAN® probes.

In some cases, a digital PCR system can be employed such as a droplet digital PCR system. The term "droplet digital PCR" refers to a digital PCR system in which the reaction area is a droplet of water in a well. Preferably, the digital PCR system is an emulsion droplet digital PCR system. The term "emulsion droplet digital PCR" refers to a digital PCR system in which the reaction area is a droplet that is formed in a water-oil emulsion. Techniques for performing droplet digital PCR and emulsion droplet digital PCR are available and include, but are not limited to, those described in <NPL>); <NPL>); and <NPL>). Droplet digital PCR systems and emulsion droplet digital PCR systems also are commercially available from sources such as, for example, the QX200™ DROPLET DIGITAL™ PCR system (Bio-Rad Laboratories, Inc. , Hercules, Calif.

There are several intrinsic advantages to ddPCR compared to traditional qPCR (<NPL>); Doi, <NUM>; <NPL>); <NPL>)). First, ddPCR allows absolute quantification without the need for normalization, calibrator or external references (<NPL>). This is because Poisson statistics allow direct estimation of template copies. Second, ddPCR provides a direct measurement expressed as number of copies of target per microliter of reaction (with confidence intervals) (Hindson, <NUM>). Third, because ddPCR is an endpoint binary assay, it is relatively insensitive to technical issues such as PCR inhibitors (Doi, <NUM>; Huggett, <NUM>; Racki, <NUM>). Fourth, unlike traditional qPCR, ddPCR has predicable technical measurement error because the underlying binomial distribution can be used to directly compute confidence intervals (<NPL>). <NUM>) ddPCR has been shown to have increased precision and sensitivity in detecting low template copies (<NPL>); <NPL>); <NPL>)). Sixth, ddPCR assays can be predictably and reliably run multiplexed. There are now hundreds of publications that underline the benefits of ddPCR and guidelines have been developed to ensure excellent data quality, precision and reproducibility for this highly sensitive technique (Huggett, <NUM>).

Due to the high sequence analogy of AR-v7 and AR-v9 recently published by Dehm's group (see, e.g., <NPL>)), the inventors investigated the expression profiles of AR-v7, AR-v9 and AR-FL in RNA-Seq already published prostate cancer patient data. The inventors acquired access to RNA-Seq data of <NUM> primary prostate cancer patients from TCGA (The Cancer Genome Atlas) and <NUM> CRPC patients from Robinson et al. (<NUM>) study. Raw sequencing reads were trimmed using Trimmomatic and aligned to human reference genome (version hg38) using STAR. Determination of expression for AR-v7 was examined based on mapped reads across junction between exon3 and CE3. Reads for AR-v9 were extracted from junction reads between exon <NUM> and CE5 (Kohli et al. For AR-fly, expression was determined through counting mapped reads across junction between exon7 and exon8 of AR gene.

In the primary prostate cancer samples, <NUM>% of the <NUM> samples were AR-FL positive, <NUM>% were AR-v7 positive and <NUM>% were AR-v9 positive. Interestingly, in the <NUM> CRPC samples, <NUM>% were AR-FL positive similar to primary prostate cancer patients. However, the expression of both AR variants was much higher in the CRPC compared to the primary samples: <NUM>% and <NUM>% were Ar-v7 and Ar-v9 positive respectively. This significant increase in the presence of AR-v7 and Ar-v9 in CRPC patients emphasizes the importance of the AR-V in the development of drug resistance and its role in mCRPC.

One or more androgen receptor full-length and/or androgen receptor variant proteins can therefore be expressed at higher levels in subjects with prostate cancer and/or in subjects with drug-resistant cancers (e.g., drug resistant prostate cancer) than in healthy persons (e.g., in subjects without prostate cancer). For example, androgen receptor full-length and/or androgen receptor variant proteins can be expressed at <NUM>% higher, or <NUM>% higher, or <NUM>% higher, or <NUM>% higher, or <NUM>% higher, or <NUM>% higher, or <NUM>% higher, or <NUM>% higher levels in subjects with prostate cancer and/or in subjects with drug-resistant cancers (e.g., drug resistant prostate cancer) than in healthy persons (e.g., in subjects without prostate cancer). In some cases, one or more androgen receptor full-length and/or androgen receptor variant proteins can be expressed at two-fold higher, or three-fold higher, or four-fold higher, or five-fold higher, or seven-fold higher, or eight-fold higher, or ten-fold higher, or twelve-fold higher, or fourteen-fold higher, or fifteen-fold higher, or seventeen-fold higher, or twenty-fold higher, or twenty-two-fold higher, or twenty-five-fold higher, or thirty-fold higher levels in subjects with prostate cancer and/or in subjects with drug-resistant cancers (e.g., drug resistant prostate cancer) than in healthy persons (e.g., in subjects without prostate cancer).

The assay methods described herein (shown in the first row) were compared to those obtained by other methods. Such a comparison is illustrated, for example, in Table <NUM>, where the features of the assay methods described herein are shown in the first row.

The methods and kits described herein can be used for any of the following:.

The assay methods described herein have been used in two prospective multi-institutional clinical trials.

Kits are also disclosed herein that are useful for detecting and/or quantifying full-length and/or variant androgen receptors.

For example, such a kit, disclosed herein (but not claimed), can include at least one primer or probe specific for one or more androgen receptor in a biological sample. One example of such a kit, disclosed herein (but not claimed), can include: at least one set of primers comprising a forward primer and a reverse primer, wherein each forward primer and reverse primer includes an oligonucleotide of between <NUM> and <NUM> nucleotides in length and of at least <NUM> contiguous nucleotides of a nucleotide sequence located in.

The kits can also include probes that can include one or more of the following sequences:.

The kits can include combinations of primer sets and/or probes in a single composition or container. Alternatively, the kits can include separate primer sets and/or probes in separate compositions or containers.

The primer sets and/or probes can be covalently attached to a solid surface or be aliquoted into wells arrayed in a solid substrate. For example, the primer sets and/or probes can be distributed on or within a microarray.

The kits can also include nucleotides, enzymes, cofactors, salts, buffers, and combinations thereof that are useful for performing methods for detecting and/or quantifying the full-length androgen receptor and/or the androgen receptor variants.

Patients can be informed of the assay results. As used herein "informing the patient" or "informing the test subject" can involve reporting test results to a hospital, medical clinic, or doctor who may provide medical care for the patient or test subject. As used herein the terms "patient" and "test subject" are used interchangeably.

For example, patients can be informed of the quantity of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), and/or androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) transcripts.

A subject or patient can have cancer (e.g., prostate cancer) and/or may be resistant to currently administered therapeutics (drug-resistant), when the quantities of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), and/or androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) transcripts are different from control quantities of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), and/or androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) transcripts. Controls can include the amounts of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), and/or androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) transcripts detected or quantified in healthy subjects, or subjects without cancer.

The methods provided herein for detecting androgen receptor variants (and full-length androgen receptors) can be combined with providing for treatment of diseases associated with expression of such androgen receptor variants (and full-length androgen receptors). For example, in some cases detection of androgen receptor variant (and/or full-length androgen receptor) expression is an indicator of drug resistance. Patients or test subjects that exhibit drug resistance (e.g., as detected by the assay methods described herein) can benefit from the provision of different treatment regimens and/or providing for administration alternative drugs or therapeutic agents.

Patients or test subjects who can be provided for treatment include cancer patients, for example, patients with prostate cancer or drug-resistant prostate cancer.

When drug-resistant androgen (variant and/or full-length) receptor expression is detected, a patient can be provided for treatment with a variety of other therapeutic agents or therapeutic procedures that may not include use of the drug to which the patient is resistant. A drug-resistant cancer can be provided for treatment with a variety of other therapies useful in the treatment of drug-resistant cancer. For example, elevated prohibitin levels have been shown to play a role in taxane-resistant cancers, and inhibition of prohibitin can reduce taxane-resistance (see e.g., <CIT>). Thus, any method for inhibiting prohibitin (e.g., <CIT>) can be used in the treatment of a taxane-resistant cancer. Exemplary inhibitors of prohibitin are known in the art and are described e.g., in <CIT>.

Other agents that prevent or reverse drug-resistance can include, but are not limited to, an inhibitor of glutathione-S-transferase π, or an inhibitor of p-glycoprotein.

Non-limiting examples of other chemotherapeutic agents that can be provided for administration to patients or test subjects include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, or uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide or trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-<NUM> (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin <NUM> and cryptophycin <NUM>); dolastatin; duocarmycin (including the synthetic analogues, KW-<NUM> and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omega1I (see, e.g., <NPL>)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, <NUM>-diazo-<NUM>-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, <NUM>-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and <NUM>-fluorouracil (<NUM>-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, <NUM>-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, <NUM>-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; <NUM>-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg. ); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; <NUM>,<NUM>',<NUM>"-trichlorotriethylamine; trichothecenes (especially T-<NUM> toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chloranbucil; GEMZAR® gemcitabine; <NUM>-thioguanine; mercaptopurine; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-<NUM>); ifosfamide; mitoxantrone; vincristine; NAVELBINEO. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-<NUM>) (including the treatment regimen of irinotecan with <NUM>-FU and leucovorin); topoisomerase inhibitor RFS <NUM>; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva@)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In addition, the methods of treatment disclosed herein can further include the use of radiation or radiation therapy. Further, the methods of treatment disclosed herein can further include the use of surgical treatments.

As described herein the provision of treatment can be varied depending on which types of AR variants are expressed in a patient. Taxanes are currently the only class of chemotherapy agents that extend survival in men with advanced prostate cancer. These drugs bind β-tubulin and stabilize cellular microtubules, leading to inhibition of microtubule-dependent intracellular trafficking and signaling, mitotic arrest, and apoptotic cell death. Although taxanes are generally considered antimitotic agents, they also inhibit tumor growth via several different mechanisms. Prostate cancer cells rely heavily on sustained androgen receptor (AR) nuclear signaling, which drives progression despite androgen-deprivation therapy. AR binds to cellular microtubules via the microtubule-associated motor protein dynein to facilitate its nuclear translocation. Taxanes can inhibit AR nuclear trafficking via stabilization of microtubules. Taxane-induced microtubule stabilization (termed drug-target engagement [DTE]) results in microtubule bundling (MTB), cytoplasmic sequestration of AR, inhibition of AR transcriptional activity, and inhibition of prostate cancer cell growth. Hence, in some cases taxane treatment can be helpful for treatment of prostate cancer.

Taxane treatment inhibits microtubule dynamics, it has been shown to differentially affect the nuclear localization of AR splice variants. The data shown herein demonstrates that AR-V7-negative patients have a greater reduction in AR percent nuclear localization when treated with taxanes compared with AR-V7-positive <NUM> patients (Table <NUM> and <FIG>). AR-FL is kept inactive in the cytoplasm, whereas the nuclear localization of AR-V7 is not affected. Accordingly, tumors that express AR-V7 are less sensitive to taxanes and taxanes may not be successfully administered to patients exhibiting AR-v7. The hinge domain that is retained in ARv567es is the minimum functional domain required for microtubule binding, although it does not bind as extensively as the entire C-terminus of AR. Hence, taxane treatment can partially impair the nuclear localization of ARv567es in vitro and detecting ARv567es expression in patients can confer taxane sensitivity in vivo.

In <NUM>% of patients with undetectable levels of either splice variant that were evaluated as described herein, but that also exhibited AR-FL expression, exceptionally high response rates to taxane treatment of <NUM>-<NUM>% were observed.

Hence, when patient samples are evaluated as described herein, expression of AR-FL in such samples indicates that a patient can successfully be provided for treatment with taxanes. Patient with samples exhibiting expression of ARv567es can in some cases be successfully provided for treatment with taxanes, but in other cases may not be successfully provided for treatment with taxanes. However, when significant quantities of AR-<NUM> are detected in patient samples, those patients should be provided for receipt of treatment other than taxanes because taxane treatment may not be effective.

The following Examples illustrate procedures and results used and obtained in the development of the invention.

This Example illustrates some of the materials and methods used in the development and methods described herein.

22Rv1 (Cat # CRL-<NUM>) and VCaP (Cat # CRL-<NUM>) cells were obtained from ATCC. ATCC authenticates human cancer cell lines using short tandem repeat analysis. These cell lines were expanded, and earlier passages were frozen in liquid nitrogen. 22Rv1 were maintained in RMPI1640 (Corning) with <NUM>% FBS, <NUM> U/mL penicillin, and 100µg/mL streptomycin (penicillin/streptomycin) in a <NUM>% CO<NUM> incubator at <NUM>. VCaP cells were cultured in DMEM (Corning) with <NUM>% FBS and penicillin/streptomycin in a <NUM>% CO<NUM> incubator at <NUM>.

pEGFP-C1-AR-FL, pEGFP-C1-AR-V7 and pEGFP-C2-AR-<NUM> plasmid DNA were used as positive controls for the development of the multiplex ddPCR assay. <NUM> ng of each plasmid was transfected in AR null HEK293T cells using FuGENE® <NUM> Transfection Reagent according to the manufacturer's instructions (Roche, Germany). Post <NUM> transfection total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany, Cat. # <NUM>), cDNA was generated from <NUM> ug of total RNA using ProtoScript First Strand cDNA Synthesis (NEB, Cat. # E6300L) and cDNA samples were used for evaluating the specificity of the methodology.

In this study, peripheral blood samples were collected from mCRPC patients in EDTA tubes and processed within <NUM> hours of the time of blood withdraw. All patients provided written informed consent. CTCs from the whole blood of mCRPC patients were enriched and isolated via two methods:.

Total RNA was extracted from the enriched CTCs pool using the RNAeasy Plus Micro kit (Qiagen) as per manufacturer's instructions. And then arrayed in <NUM>-well format for PCR using commercially available multiplexed master mixes of PCR enzyme/buffer from the One-Step RT ddPCR Advanced Kit for Probes (Bio-Rad), amplicons for a GUSB control DNA region, and primers that specifically detect the AR- FL and AR-variants.

We included <NUM> healthy male subjects to determine background levels of AR- FL, AR-V7 and AR-v567es transcripts in peripheral whole blood and whole blood processed in a similar manner as for CTC enrichment from the blood of prostate cancer patients. Samples from healthy subjects were obtained and stored under the same conditions as for patient samples to minimize any bias.

Each primer set was designed to recognize unique and distinguished regions of each of the variants using Primer3Plus based on the direction of the ddPCR Application Guide Bulletin <NUM> (Bio-Rad). AR-FL assay recognizes unique junction between exon <NUM> and <NUM>, AR-v7 assay recognizes the junction of exon <NUM> and cryptic exon <NUM>, and AR-v567es assay recognizes the junction between exon <NUM> and exon <NUM>. The primers were designed such at least one primer from a pair spans on the exon-exon junction to avoid unspecific amplification from other variants and synthesizes from genomic DNA. Specificity of the primer design was assessed by a Nucleotide BLAST (blastn) search against the up-to-date version the human genome reference (hg38) database and IGV (Integrative Genomics Viewer). The primers and probes were designed and purchased from Bio-Rad for use in all droplet digital PCR (ddPCR) reactions as shown in Table <NUM>.

Droplet Digital PCR (ddPCR) is a digital PCR method based on the water-oil emulsion droplet technology (<NPL>); <NPL>)). The Droplet Digital PCR System partitions nucleic acid samples into <NUM>,<NUM> nanoliter-sized droplets and PCR amplification is carried out within each droplet.

AR-FL, AR-V7 and AR-v567es transcript quantifications were carried out on a QX200 Droplet Digital PCR (ddPCR) system with automated droplet generation (Bio-Rad Laboratories). Reactions were carried out in ddPCR Plates <NUM>-Well, Semi-Skirted (twin. tec PCR, Eppendorf). Each well contained <NUM>. 5µl of ddPCR Supermix for Probes, <NUM>. 2ul Reverse Transcriptase and <NUM>. 1ul of <NUM> DTT and other components of a One-Step RT-ddPCR Advanced Kit for Probes (Cat. # <NUM>-<NUM>, from Bio-Rad), <NUM>µl of target-specific primers, and <NUM>µl of sample RNA, for a total volume of <NUM>µl. Plates were sealed, spun down and loaded into a QX200 automated droplet generator (Bio-Rad). Immediately after droplet generation, <NUM>-well plates containing droplet-partitioned samples were heat-sealed and PCR was carried out on a C1000 Touch Thermal Cycler (Bio-Rad) using the following cycling protocol: reverse transcription at <NUM> for <NUM>, enzyme activation at <NUM> for <NUM> minutes followed by <NUM> cycles of <NUM> for <NUM> seconds (for denaturation) and <NUM> for <NUM> seconds (for annealing/extension), followed by a final <NUM>-minute incubation at <NUM> for enzyme deactivation. Ramp rate was <NUM> per second. Plates were then kept at <NUM> for at least <NUM> hours prior to being analyzed in the BioRad QX200 droplet reader because this has been shown to improve acceptable droplet counts. Included on each plate were positive and negative "plate controls": no template control (NTC), HEK <NUM> cells transfected with either AR-FL, AR-V7 or AR-v567es. Purified nuclease-free water was used as negative, no-template control (NTC). Plates were sealed on the robotic platform using a plate sealing stage. All robotic procedures were performed in a pre-PCR clean area.

Data acquisition and analysis was performed with the system's pre-installed software QuantaSoft from Bio-Rad. The fluorescence amplitude threshold, distinguishing the positive from the negative droplets was set manually by the technician in between the fluorescence amplitude of the positive and negative droplets taking into consideration all the positive and negative controls within each plate.

Each plate and each well contained controls to ensure assay validity and performance. Wells or plates that did not meet the expected criteria were flagged and reran. Plates were flagged for evaluation and/or reran if any of the following conditions was not met: <NUM>) positive droplets in NTC; <NUM>) positive droplets in the no-mastermix control; and <NUM>) an average of less than <NUM>,<NUM> droplets/well across the plate.

AR-FL and AR-Vs expression was detected and/or quantified using ddPCR. The ddPCR method can reliably measure, with high precision, extremely low concentrations of specific DNA sequences even when a complex mixture of templates is present. Moreover, the ddPCR method does not require development of or use of a standard curve. Digital PCR is a method of absolute nucleic acid quantification based on the partitioning a qPCR reaction sample into tens of thousands of nano-reactions (droplets) of defined volume. Each droplet either contains or does not contain a template molecule (Vogelstein, <NUM>; Pinheiro, <NUM>; <NPL>)). After PCR, droplets that contained a template will have a fluorescent signal (positive droplets) that distinguishes them from the droplets without a template (negative droplets). It is the ratio of detected "positive" droplets to total droplets that allows the number of target molecules per droplet to be calculated from a Poisson distribution.

The analytical specificity of the ddPCR assay was determined by transfecting HEK293T cells with plasmids encoding AR-FL, AR-v7 or AR-v567es. The HEK293T cells express very low endogenous levels of AR-FL and are negative for AR-v7 and AR-v567 transcripts. Each primer pair and probe was designed to be at the position of exon junctions to ensure specificity for each respective transcript and avoid non-specific signals from other variants. See, e.g., <FIG>.

<FIG> shows signal detection expressed as copies/sample, specific for each transcript, while the water control was completely negative for all three transcripts. No variant signal was detected in the non-transfected HEK293T cells, while low levels of endogenous AR-FL was present, as expected. In addition, when we performed this assay using genomic DNA as input, no signal was detected, confirming the specificity of the assay.

<FIG> illustrates the analytical sensitivity of the assay as determined for each individual AR splice variant using a calibration curve that was generated using serial dilutions of the respective DNA plasmids (<NUM>, <NUM>, <NUM> ng) in triplicate for each concentration. These results showed linearity over the entire quantification range and correlation coefficients greater than <NUM> in all cases, indicating a precise log-linear relationship. <FIG> shows that the assay does not detect genomic DNA.

The sensitivity of the ddPCR assay was also determined by spiking <NUM>, <NUM>, <NUM> or <NUM> VCAP or 22RV prostate cancer cells into <NUM> million PBMCs isolated via Ficoll gradient separation from the peripheral blood of a male healthy donor. Following RNA extraction, the total RNA was split into three different reactions for the detection of each the AR variants. As shown in <FIG>, the AR-FL and AR-v7 mRNA transcripts were detected in the VCaP spiked cells at levels as low as the quantity of RNA in a single cell. As also illustrated in <FIG>, the AR-v567es transcript was not detected in the VCaP cells.

The limit of detection of these androgen receptors was also evaluated by isolating single cells using the Cell Celector system from ALS (<FIG>). AR-FL and AR-v7 mRNA transcripts were detected in RNA obtained from just half a VCaP cell.

As shown in <FIG>, the expression levels of the AR-FL and AR-v7 were varied among the different single VCaP and 22RV cells tested, with some cells expressing higher levels of AR-FL than AR-v7 and vice versa emphasizing that the population of CTCs is heterogeneous.

Intra-assay variance (repeatability) of the ddPCR assay was evaluated by repeatedly analyzing 10ng of RNA from transfected AR-FL, ARv7 and AR-v567es DNA plasmids in HEK293T cell line and a non-transfected HEK293T null cell line in <NUM> parallel experimental set-ups.

Each experimental replicate was carried out under the following repeatability conditions. The same RNA batch from the transfected plasmids expressing the AR-FL, AR-v7 and AR-v567es, the same pre-mix from the One Step kit, cartridges from the same batch, the same instrument, but randomly positioned on the <NUM>-well PCR plate. Intra-assay variance was expressed as the standard deviation (SD) of the Copies/µl. The intra-assay variance in Copies/µl for AR-FL, ranged from <NUM> to <NUM>. The intra-assay variance in Copies/µl for AR-v7 ranged from <NUM> to <NUM>. The intra-assay variance in Copies/µl for AR-v567es ranged from <NUM> to <NUM>. See Table <NUM>.

As shown the Intra-assay variance expressed as within-run coefficient of variation (CV) of copies/µL ranged for AR-FL, from <NUM>% to <NUM>%, for AR-v7 from <NUM>% to <NUM>%, and for AR-v567es from <NUM>% to <NUM>%.

The Inter-assay variance (reproducibility) was also evaluated by analyzing the same DNA plasmids of AR-FL, AR-v7, and AR-v567es transfected in the HEK293 cell line on <NUM> separate assays performed on <NUM> different days. The results were expressed as between-run standard deviations (SDs). As shown in the chart below, between-run SDs of the copies variance for AR-FL ranged from <NUM> to <NUM>, for AR-v7 ranged from <NUM> to <NUM>, for AR-v567es ranged from <NUM> to <NUM>, while CVs were for AR-FL from <NUM> to <NUM>%, for AR-v7 from <NUM> to <NUM>%, for AR-v567es from <NUM> to <NUM>%. See Table <NUM>.

This Example illustrates validation of the assay procedures as described in the foregoing Examples within healthy volunteers and metastatic castration-resistant prostate cancer (mCRPC) patients.

The specificity of the assay was tested in using peripheral blood mononuclear cell (PBMC) fractions of peripheral blood samples from healthy male donors. Using the Ficoll-hypaque density gradient separation method PBMCs were isolated from <NUM> healthy male individuals.

As shown in <FIG>, all the healthy donor control samples were negative for both AR-variants, but expression of the reference gene GUSB was detected. Only very low levels of the AR-FL were detected in normal, healthy persons.

The performance of the ddPCR assay was evaluated in circulating tumor cells (CTCs) and PBMCs isolated from the peripheral blood of six mCRPC samples. CTCs were isolated from <NUM>- <NUM> of peripheral blood through use of the antigen-agnostic CD45 negative depletion Rosette Sep kit. In parallel, <NUM>-2mls of matching blood from the six mCRPC patient was processed using the Ficoll gradient centrifugation for the isolation of the PBMC fraction.

As illustrated in <FIG>, heterogeneous expression of AR-FL, AR-v7 and AR-v567 transcripts was detected in the CTCs from these patient samples. The PBMC fraction from the same patient samples expressed very low levels of AR-FL, while the AR-V7 and AR-v567es were not detectable in PBMC samples (<FIG>).

The reproducibility of the ddPCR assay in CTCs from mCRPC patient samples was evaluated.

Nearly identical results were obtained for the expression of each AR transcript when the CTCs from the same patient sample was split and run twice. CTCs were isolated as described in the foregoing Examples from <NUM>-<NUM> of peripheral blood by the CD45 negative depletion method. The RNA was extracted and was split into two fractions. The libraries and the ddPCR experimental setup for each of the two runs were processed by two different operators.

<FIG> shows the expression levels of the AR-FL, AR-v7 and AR-v567es RNAs in each given sample, showing that the expression levels were similar between the two runs.

The absolute copy number of each of the AR-variants was quantified in each of the patient samples and used to determine expression pattern and prevalence of each of these variants in CTCs using the ddPCR assay described herein.

As shown in <FIG>, AR-FL was the predominantly expressed transcript with a median and mean respectively of [x;y copies/ul] and range of [x;y] while AR-V7 was expressed at a median and mean of [x,y] and range of [x;y] and AR-v567es has a median and mean of [x,y] and range of [x;y].

Table <NUM> below shows that of the <NUM> mCRPC samples that were analyzed, <NUM>/<NUM> (<NUM>%) were AR-FL positive, <NUM>/<NUM> (<NUM>%) were ARV7-positive, <NUM>/<NUM> (<NUM>%) were AR-v567es-positive, <NUM>/<NUM> (<NUM>%) had both variants, and <NUM> of <NUM> (<NUM>%) expressed all three AR-FL, AR-V7 and AR-v567es.

The epithelial cell adhesion molecule (EpCAM) is the most widely used antigen for the positive identification of CTCs in solid tumors, including in prostate cancer cases. However, as EpCAM is downregulated during epithelial to mesenchymal transition (EMT), which is a process that precedes metastasis, its validity in the molecular characterization of CTC from metastatic patients is questionable.

Transferrin receptor (TfR) is highly expressed in prostate cancer and overexpressed in metastatic CRPC patients. As TfR appears to not be affected by EMT the inventors tested TfR as an alternative positive selection antigen for mCRPC CTCs.

TfR protein expression was first analyzed by immunofluorescence in a panel of prostate cancer cell lines and in healthy donor leukocytes using methods described by <NPL>)). While all prostate cancer cells lines analyzed were positive for TfR expression, none of the leukocytes expressed the receptor. Moreover, TfR expression was detected also in EpCAM negative prostate cancer cell lines.

To determine the clinical applicability of this novel CTC identifier, TfR expression was determined in CTCs isolated from peripheral blood of <NUM> metastatic CRPC patients using the Cell Celector technology. Two pools of TFR+ and EpCAM+ CTCs were also subjected to the ddPCR assay.

The results are shown in Table <NUM>, and in <FIG>.

As illustrated in Table <NUM> and in <FIG>, higher enrichment for ARv567es and ARV7 expressing cells was observed in the TfR+ CTCs. No significant difference was observed in the detection of AR-FL transcripts within TFR-CTC positive cells (<NUM>% express AR-FL) and the EpCAM-CTC positive cells (<NUM>% express AR-FL).

However, there was a significant difference in the detection of both AR-V7 and Ar-v567es within TFR-CTCs and EpCAM-CTCs. As shown, <NUM>% of TFR-CTCs express AR-V7 while only <NUM>% of EpCAM-CTCs express AR-V7. The AR-v567es variant is expressed in <NUM>% of TFR-CTCs, while only <NUM>% of EpCAM-CTCs express AR-v567es.

These data indicate that TfR is a promising biomarker for the detection of CTCs in mCRPC patients, and potentially superior to EpCAM. Hence, rather than relying solely on EpCAM+ enrichment, TfR can be useful for CTC enrichment from prostate cancer patients.

In addition to prostate cancer patients, the applicants have successfully used TfR to identify CTCs from the peripheral blood of non-small cell lung cancer (NSCLC) patients. Currently, there is no existing assay able to reliably and consistently identify CTCs in NSCLC patients, including the FDA-cleared CellSearch (slide <NUM>). The applicants have used TfR labeling and have identified CTCs from both early-stage (stage I-IIIA) and metastatic (stage IV) NSCLC patients. The TfR+-cells were deemed to be CTCs based on co-expression of pan-cytokeratin (epithelial cell marker) and TTF1 (established marker used clinically by pathologists to identify adenocarcinoma of the lung) (slide <NUM>).

Applicants believe that the methods described herein describe the first specific dd-PCR assay that reliably and concomitantly detects the expression of both AR-V7 and AR-v567 variants as well AR-FL expression in CTCs from mCRPC patients.

The assay specificity relates to the design of primers that lie on unique exon-exon spanning regions of each variant that avoid interference from other variants. Such interference can be present in currently available assay methods due to sequence similarities, such as that of AR-v9 with other variants.

There have four recent publications on the dd-PCR assay for the detection of the AR-v7, and only one of which has any specificity for the detection of the AR-v7 from the whole blood of prostate patient samples, while the other three are non-specific due to the lack of specificity in the primer design and because those assays detect both the AR-v7 and the AR-v9 variants. The inability to reliably distinguish AR-v7 and AR-v9 variant signals by such currently available assay methods may have led to misleading and inaccurate quantification of these variants, with an inappropriate correlation to patient outcome.

There has been only report (Hornberg <NUM>) on the quantification of both AR-v567 and AR-v7 from prostate patient tissues by qRT-PCR, in which the AR-v567 primers were deemed to be specific, but the role of AR-v7 was unclear due to AR-v9 detection (and the inability to distinguish AR-v9 from other variants). In that study, AR-v567 was prevalent in <NUM>% of CRPC samples, but such expression data were acquired using much greater amounts of RNA (e.g., <NUM> ng of RNA) extracted from bone metastases of each patient. Hence, the source of the RNA and reliability of the results of such an assay are different (and less reliable) than the methods described herein.

The assay methods described in this application are therefore an improvement over assay methods previously described and/or currently available.

Patients with metastatic castration-resistant prostate cancer (mCRPC) have several treatment options; however, intrinsic and acquired resistance to various treatment modalities is common. The association was evaluated between AR-V7 and ARv567es splice variant expression and response in patients receiving taxanes in the TAXYNERGY study (NCT01718353). TAXYNERGY evaluated the benefit of an early switch from docetaxel to cabazitaxel or vice versa in patients with chemotherapy-naive mCRPC patients. Absence of both variants at baseline was associated with the best prostate-specific antigen response and progression-free survival in patients receiving taxanes. AR nuclear localization did not change following taxane treatment in AR-V7-positive or double-negative patients, suggesting AR-V7 may be dominant over ARv567es. These results indicate that absence of AR splice variants may be associated with a superior response to taxane treatment in mCRPC patients.

TAXYNERGY was a non-comparative randomized Phase II study that enrolled chemotherapy-naive patients with progressive mCRPC. Patients were randomly assigned <NUM>:<NUM> to initial treatment with docetaxel <NUM>/m<NUM> or cabazitaxel <NUM>/m<NUM> every <NUM> weeks, where the first administration is referred to as cycle <NUM>, the second administration is referred to as cycle <NUM>, etc. Patients achieving at least a <NUM>% PSA decline from baseline by cycle <NUM>, day <NUM> (C5D1) continued to receive the same taxane, whereas those who did not achieve at least a <NUM>% PSA decline were switched to the alternative taxane at C5D1. Treatment continued until disease progression, death, unacceptable toxicity, or withdrawal of consent (<NPL>)).

The protocol complied with recommendations of the 18th World Health Congress (Helsinki, <NUM>) and all applicable amendments. The study was approved by the institutional review board at each participating center and conducted in compliance with guidelines for Good Clinical Practice. Patients provided written informed consent before participation.

As part of this study, peripheral blood samples were collected from each patient at baseline prior to initiating protocol treatment and at various time points on and after treatment, in EDTA tubes and shipped to Weill Cornell Medicine within <NUM> hours of the time of blood draw (Antonarakis <NUM>). CTCs from the whole blood of patients with mCRPC were captured using the prostate-specific membrane antigen based geometrically enhanced differential immunocapture microfluidic device as previously described (<NPL>); <NPL>)). All patients provided written informed consent.

ddPCR is a digital PCR method based on water-oil emulsion droplet technology. The ddPCR system partitions nucleic acid samples into <NUM>,<NUM> nanoliter-sized droplets and PCR amplification is carried out within each droplet. Unlike traditional qPCR, ddPCR allows for absolute quantification of the transcript without the need for normalization or external reference genes (<NPL>); <NPL>); <NPL>); <NPL>)).

Total RNA was extracted from the enriched CTCs pool using the RNAeasy Plus Micro kit (Qiagen) as per manufacturer's instructions. After PCR, droplets that contained a template had a fluorescent signal (positive droplets) that distinguished them from the droplets without a template (negative droplets). The number of target molecules was calculated from the ratio of detected positive droplets to total droplets, using Poisson distribution analysis. CTC-derived mRNA was used as input for the ddPCR reactions arrayed in <NUM>-well format using commercially available multiplexed master mixes of PCR enzyme/buffer from the One-Step RT ddPCR Advanced Kit for Probes (Bio-Rad). Primers and probes specific for AR-FL, ARv567es, and AR-V7 were used to generate amplicons for each transcript and can be found in Table <NUM>. AR-FL, ARv567es, and AR-V7 transcript quantifications were carried out on a QX200 ddPCR system with automated droplet generation (Bio-Rad Laboratories), as described herein. As is standard practice, no threshold was applied to the ddPCR values (<NPL>); <NPL>); <NPL>)). Transcript copy numbers for each patient can be found in Table <NUM>.

The assay is highly specific for each transcript, which was confirmed using HEK293T cells transfected with plasmids encoding AR-FL, AR-V7 or ARV567es. In these validation assays, each primer set specifically amplified its intended target. Positive and negative controls were included in every assay to ensure optimal primer performance. The primers/probes used for AR-V7 do not co-amplify the highly homologous AR-V9 variant transcript. The assay sensitivity was assessed in spike-in experiments demonstrating single-cell AR-V detection (<FIG>). Briefly, one or more cells of the human prostate cancer cell line 22RV1, which expresses endogenous AR-FL and AR-V7, was spiked into healthy donor (HD) blood. The cell mixtures were processed through the GEDI device, following the same protocol as for the patient sample processing used herein. The results showed that the assay can reliably and repeatedly detect AR-FL and AR-V7 transcripts from a single prostate cancer cell in the presence of healthy donor peripheral blood mononuclear cells. No AR-V transcripts were detected in healthy donor (HD) blood run through the GEDI, while AR-FL was detected in <NUM>/<NUM> HD samples (<FIG>).

PSA levels were measured before treatment administration at each cycle and at the end-of-treatment visit, and then every three months at each follow-up visit until progression, death or study cut-off. PSA response was recorded as at least a <NUM>% (PSA50) reduction from baseline either by C5D1 (prior to switch) or at any point during the entire treatment continuum. Progression-free survival (PFS) was defined as the time between randomization and the first documentation of radiographic tumor progression (using RECIST <NUM>), clinical progression (including skeletal-related events, increasing pain requiring escalation of narcotic analgesics, urinary obstruction, etc.), PSA progression, or death from any cause. Progression-free survival was required to be confirmed at least <NUM> weeks after initial assessment.

Descriptive statistics were used to present the results: mean, standard deviation, median, range, number, and percentage of patients. To relate the presence of splice variants to response, standard chi-square procedures were used. To relate the presence of splice variants to progression-free survival (PFS), Kaplan-Meier, Cox regression, and log-rank techniques were employed.

Of <NUM> patients enrolled, <NUM> received taxane treatment. No new safety concerns were identified (<NPL>)). AR splice variant expression data at baseline and treatment response data were available for <NUM> patients. For the nine excluded patients, reasons for exclusion included no PSA results (n = <NUM>), non-evaluable CTC mRNA at baseline (n = <NUM>), and randomized but not treated patients (n = <NUM>). Median age was <NUM> years (range <NUM>-<NUM>); <NUM>%, <NUM>% and <NUM>% had an Eastern Cooperative Oncology Group performance status of <NUM>, <NUM> and <NUM>, respectively; and <NUM>% (<NUM> patients) had received prior AR-targeted therapy (Table <NUM>).

Baseline characteristics for this subgroup of patients from TAXYNERGY were generally similar to those published for the overall patient population in the TAXYNERGY study (<NPL>)). Thirty-six patients were randomized to initial treatment, eighteen received initial treatment with docetaxel, and eighteen received initial treatment with cabazitaxel.

Among the <NUM> patients, <NUM>% (<NUM> patients) and <NUM>% (<NUM> patients) were AR-V7-positive and ARv567es-positive as measured by ddPCR at baseline, respectively. Forty-nine (<NUM>%) were positive for either or both splice variants, and only five (<NUM>%) were double negative (<FIG>). ddPCR splice variant expression appeared to be numerically more frequent in those patients who had received prior AR-targeted agents. Prior AR-targeted agents had been given in <NUM>% vs <NUM>% of patients who were ARv567es-negative vs ARv567es-positive, and <NUM>% vs <NUM>% of patients who were AR-V7-negative vs AR-V7-positve. Baseline characteristics by splice variant expression are shown in Table <NUM>. Of note, patients expressing either AR-V-<NUM> or ARv567es splice variant had a numerically higher median PSA level, and ARv567es-positve patients had a numerically higher frequency of visceral metastases (Table <NUM>).

The inventors' analysis of TAXYNERGY results, revealed that a significant correlation exists between PSA response rate to taxane chemotherapy and changes in CTC AR nuclear localization (%ARNL). Patients with biochemical responses to taxanes had significant decreases in percent CTC AR nuclear localization at eight days after initial taxane treatment (C1D8) compared to the first day of initial taxane treatment (C1D1). As the ARNL immunofluorescence assay does not differentiate between AR-FL or AR-V, the inventors sought to correlate AR-V mRNA expression at baseline with changes in percent CTC AR nuclear localization (%ARNL) at eight days after initial taxane treatment (C1D8) compared to the first day of initial taxane treatment (C1D1). Twenty four of the <NUM> patients provided %ARNL data at both C1D1 and C1D8 (Tables <NUM> and <NUM>; <FIG>).

As many of the samples co-expressed both variants, to determine the relative impact of each variant, the samples were initially categorized into four groups (double positive, double negative, AR-V7-positive/ARv567es-negative, and AR-V7-negative/ARV567es-positive). Of these <NUM> patients, thirteen were double positive, three were double negative, three were AR-V7-positive/ARv567es-negative and five were AR-V7-negative/ARv567es-positive.

Patients who were AR-V7-positive /ARv567es-negative had a <NUM>% decrease in %ARNL by C1D8, compared with a <NUM>% decrease in patients who were AR-V7-negative /ARv567es-positive by C1D8 (not shown), while double-positive patients had a change of <NUM>% by C1D8 (not shown).

These data taken together, show that AR-V7 can have a dominant role in driving taxane resistance. Due to the small number of patients in each group and since the presence of AR-V7 seemed to have a dominant effect over ARv567es, results are presented in three groups: AR-V7-positive (regardless of ARv567es status), ARv567es-positive /AR-V7-negative, and double negative in <FIG>, and Table <NUM>.

Patients who were double negative for AR-V7 and ARv567es expression had a <NUM>% decrease in %ARNL by C1D8, compared with a <NUM>% decrease in patients who were AR-V7-negative/ARv567es-positive by C1D8, and compared with a negligible <NUM>% decrease in patients who were AR-V7-positive by C1D8 (p = <NUM> for trend) (Table <NUM>). Comparison of the change in %ARNL between AR-V7-positive vs AR-V7-negative patients, regardless of ARv567es expression, revealed a <NUM>% decrease in %ARNL in AR-V7-negative patients by C1D8 vs a <NUM>% decrease in AR-V7-positive patients by C1D8 (p=<NUM>) (Table <NUM>).

Splice variant expression and PSA outcomes are presented in Table <NUM> and Table <NUM>. PSA<NUM> response at cycle <NUM>, day <NUM> (C5D1) was observed in <NUM>% of AR-V7-negative patients vs <NUM>% of AR-V7-positive patients (p = <NUM>) (Table <NUM>). Similar trends were observed for PSA<NUM> response at any time during the study. For example, PSA<NUM> response at C5D1 was observed at any time in <NUM>% of AR-V7-negative patients vs <NUM>% of AR-V7-positve patients (p = <NUM>).

PSA<NUM> responses at cycle <NUM>, day <NUM> (C5D1) were observed in <NUM>% of ARv567es-positive patients (regardless of AR-V7 status) vs <NUM>% of ARv567es-negative patients (p=<NUM>).

For PSA<NUM> at any time (Table <NUM>), responses were observed in <NUM>% of ARv567es-positive patients vs <NUM>% of ARv567es-negative patients (p=<NUM>). However, when patients were divided into three subgroups, in order to model a dominant role of AR-V7, <NUM>% of AR-V7-negative/ ARv567es-negative patients had a greater than <NUM>% PSA decline by Cycle <NUM> vs <NUM>% in AR-V7-negative/ ARv567es-positve patients (p=<NUM>) (Table <NUM>).

In double negative patients, all patients had a greater than <NUM>% PSA decline at any time in the study vs <NUM>% of AR-V7-negative/ARv567es-positive patients (p = <NUM>) (Table <NUM>). Median AR-V7 and ARv567es copy numbers at baseline were numerically lower in PSA responders than in PSA non-responders; however, the difference was not statistically significant, and overall variability was high (Table <NUM>).

Median PFS was <NUM> months for double negative patients compared with <NUM> months for AR-V7-negtive/ARv567es-positive (p = <NUM>), and <NUM> months for AR-V7-positive patients (p = <NUM>). For the AR-V7-negative vs ARV7-positive comparison, median PFS was <NUM> vs <NUM> months (hazard ratio = <NUM>, p = <NUM>). The p-value for the trend across AR-V7-negative / ARv567es-negative, AR-V7-negative / ARv567es-positive and AR-V7-positive was <NUM>. For the ARv567es-negative vs ARv567es-positive comparison, median PFS was <NUM> vs <NUM> months (hazard ratio = <NUM>, p = <NUM>) (<FIG>).

The results provided herein illustrate the association of treatment outcomes (PSA response and PFS) with the expression of ARv567es and AR-V7 as measured by ddPCR at baseline and after taxane treatment. Previous studies using RT-PCR to measure AR levels have not shown that AR-V7 presence is associated with primary taxane resistance (<NPL>)). However, as described herein, outcomes of AR-V7 and ARv567es double negative mCRPC patients that were enrolled in the TAXYNERGY study had PSA response rates that were numerically superior to ARv567es-positive / AR-V7-negative mCRPC patients compared to AR-V7-positive mCRPC patients. PFS was longest in double negative patients who did not express either of the AR-V7 and ARv567es splice variants. PFS was longer with taxane therapy in AR-V7-negative patients compared with AR-V7-positive patients. Absence of AR splice variants as measured by ddPCR was associated with superior response and PFS to cabazitaxel or docetaxel in patients with mCRPC.

This Example illustrates improved methods for identifying CTCs from lung cancer patients. No CTC identification methodology is currently used in non-small cell lung cancer (NSCLC) patients. For example, the CellSearch® method detects and enumerates CTCs that are CD45-negative, EpCAM-positive, and positive for cytokeratins <NUM>, <NUM>, and/or <NUM>. CellSearch® has been FDA-approved for CTC detection in metastatic breast, prostate and colon cancer patients, but not in metastatic lung cancer patients. Hence, CTC identification and molecular characterization in NSCLC is an unmet clinical need.

Samples were obtained from NSCLC patients and CTCs were enriched using an antigen agnostic process that included RosetteSep CD45 depletion, staining for TfR, staining for CK (a standard intracellular CTC identifier), staining for TTF1 (standard marker for epithelial cells of lung origin), staining for CD45 (leukocyte marker), and staining with DAPI (nuclear marker).

All TfR-positive CTCs were also TTF1-positive, confirming the lung origin of the cell population in the samples.

CK is a standard intracellular CTC identifier. As shown in Table <NUM> and <FIG>, there is concordance between TfR and cytokeratin (CK) expression in CTCs. Note that CK as an intracellular marker cannot provide CTC enrichment when it is used alone.

Tables 12a and 12b shows that CK and TfR are generally co-expressed in CTCs.

<FIG> illustrates that TfR-positive labeling identifies CTCs across cancer stage, and across EGFR or K-Ras mutation status in early-stage NSCLC patients.

Approximately <NUM>-<NUM>% of patients with non-small cell lung cancer in the United States and <NUM>% in Asia have an EGFR positive mutation. KRAS gene mutations are found in <NUM> to <NUM> percent of all lung cancer cases but are more frequent in white populations than in Asian populations. For example, <NUM> to <NUM> percent of whites with lung cancer have KRAS gene mutations, whereas <NUM> to <NUM> percent of Asians with lung cancer have KRAS gene mutations.

These results show that TfR identifies CTCs in early stage NSCLC, a clinical scenario where the standard EpCAM-based CellSearch® has always performed poorly.

This Example illustrates improved methods for identifying CTCs from pancreatic cancer patients. CellSearch® has been FDA-approved for CTC detection in metastatic breast, prostate and colon cancer patients, but it detects fewer CTCs than are actually present in samples from pancreatic cancer patients (see, e.g., <NPL>)). Hence, CTC identification and molecular characterization in pancreatic cancer patients is an unmet clinical need.

Samples were obtained from pancreatic cancer patients and CTCs were enriched using an antigen agnostic process that included RosetteSep CD45 depletion. Forty-three samples from <NUM> patients were evaluated. Matched samples were separately stained for TfR and for EpCAM and the numbers of cells expressing TfR and EpCAM was evaluated.

One patient (patient <NUM>) was further evaluated. Patient <NUM> had nine cycles of FOLFIRINOX but had a pathological progression in December <NUM>, so she was switched to Gem/ Abraxane and now has stable disease. Samples were obtained at different time points and evaluated using the transferrin receptor methods described herein to detect and quantify CTCs.

<FIG> shows the numbers of pancreatic CTCs from matched samples that express TfR and EpCAM. The median number of TfR-positive and EpCAM-negative cells detected was <NUM> (range : <NUM>-<NUM>) while the median number of EpCAM-positive and TfR-negative cells detected was <NUM> (range: <NUM>-<NUM>). As shown in <FIG>, TfR-positive labeling identifies that more CTCs are generally present in these samples than EpCAM labeling does in these matching patient samples.

<FIG> shows that more CTCs were detected using transferrin receptor as the marker for pancreatic CTCs in samples obtained in December <NUM> when the patient was undergoing pathological progression, than were detected before significant pathological progression began (in September <NUM>). Significantly, use of transferrin receptor was more effective for quantifying pancreatic CTCs than was EpCAM (see <FIG>).

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a nucleic acid" or "a primer" or "a cell" includes a plurality of such nucleic acids, primers, or cells (for example, a solution or dried preparation of nucleic acids or primers, or a population of cells), and so forth. In this document, the term "or" is used to refer to a nonexclusive or, such that "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated.

Claim 1:
A method comprising:
a. capturing circulating cancer cells from a sample from a test subject;
b. extracting mRNA to provide a RNA sample; and
c. detecting and quantifying each of full-length androgen receptor (AR-FL), androgen receptor variant <NUM> (AR-V7), and androgen receptor variant <NUM>,<NUM>,<NUM> (AR-V567) in parallel digital droplet PCR assays;
wherein:
(i) the parallel digital droplet PCR assay(s) for detecting and quantifying for AR-FL optionally comprises the following set of primers or probes: a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>, and optionally a probe according to the sequence as set forth in SEQ ID NO: <NUM>;
(ii) the parallel digital droplet PCR assay(s) for detecting and quantifying for AR-V7 comprises the following set of primers or probes: a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>, and optionally a probe according to the sequence as set forth in SEQ ID NO: <NUM>; and
(iii) the parallel digital droplet PCR assay(s) for detecting and quantifying for AR-v567es optionally comprises the following set of primers or probes: a primer according to the sequence as set forth in SEQ ID NO: <NUM> and a primer according to the sequence as set forth in SEQ ID NO: <NUM>, and optionally a probe according to the sequence as set forth in SEQ ID NO: <NUM>.