US20230304103A1 - Systems and methods for detecting genetic alterations - Google Patents
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Definitions
- the invention relates generally to the field of precision medicine, specifically cancer prediction, diagnostics or prognostics, and, more specifically of Gene RADAR and PCR methods for predicting resistance to androgen receptor-targeted therapies in a prostate cancer patient by the detection of genetic alterations including RNA-based androgen receptor (AR) splicing variants (AR-Vs), including but not limited to, AR-V7, AR-V567es, AR-V9, together with additional genetic alterations including, but not limited to, DNA- and/or RNA-based mutation, indel, copy number variation, gene fusions from biofluid samples, e.g., plasma, serum, urine, and saliva etc.
- AR RNA-based androgen receptor
- Prostate cancer is the most commonly diagnosed cancer and the 3rd most common cause of cancer-related death in the United States. About 10%-20% of men with prostate cancer will advance to castration resistant prostate cancer (CRPC). Expression of AR-Vs has been shown to correlate with disease progression and shortened survival. Treatment of CRPC is supported by the efficacy of newer AR-directed therapies with: (i) androgen synthesis inhibitors; (ii) AR-signaling inhibitors. Understanding the mechanisms of response and resistance to AR-directed agents may help improve treatment outcome.
- RNA-based splicing variant such as AR-V7
- DNA-based mutations such as AR-T878A, AR-F876L
- Circulating tumor cells are emerging as a source of information in therapy-resistant prostate cancer patients.
- CTC can be obtained through non-invasive means, opening the door to serial assessments of the disease state and examine tumor cell response to therapy.
- Recent CTC studies suggested that detection of AR-V7 in CTCs from men with metastatic castration resistant prostate cancer (mCRPC) was associated with resistance to both enzalutamide and abiraterone, as evidenced by inferior PSA50 response rates, profession free survival (PFS) and overall survival (OS). These results suggest that the AR-V7 status may be used as a predictive (i.e. for treatment selection) or prognostic non-invasive biomarker in patients treated with AR-targeting agents.
- mCRPC men with metastatic castration resistant prostate cancer
- PFS profession free survival
- OS overall survival
- CTC in mCRPC prediction
- one key limitation of using CTC in mCRPC prediction is the lack of detectable CTCs (less than 5 CTCs per 7.5 ml) in the majority of mCRPC patients, thus limiting the potential of CTC-based detection of RNA splicing variants such as AR-V7. Therefore, it is desirable to have a new method for CRPC prediction without the limitation of using CTCs.
- ssRNA single strand RNA
- dsDNA double strand DNA
- the present invention further provides that the barcoded dsDNA is obtained by reverse transcribing ssRNA to dsDNA after ss RNA is ligated to one or more oligonucleotides.
- the genetic alterations to be detected comprising one or more gene splice variants (AR-Vs), mutations, indels, copy numbers changes, fusions and combination thereof.
- the biofluid samples are selected from a group consisting of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascetic fluid, tumor cyst fluid, amniotic fluid, and a combination thereof.
- the barcoded DNA mixture is subsequently analyzed by Next Generation Sequencing, Polymerase Chain Reaction, and/or array-based technologies.
- the present invention further comprises the detection of presence and absence of a genetic alteration is indicative of a disease and the disease is one or more cancers, for example, a genetic alteration is from an androgen receptor gene mutation which predicts prostate cancer.
- the present invention provides a method for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient, comprising: assaying the presence or absence of CRPC-related genetic alterations in a biofluid from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- AR resistance to androgen receptor
- ssRNA single strand RNA
- dsDNA double strand DNA
- the system can be a closed system and an automated system.
- the present invention further provides a platform for detecting multiple androgen receptor variants in a patient, comprising: (a) a kit of reagents for circulating nucleic acid extraction and oligonucleotides targeting one or more gene alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- the present invention further provides a kit and device for the detection genetic alterations.
- the disclosed method comprises assaying the presence or absence of one or more androgen receptor gene splice variants (AR-Vs) and additional genetic alterations such as mutation, indels, copy number variation, gene fusions etc. in a biofluid sample from the patient.
- AR-Vs androgen receptor gene splice variants
- the biofluid sample is a sample of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascetic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
- the step of assaying comprises extracting RNA from the biofluid sample and subsequently reverse transcribing the extracted RNA into a complementary DNA.
- the step of assaying comprised extracting both DNA and RNA from the biofluid sample simultaneously and then reverse transcribing the extracted RNA to a complementary DNA.
- the resultant complementary DNA is subsequently measured by Next Generation Sequencing, Polymerase Chain Reaction including qPCR and digital PCR, array-based technologies, and other related technologies.
- the method is for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient.
- the method comprises assaying the presence or absence of CRPC-related genetic alterations in a biofluid sample from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- a platform for detecting an androgen receptor variant in a patient comprising: (a) a kit comprising reagents for circulating nucleic acid extraction, including DNA, RNA, small RNA including microRNA, and other biological materials and oligos targeting the androgen receptor genetic alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- the bioinformatics analysis can separate the DNA and RNA reads based on the RNA-specific barcodes and integrate the RNA and DNA information to detect genetic alteration comprising gene slicing variations, SNV, Indel, CNV, with increased sensitivity and specificity.
- FIG. 1 shows a Gene RADAR (RNA and DNA single molecular digital Reading) assay flowchart depicting the steps from biofluid to wet lab testing and data analysis of RNA- and DNA-derived genetic alterations.
- Gene RADAR RNA and DNA single molecular digital Reading
- FIG. 2 shows a plot depicting the measured vs. expected percent of AR-V7 reads among all RNA reads. The titration experiment was performed in 6 levels of AR-V7 percentage: 1.56, 3.13, 6.25, 12.5, 25, and 50.
- FIG. 3 shows the number of AR-V7 and AR-FL junction reads detected from clinical plasma samples by NGS.
- V-cap cell line was used as positive control.
- FIG. 4 shows an IGV (Integrative Genomics Viewer) screen shot describing the multiple AR splicing variants detected from one clinical plasma sample.
- the red arches represent the junction reads specific to the particular AR splicing variants.
- FIGS. 5 A- 5 B show an example of TMPRSS2-ERG fusion detected in a prostate cancer patient plasma.
- FIG. 6 shows the Gene RADAR Bioinformatics analysis workflow.
- FIG. 7 shows an example of AR copy number gains detected in patient plasma samples.
- FIG. 8 shows an example of PTEN deletion detected in patient plasma samples.
- FIG. 9 shows an example of RNA- and DNA-based mutation detection.
- FIG. 10 shows a case report: AR-V7 and AR mutation detection in an Abiraterone-resistance mCRPC patient.
- the present invention provides a method for detecting a genetic alteration from a biofluid.
- the present invention provides an extraction method for obtaining nucleic acids from the biofluid samples.
- the samples include but are not limited to blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, and a combination thereof.
- the present invention further provides that dividing of samples into two portions, one portion is double stranded DNA and the other portion is single stranded RNA.
- the method for preparation is known to one of the skills in the art.
- the present invention provides a method for barcoding an oligonucleotide tag on the RNA sample by reserve transcribing it to cDNA and dsDNA.
- the oligo nucleotide consists of 5, 10, 12, 14, 15, 20 nucleic acid bases.
- the oligo nucleotide can be designed for fitting the identification in further analysis.
- the barcoding process is by ligation an oligonucleotide to ssRNA.
- the tagged dsDNA is mixed with the other portion of the dsDNA extract to use for further genetic analysis including but not limited to sequencing assay such as Next Generation Sequencing, Polymerase Chain Reaction, array-based technologies and/or cytogenetics assay.
- sequencing assay such as Next Generation Sequencing, Polymerase Chain Reaction, array-based technologies and/or cytogenetics assay.
- the genetic alterations include but are not limited to gene splice variants (AR-Vs), mutations, indels, copy number changes, fusions and combination thereof.
- AR-Vs gene splice variants
- the method of detecting the alterations is used to detect the changes of above.
- the present invention further comprises the detection of presence and absence of a genetic alteration is indicative of a disease and the disease is one or more cancers.
- genetic alteration detection is to detect an androgen receptor gene variation.
- the present invention provides a method for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient.
- AR resistance to androgen receptor
- the embodiment further comprises assaying the presence or absence of CRPC-related genetic alterations in a biofluid from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- ssRNA single strand RNA
- dsDNA double strand DNA
- the system can be an opened or closed system. And both systems can be automated system.
- the system can be in a device setting.
- the present invention further provides a platform for detecting multiple androgen receptor variants in a patient, comprising: (a) a kit of reagents for circulating nucleic acid extraction and oligos targeting one or more gene alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- the present invention further provides a kit the detection genetic alterations.
- the present invention is based, in part, on a novel method for identifying biomarker that enables prospective prediction of resistance to AR targeted therapies in a patient afflicted with mCRPC.
- the novel method employs a CTC-independent approach to isolate RNA and DNA from a biofluid sample.
- cfRNA cell-free RNA
- extracellular vesicles there are abundant cell-free RNA (cfRNA) and extracellular vesicles in all people including patients and the abundance of cfRNA and extracellular vesicles is independent of the number of CTCs.
- the novel method enables the detection of genetic alterations in more patients than CTC-based methods.
- the present disclosure allows the detection of RNA splice variant of many genes, in addition to Androgen Receptor as an example used here, from biofluids in specimens of various sources.
- the disclosed novel method streamlines the extraction of circulating RNA from biofluids, reverse transcription of targeted region of interest and NGS library preparation and sequencing. In contrast to existing methods known in the art, this streamlined process enables the non-invasive detection of RNA splice variant of interest by using biofluid samples in a quick, simplified, and consistent manner.
- the disclosed method also allows for the reverse transcribed RNA (cDNA) to be included in the mainstream cfDNA library preparation and target enrichment protocol thereby allowing consolidated result of both DNA and RNA from a single biofluid sample.
- cfDNA and cfRNA are extracted simultaneously, and then the extracted sample are used for reverse transcription and further used for the library processing and sequencing.
- the extracted RNA is barcoded according to methods known in the art and reverse transcribed in a single step before the steps of library preparation and sequencing.
- the present disclosure provides a method for detecting castration resistant prostate cancer in a patient comprising assaying the presence or absence of one or more types of genetic alterations at both RNA and DNA levels, such as androgen receptor gene RNA splice variants (AR-Vs) and RNA/DNA-based mutation detection in a biofluid sample from the patient; wherein the presence of such genetic alterations indicates the presence of the castration resistant prostate cancer in the patient.
- the castration resistance is de novo resistance.
- the castration resistance preexists.
- the prostate cancer is metastatic castration resistant prostate cancer (mCRPC).
- RNA alone is extracted from circulating RNA inside and outside of extracellular vesicles in a biofluid sample for further assay.
- RNA is extracted together with DNAs from circulating nucleic acid and nucleic acid-containing extracellular vesicles in a biofluid sample. In the latter embodiments, no extra step of RNA purification is needed and therefore the process is simplified.
- the sources of nucleic acids are extracellular vesicles (EVs), including exosomes and microvesicles, which have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs and considered to be a minimally invasive novel source of materials for molecular diagnostics.
- EVs extracellular vesicles
- exosomes and microvesicles which have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs and considered to be a minimally invasive novel source of materials for molecular diagnostics.
- EVs are membranous, cell-derived, mixed populations of vesicles, ranging from approximately 40-5000 nm in diameter, which are released by a variety of cells into the intercellular microenvironment and various extracellular biofluids.
- Methods for procuring a microvesicle fraction from a biofluid sample are described in scientific publications and patent applications (Chen et al., 2010; Miranda et al., 2010; Skog et al., 2008). See also WO 2009/100029, WO 2011009104, WO 2011031892, and WO 2011031877.
- methods of microvesicle procurement by differential centrifugation are described in a paper by Raposo et al. (Raposo et al., 1996), a paper by Skog et al. (Skog et al., 2008) and a paper by Nilsson et. al. (Nilsson et al., 2009).
- microvesicles can be identified and isolated from a subject's bodily fluid by a microchip technology that uses a microfluidic platform to separate tumor-derived microvesicles (Chen et al., 2010).
- Methods for nucleic acid extraction are generally based on procedures well-known in the art plus proprietary procedures developed in-house. Persons of skill will select a particular extraction procedure as appropriate for the particular biological sample. Examples of extraction procedures are provided in patent publications WO/2009/100029, US 20100196426, US 20110003704, US 20110053157, WO 2011009104, WO 2011031892, US20130131194 and US20110151460. Each of the foregoing references is incorporated by reference herein for its teaching of these methods.
- biofluids contain circulating nucleic acids and/or nucleic acid-containing EVs.
- these biofluids include blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
- the biofluid sample is obtained from a subject who has been diagnosed with prostate cancer based on tissue or liquid biopsy and/or surgery or clinical grounds. In some embodiments, the biofluid sample is obtained from a subject showing a clinical manifestation of prostate cancer advancing to CRPC, including without limitation, rising PSA levels prior to diagnosis, after initial surgery or radiation, or despite hormone therapy. In some embodiments, the biofluid sample is obtained from a subject who has been on hormone therapy or who has had a bilateral orchiectomy and whose testosterone levels have dropped to less than 50 ng/dl, and who shows evidence of disease progression in the form of rising PSA levels or bone or soft tissue metastases.
- the biofluid sample is obtained from a subject who has been undergoing primary hormone therapies, which are the LHRH agonists, for example, leuprolide (Lupron) or goserelin (Zoladex).
- primary hormone therapies which are the LHRH agonists, for example, leuprolide (Lupron) or goserelin (Zoladex).
- the biofluid sample is obtained from a healthy subject or a subject deemed to be at high risk for prostate cancer and/or metastasis of existing prostate cancer based on known clinically established criteria including, for example, age, race, family and history.
- the AR splicing variant detected in the present disclosure has a functional DNA binding domain, but not a functional ligand binding domain.
- the AR splicing variant includes the entire or at least a functional portion of exon 1 encoding the N-terminal domain and the entire or at least a functional portion of exons 2 and 3 encoding the DNA binding domain, but does not include at least a functional portion of exon 4 encoding the short hinge region or at least a functional portion of exons 4-8 encoding the ligand binding domain.
- the AR splicing variant that substantially consists of exons 1-3 may also include a non-functional portion of the nucleic acid sequence from a genomic region or exons 4-8. It is contemplated that the splicing process may give rise to such AR splicing variants that retain DNA binding function but not ligand binding function, indicative of a castrate-resistant prostate cancer.
- the androgen receptor gene splice variant is a truncated AR with C-terminal loss and therefore lacking LBD.
- the LBD-lacking truncated AR variant is AR-V7, AR-V9, ARv567es or other known or novel splicing variants.
- the AR genetic alteration is AR-T878A or other related mutation, copy number gain, RNA over expression, among others.
- the present disclosure provides a method of detecting genetic alterations related to resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient comprising assaying the presence or absence of one or more androgen receptor genetic alterations in a biofluid sample from the patient; wherein the presence of the one or more AR-Vs indicates the presence of resistance to AR-targeted therapy in the prostate cancer patient.
- the castration resistance is de novo resistance. In other embodiments, the castration resistance preexists.
- the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).
- the prediction of resistance to AR-targeted therapy informs a subsequent treatment decision.
- Androgens in the form of testosterone or the more potent dihydrotestosterone (DHT) have been well-defined drivers of progression of prostate cancer and differentiation of the prostate gland.
- DHT dihydrotestosterone
- the backbone of treatment for advanced prostate cancers was established decades ago when castration in the form of surgical orchiectomy achieved significant prostate tumor regression. Since then, substitution to chemical castration has been employed mostly due to patient preference.
- Androgen Deprivation Therapy ADT has therefore become the standard systemic treatment for locally advanced or metastatic prostate cancer. While ADT is almost always effective in most patients, disease progression to castration resistance inevitably occurs.
- the androgen receptor axis is a validated target for the treatment of castration-resistant prostate cancer.
- Several perturbations in this pathway are postulated to lead to androgen-independent growth, including androgen receptor mutation and amplification as well as the autocrine production of testosterone.
- abiraterone exerts antitumor activity by inhibition of the 17,20-lyase pathway, crucial to testosterone synthesis.
- Enzalutamide binds to the androgen receptor and prevents its translocation into the nucleus.
- Abiraterone is also approved for patients in the prechemotherapy setting based on results of recent clinical trials.
- barcode or barcoding with random oligonucleotide sequences such as 5, 10 12, 14, 15 nucleotides to uniquely tag individual target DNA molecules can be used.
- Such application increases the accuracy and reduce sequencing.
- it can be used for PCR or NGS analysis to identify individual molecules (DNA or RNA fragments) in samples.
- the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
- patient preferably refers to a human, but also encompasses other mammals. It is noted that, as used herein, the terms “organism,” “individual,” “subject,” or “patient” are used as synonyms and interchangeably.
- genetic alteration comprise gene splice variants, SNV, InDel, CNV, fusion and combination thereof.
- barcoding means using one or more oligonucleotides as tags/markers to incorporate into a dsDNA. Such tagged dsDNA is used for future analysis.
- AR-targeted therapy in the context of the methods described herein encompasses any therapy that directly or indirectly inhibits the AR signaling pathway, including, for example, through inactivation of androgen production, inactivation of androgen binding to the AR or inactivation of the AR directly. Any therapy that disrupts the AR signaling axis and inhibits the signaling pathway is therefore included in the term AR-targeted therapy.
- An example of direct inhibition, enzalutamide, also known as MDV3100, is one of the most frequently studied AR antagonists. Enzalutamide targets several steps in the AR-signaling pathway.
- enzalutamide Due to its increased binding affinity for the AR, it is able to block androgens from binding to the receptor, preventing nuclear translocation of the AR, DNA binding, and co-activator recruitment of the ligand-receptor complex.
- One of the more notable characteristics of enzalutamide is that it is able to bind and inhibit not only wild type but also mutant AR, which means point mutations of the AR that commonly occur after the progression of PCa which causes castration resistance.
- Another emerging agent in the treatment of CRPC is abiraterone acetate. While enzalutamide targets the AR directly, abiraterone acts by indirectly inhibiting the AR signaling pathway.
- CYP17 an enzyme of the cytochrome P450 family, is inhibited by abiraterone.
- CYP17 plays a critical role in testosterone synthesis. Accordingly, inhibition of causes inhibition of testosterone synthesis, limiting the amount of androgens circulating in the body, thus also limiting the action of the AR. While castration is able to decrease testosterone and DHT synthesis, it does not remove all possible sources of androgens within the body, such as intratumoral or adrenal androgens. As will be understood by those skilled in the art, any mechanism of androgen inhibition is a potential avenue for AR-targeted therapy.
- the term “resistance” in the context of AR-targeted therapy or chemotherapy, including taxanes, means that the subject does not show a response to the therapy based on an underlying ability of tumor cells to escape the effect of the therapeutic agent. Resistance includes de novo resistance and acquired resistance. Cancer patients that exhibit de novo resistance do not respond to chemotherapy from the start. However, in acquired resistance, the cancer cells initially respond to a chemotherapeutic drug but eventually acquire resistance to it. The cells might also show cross-resistance to other structurally and mechanistically unrelated drugs—a phenomenon commonly known as multidrug resistance (MDR). Owing to the acquisition of MDR, treatment regimens that combine multiple agents with different targets are no longer effective.
- MDR multidrug resistance
- the example describes the detailed steps of assaying the presence or absence of one or more androgen receptor gene splice variants (AR-Vs) in a biofluid sample with spiked in AR-Vs and AR-FL (WT) oligos in the cfDNA extracted from the biofluid sample.
- AR-Vs androgen receptor gene splice variants
- RNA and DNA single molecular digital Reading we used the Gene RADAR (RNA and DNA single molecular digital Reading) workflow, as shown in FIG. 1 , to process about 5 ml plasma of healthy donor.
- Gene RADAR RNA and DNA single molecular digital Reading
- FIG. 1 we used optimized nucleic acid extraction procedure to extract RNA and DNA from plasma.
- cfDNA was size selected using Agencourt AMPure XP beads (Beckman Coulter) and subsequently quantified using Qubit 3.0 fluorometer.
- AR-FL DNA fragment of 139 bp and AR-V7 DNA fragment of 125 bp were synthesized and pre-annealed, and then a mixer of AR-FL DNA fragment and AR-V7 DNA fragment equivalent to the copy number of cfDNAs were spiked into cfDNA samples.
- a mixer of AR-FL DNA fragment and AR-V7 DNA fragment equivalent to the copy number of cfDNAs were spiked into cfDNA samples.
- cfDNA fragments were subsequently converted to digital sequence libraries according to the kit manufacturer's instructions with modification. Briefly, DNA was processed to end repaired, 5′-phosphorylated, and 3′-dA-tailed dsDNA fragments. Then DNA fragments were ligated to dsDNA adapters with 3′-dTMP overhangs. The libraries were amplified and then subsequently enriched for target genes using biotinylated custom baits of DNA probes and IDT xGen lockdown reagents. Enriched libraries were sequenced on NextSeq 500 (Illumina). As shown in FIG. 2 , the measured percentage of AR-V7 reads is highly correlated with the expected percentage of AR-V7 reads.
- This example partly illustrates one embodiment of the disclosed method of detecting AR-V7 and AR-FL in castration-resistant prostate cancer patients.
- circulating nucleic acid is extracted from blood plasma samples using optimized nucleic acid extraction procedure. RNA in the above extraction is then reverse transcribed into cDNA.
- the first layer RNA molecular barcoding scheme includes adding RNA specific tags plus random molecular barcodes. After cDNA is converted as dsDNA (double-stranded DNA), it was the same as regular ds cfDNA. We then added the DNA level barcodes by ligation of adaptors.
- cDNA Synthesis Mix is prepared by adding sequentially 10 ⁇ RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then heat inactivated. Then RNA was removed. The cDNA synthesis reaction can be stored at ⁇ 30° C. to ⁇ 10° C.
- the following reagents are added to a 0.2-mL tube with 1st strand cDNA from above, DNA polymerase and AR specific primers (contain molecular barcode information).
- the second strand synthesis reaction is performed as follows: 37° C. for 60 min.
- the reaction is then cleaned up using a 2 ⁇ AMPure XP beads according to the manufacturer's instructions.
- Quantification of DNA sample is performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution is analyzed using Agilent High Sensitivity DNA chips.
- cfDNA fragments were subsequently used in PCR test or converted to NGS digital sequence libraries according to the commercial kit manufacturer's instructions with modification. Briefly, DNA was processed to end repaired, 5′-phosphorylated, and 3′-dA-tailed dsDNA fragments. Then DNA fragments were ligated to dsDNA adapters with 3′-dTMP overhangs. The adapters contain sample/molecular barcode information. The libraries were amplified, quantified, and then pooled together. The pooled libraries then subsequently enriched for target genes using biotinylated custom baits of DNA probes and IDT xGen lockdown reagents (IDT) according to the manufacturer's instructions with modification.
- IDT xGen lockdown reagents IDT xGen lockdown reagents
- Quantification of enriched libraries was performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution was analyzed using Agilent High Sensitivity DNA chips. Certain number of enriched libraries were denatured, diluted and loaded on NextSeq 500 (Illumina) for sequencing.
- FIG. 3 shows the analysis result, the AR-V7 and AR-FL specific junction read count. The result shows that AR-V7 junction reads were detected in 6 out of 7 samples. And no AR-V7 junction reads were detected in an AR-V7 negative control sample, and high level of AR-V7 junction reads was detected in the AR-V7 positive control sample (Vcap cell line).
- circulating nucleic acid was extracted from blood plasma samples using optimized nucleic acid extraction procedure.
- RNA in the above extraction is then reverse transcribed into cDNA.
- primer, dNTP mix are mixed in a 0.2 mL PCR tube with total nucleic acid extraction.
- the following cDNA Synthesis Mix was prepared by adding sequentially 10 ⁇ RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then heat inactivated. Then RNA was removed.
- the cDNA synthesis reaction can be stored at ⁇ 30° C. to ⁇ 10° C.
- Second strand cDNA synthesis, and following library preparation, Enrichment, Sequencing on illumine NextSeq 500 procedure were same as Example 2.
- FIG. 4 shows one example of detecting multiple AR splicing variants (AR-V1/V2, AR-V7, AR-V9 and AR-FL) from one clinical plasma sample.
- the read arches represented the junction reads across two exons.
- This example partly illustrates one embodiment of the disclosed method of detecting gene fusion or other gene splicing isoforms in prostate cancer patient biofluid samples.
- circulating nucleic acid was extracted from blood plasma samples using optimized nucleic acid extraction procedure.
- RNA in the above extraction was then reverse transcribed into cDNA.
- a 0.2 mL PCR tube with total nucleic acid extraction random hexamer all Predicine-designed fusion gene specific primers with a molecular barcode, dNTP mix are mixed.
- the following cDNA Synthesis Mix was prepared by adding sequentially 10 ⁇ RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then was heat inactivated. Then RNA was removed.
- the cDNA synthesis reaction can be stored at ⁇ 30° C. to ⁇ 10° C.
- the following reagents were added to a 0.2-mL tube with 1st Strand cDNA from above, DNA polymerase and Predicine-designed splicing/fusion gene specific primers (contain molecular barcode information).
- the second strand synthesis reaction was performed as follows: 37° C. for 60 min. The reaction was then cleaned up using a 2 ⁇ AMPure XP beads according to the manufacturer's instructions. Quantification of DNA sample is performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution was analyzed using Agilent High Sensitivity DNA chips.
- FIGS. 5 A- 5 B show an example of TMPRSS2-ERG gene fusion was identified in a prostate cancer patient plasma.
- FIG. 5 A shows the gene model of the TMPRSS2-ERG fusion.
- FIG. 5 B shows the IGV screen shot of sequencing data from a patient plasma sample. The plot shows two types of fusion junction reads were identified.
- This example partly illustrates one embodiment of the disclosed method of detecting DNA copy number gains while measuring other RNA level variants at the same time from the patient biofluid sample.
- DNA copy number change is one of the important genomic abnormalities causing cancers. It is important to detect DNA copy number changes while detecting other RNA level variations in cancer patients.
- Gene RADAR RNA and DNA single molecular digital Reading bioinformatics analysis workflow, as shown in FIG. 6 , to analyze both DNA and RNA reads.
- the sequencing reads were first separated as RNA reads (with RNA specific tags arcodes) and DNA reads (without RNA specific tags/barcodes).
- For DNA reads we aligned the reads to human reference genome, and then calculate the consensus sequences based on molecular barcodes and alignment start and end location of the fragments using in-house developed algorithm.
- RNA reads we first aligned the reads to human reference genome and transcriptome, calculated the consensus sequences based on molecular barcodes and alignment start and end location of the fragments using in-house developed algorithm. We counted the junction reads for splicing junctions of interest based on aligned consensus sequences. We detected SNV and Indels from RNA data. Finally, we integrated the DNA and RNA analysis results and received comprehensive variant analysis results.
- FIG. 7 shows an example of detecting copy number changes following the analysis workflow shown in FIG. 6 .
- FIG. 7 shows Case-3 has significant AR copy number gain while Case-4 has less significant AR copy number gains, and other two patients had their calculated AR copy numbers close to the normal control samples (shown in grey color).
- FIG. 8 Shows an Example of PTEN Deletion Detected in Plasma Samples of CRPC Patient
- Example 8 A Case Report: AR-V7 and AR Mutation Detection in the Abiraterone-Resistance mCRPC Patient Plasma
- This case is about a prostate cancer patient, who took Abiraterone for a period of time and got drug resistance.
- time 1 before taking a new drug, the measured AR-V7 and DNA mutation using our Gene RADAR technology.
- the NGS data was analyzed by following the workflow shown in FIG. 6 .
- the NGS result shows AR-V7 positive and AR T878A point mutation with 7% mutation allele frequency at time point 1.
- the new drug aims to suppress the AR expression (including AR-V7 expression).
- the new drug after taking the new drug for about two weeks, the took another measurement.
- the NGS analysis result shows that AR-V7 was negative and the mutation allele frequency of AR T878A point mutation was increased to 21% ( FIG. 10 ). This result provided very good guidance to the drug treatment.
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Abstract
Disclosed are systems and methods for detecting genetic alterations comprising androgen receptor gene splice variants (AR-Vs), mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient. The systems and methods are similarly applicable to the detection of gene alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof of other genes of interest. The streamlined methods improve the consistency and simplicity of non-invasive detections of biomarkers.
Description
- This application is a divisional of U.S. application Ser. No. 16/093,636, filed Oct. 14, 2018, which is a National Stage Entry of International Application No. PCT/US2017/027830, filed Apr. 16, 2017, which claims the benefit of U.S. Provisional Application No. 62/480,288, filed Mar. 31, 2017, and U.S. Provisional Application No. 62/323,547, filed Apr. 15, 2016, each of which is incorporated by reference herein in its entirety.
- The invention relates generally to the field of precision medicine, specifically cancer prediction, diagnostics or prognostics, and, more specifically of Gene RADAR and PCR methods for predicting resistance to androgen receptor-targeted therapies in a prostate cancer patient by the detection of genetic alterations including RNA-based androgen receptor (AR) splicing variants (AR-Vs), including but not limited to, AR-V7, AR-V567es, AR-V9, together with additional genetic alterations including, but not limited to, DNA- and/or RNA-based mutation, indel, copy number variation, gene fusions from biofluid samples, e.g., plasma, serum, urine, and saliva etc.
- Prostate cancer is the most commonly diagnosed cancer and the 3rd most common cause of cancer-related death in the United States. About 10%-20% of men with prostate cancer will advance to castration resistant prostate cancer (CRPC). Expression of AR-Vs has been shown to correlate with disease progression and shortened survival. Treatment of CRPC is supported by the efficacy of newer AR-directed therapies with: (i) androgen synthesis inhibitors; (ii) AR-signaling inhibitors. Understanding the mechanisms of response and resistance to AR-directed agents may help improve treatment outcome.
- Some data suggests that genetic alterations in AR gene, including, RNA-based splicing variant such as AR-V7, and DNA-based mutations such as AR-T878A, AR-F876L, may drive resistance in CRPC. Expression of AR-Vs has been shown to correlate with disease progression and shortened survival and AR-V7 is most abundant in CRPC specimens. Considering that truncated AR-Vs with C-terminal loss (splice variants) lack a functional ligand binding domain (LBD) and are constitutively active, C-terminal AR-directed therapies may not be effective and novels agents are needed that target mutated ARs including AR-Vs.
- Circulating tumor cells (CTCs) are emerging as a source of information in therapy-resistant prostate cancer patients. CTC can be obtained through non-invasive means, opening the door to serial assessments of the disease state and examine tumor cell response to therapy. Recent CTC studies suggested that detection of AR-V7 in CTCs from men with metastatic castration resistant prostate cancer (mCRPC) was associated with resistance to both enzalutamide and abiraterone, as evidenced by inferior PSA50 response rates, profession free survival (PFS) and overall survival (OS). These results suggest that the AR-V7 status may be used as a predictive (i.e. for treatment selection) or prognostic non-invasive biomarker in patients treated with AR-targeting agents.
- However, one key limitation of using CTC in mCRPC prediction is the lack of detectable CTCs (less than 5 CTCs per 7.5 ml) in the majority of mCRPC patients, thus limiting the potential of CTC-based detection of RNA splicing variants such as AR-V7. Therefore, it is desirable to have a new method for CRPC prediction without the limitation of using CTCs.
- The present invention provides a method for detecting a genetic alteration from a biofluid comprises: a) obtaining nucleic acids from the biofluid and preparing two portions of sample wherein one portion comprises s single strand RNA (ssRNA) and the other portion comprises a double strand DNA (dsDNA), b) barcoding ss RNA and converting ssRNA to dsDNA wherein the dsDNA is a barcoded dsDNA, and c) mixing the barcoded dsDNA and the dsDNA portion for further genetic alteration analysis.
- The present invention further provides that the barcoded dsDNA is obtained by reverse transcribing ssRNA to dsDNA after ss RNA is ligated to one or more oligonucleotides. The genetic alterations to be detected comprising one or more gene splice variants (AR-Vs), mutations, indels, copy numbers changes, fusions and combination thereof.
- The biofluid samples are selected from a group consisting of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascetic fluid, tumor cyst fluid, amniotic fluid, and a combination thereof.
- The barcoded DNA mixture is subsequently analyzed by Next Generation Sequencing, Polymerase Chain Reaction, and/or array-based technologies.
- The present invention further comprises the detection of presence and absence of a genetic alteration is indicative of a disease and the disease is one or more cancers, for example, a genetic alteration is from an androgen receptor gene mutation which predicts prostate cancer.
- The present invention provides a method for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient, comprising: assaying the presence or absence of CRPC-related genetic alterations in a biofluid from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- The present invention further provides a system for detecting a genetic alteration from a biofluid comprises: a) obtaining nucleic acids from the biofluid and preparing two portions of sample wherein one portion comprises s single strand RNA (ssRNA) and the other portion comprises a double strand DNA (dsDNA), b) barcoding ss RNA and converting ssRNA to dsDNA wherein the dsDNA is a barcoded dsDNA, and c) mixing the barcoded dsDNA and the dsDNA portion for further genetic alteration analysis.
- The system can be a closed system and an automated system.
- The present invention further provides a platform for detecting multiple androgen receptor variants in a patient, comprising: (a) a kit of reagents for circulating nucleic acid extraction and oligonucleotides targeting one or more gene alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- The present invention further provides a kit and device for the detection genetic alterations.
- Disclosed are systems and methods for detecting genetic alterations in castration resistant prostate cancer patient. In one aspect, the disclosed method comprises assaying the presence or absence of one or more androgen receptor gene splice variants (AR-Vs) and additional genetic alterations such as mutation, indels, copy number variation, gene fusions etc. in a biofluid sample from the patient.
- In some embodiments, the biofluid sample is a sample of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascetic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
- In some embodiments, the step of assaying comprises extracting RNA from the biofluid sample and subsequently reverse transcribing the extracted RNA into a complementary DNA.
- In other embodiments, the step of assaying comprised extracting both DNA and RNA from the biofluid sample simultaneously and then reverse transcribing the extracted RNA to a complementary DNA.
- In some embodiments, the resultant complementary DNA is subsequently measured by Next Generation Sequencing, Polymerase Chain Reaction including qPCR and digital PCR, array-based technologies, and other related technologies.
- In another aspect, the method is for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient. In some embodiments, the method comprises assaying the presence or absence of CRPC-related genetic alterations in a biofluid sample from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- Also disclosed is a platform for detecting an androgen receptor variant in a patient, comprising: (a) a kit comprising reagents for circulating nucleic acid extraction, including DNA, RNA, small RNA including microRNA, and other biological materials and oligos targeting the androgen receptor genetic alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- In other embodiments, the bioinformatics analysis can separate the DNA and RNA reads based on the RNA-specific barcodes and integrate the RNA and DNA information to detect genetic alteration comprising gene slicing variations, SNV, Indel, CNV, with increased sensitivity and specificity.
-
FIG. 1 shows a Gene RADAR (RNA and DNA single molecular digital Reading) assay flowchart depicting the steps from biofluid to wet lab testing and data analysis of RNA- and DNA-derived genetic alterations. -
FIG. 2 shows a plot depicting the measured vs. expected percent of AR-V7 reads among all RNA reads. The titration experiment was performed in 6 levels of AR-V7 percentage: 1.56, 3.13, 6.25, 12.5, 25, and 50. -
FIG. 3 shows the number of AR-V7 and AR-FL junction reads detected from clinical plasma samples by NGS. V-cap cell line was used as positive control. -
FIG. 4 shows an IGV (Integrative Genomics Viewer) screen shot describing the multiple AR splicing variants detected from one clinical plasma sample. The red arches represent the junction reads specific to the particular AR splicing variants. -
FIGS. 5A-5B show an example of TMPRSS2-ERG fusion detected in a prostate cancer patient plasma. -
FIG. 6 shows the Gene RADAR Bioinformatics analysis workflow. -
FIG. 7 shows an example of AR copy number gains detected in patient plasma samples. -
FIG. 8 shows an example of PTEN deletion detected in patient plasma samples. -
FIG. 9 shows an example of RNA- and DNA-based mutation detection. -
FIG. 10 shows a case report: AR-V7 and AR mutation detection in an Abiraterone-resistance mCRPC patient. - The present invention provides a method for detecting a genetic alteration from a biofluid. The present invention provides an extraction method for obtaining nucleic acids from the biofluid samples.
- In one embodiment, the samples include but are not limited to blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, and a combination thereof.
- The present invention further provides that dividing of samples into two portions, one portion is double stranded DNA and the other portion is single stranded RNA. The method for preparation is known to one of the skills in the art.
- The present invention provides a method for barcoding an oligonucleotide tag on the RNA sample by reserve transcribing it to cDNA and dsDNA. In one embodiment, the oligo nucleotide consists of 5, 10, 12, 14, 15, 20 nucleic acid bases. In another embodiment, the oligo nucleotide can be designed for fitting the identification in further analysis. In another embodiment, the barcoding process is by ligation an oligonucleotide to ssRNA.
- In another embodiment, the tagged dsDNA is mixed with the other portion of the dsDNA extract to use for further genetic analysis including but not limited to sequencing assay such as Next Generation Sequencing, Polymerase Chain Reaction, array-based technologies and/or cytogenetics assay.
- The genetic alterations include but are not limited to gene splice variants (AR-Vs), mutations, indels, copy number changes, fusions and combination thereof. The method of detecting the alterations is used to detect the changes of above.
- The present invention further comprises the detection of presence and absence of a genetic alteration is indicative of a disease and the disease is one or more cancers. One embodiment, genetic alteration detection is to detect an androgen receptor gene variation.
- In one embodiment, the present invention provides a method for predicting resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient.
- The embodiment further comprises assaying the presence or absence of CRPC-related genetic alterations in a biofluid from the patient; wherein the presence or absence of DNA- and/or RNA-based genetic alterations indicates the presence of resistance to AR targeted therapy in the prostate cancer patient.
- The present invention further provides a system for detecting a genetic alteration from a biofluid comprises: a) obtaining nucleic acids from the biofluid and preparing two portions of sample wherein one portion comprises s single strand RNA (ssRNA) and the other portion comprises a double strand DNA (dsDNA), b) barcoding ss RNA and converting ssRNA to dsDNA wherein the dsDNA is a barcoded dsDNA, and c) mixing the barcoded dsDNA and the dsDNA portion for further genetic alteration analysis.
- The system can be an opened or closed system. And both systems can be automated system. The system can be in a device setting.
- The present invention further provides a platform for detecting multiple androgen receptor variants in a patient, comprising: (a) a kit of reagents for circulating nucleic acid extraction and oligos targeting one or more gene alterations; and (b) bioinformatics analysis solution to decipher DNA and RNA-derived information.
- The present invention further provides a kit the detection genetic alterations.
- In further embodiment, the present invention is based, in part, on a novel method for identifying biomarker that enables prospective prediction of resistance to AR targeted therapies in a patient afflicted with mCRPC. The novel method employs a CTC-independent approach to isolate RNA and DNA from a biofluid sample. There are abundant cell-free RNA (cfRNA) and extracellular vesicles in all people including patients and the abundance of cfRNA and extracellular vesicles is independent of the number of CTCs. Hence, the novel method enables the detection of genetic alterations in more patients than CTC-based methods.
- In one aspect, the present disclosure allows the detection of RNA splice variant of many genes, in addition to Androgen Receptor as an example used here, from biofluids in specimens of various sources. In some embodiments, the disclosed novel method streamlines the extraction of circulating RNA from biofluids, reverse transcription of targeted region of interest and NGS library preparation and sequencing. In contrast to existing methods known in the art, this streamlined process enables the non-invasive detection of RNA splice variant of interest by using biofluid samples in a quick, simplified, and consistent manner.
- In another aspect, the disclosed method also allows for the reverse transcribed RNA (cDNA) to be included in the mainstream cfDNA library preparation and target enrichment protocol thereby allowing consolidated result of both DNA and RNA from a single biofluid sample. For example, cfDNA and cfRNA are extracted simultaneously, and then the extracted sample are used for reverse transcription and further used for the library processing and sequencing. In the process, there is no need to separate cfDNA from the cfRNA in the biofluid nucleic acid extraction. In some instances, to distinguish the DNA- and RNA-derived signals, the extracted RNA is barcoded according to methods known in the art and reverse transcribed in a single step before the steps of library preparation and sequencing.
- In some embodiments, the present disclosure provides a method for detecting castration resistant prostate cancer in a patient comprising assaying the presence or absence of one or more types of genetic alterations at both RNA and DNA levels, such as androgen receptor gene RNA splice variants (AR-Vs) and RNA/DNA-based mutation detection in a biofluid sample from the patient; wherein the presence of such genetic alterations indicates the presence of the castration resistant prostate cancer in the patient. In some embodiments, the castration resistance is de novo resistance. In other embodiments, the castration resistance preexists. In some particular embodiments, the prostate cancer is metastatic castration resistant prostate cancer (mCRPC).
- In some embodiments, RNA alone is extracted from circulating RNA inside and outside of extracellular vesicles in a biofluid sample for further assay. In other embodiments, RNA is extracted together with DNAs from circulating nucleic acid and nucleic acid-containing extracellular vesicles in a biofluid sample. In the latter embodiments, no extra step of RNA purification is needed and therefore the process is simplified.
- In some embodiments, the sources of nucleic acids are extracellular vesicles (EVs), including exosomes and microvesicles, which have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs and considered to be a minimally invasive novel source of materials for molecular diagnostics. See Jia et al., “Emerging technologies in extracellular vesicle-based molecular diagnostics”, Expert Rev. Mol. Diagn. 1-15 (2014). EVs are membranous, cell-derived, mixed populations of vesicles, ranging from approximately 40-5000 nm in diameter, which are released by a variety of cells into the intercellular microenvironment and various extracellular biofluids. Methods for procuring a microvesicle fraction from a biofluid sample are described in scientific publications and patent applications (Chen et al., 2010; Miranda et al., 2010; Skog et al., 2008). See also WO 2009/100029, WO 2011009104, WO 2011031892, and WO 2011031877. For example, methods of microvesicle procurement by differential centrifugation are described in a paper by Raposo et al. (Raposo et al., 1996), a paper by Skog et al. (Skog et al., 2008) and a paper by Nilsson et. al. (Nilsson et al., 2009). Methods of anion exchange and/or gel permeation chromatography are described in U.S. Pat. Nos. 6,899,863 and 6,812,023. Methods of sucrose density gradients or organelle electrophoresis are described in U.S. Pat. No. 7,198,923. A method of magnetic activated cell sorting (MACS) is described in a paper by Taylor and Gercel-Taylor (Taylor and Gercel-Taylor, 2008). A method of nanomembrane ultrafiltration concentration is described in a paper by Cheruvanky et al. (Cheruvanky et al., 2007). Further, microvesicles can be identified and isolated from a subject's bodily fluid by a microchip technology that uses a microfluidic platform to separate tumor-derived microvesicles (Chen et al., 2010).
- Methods for nucleic acid extraction are generally based on procedures well-known in the art plus proprietary procedures developed in-house. Persons of skill will select a particular extraction procedure as appropriate for the particular biological sample. Examples of extraction procedures are provided in patent publications WO/2009/100029, US 20100196426, US 20110003704, US 20110053157, WO 2011009104, WO 2011031892, US20130131194 and US20110151460. Each of the foregoing references is incorporated by reference herein for its teaching of these methods.
- Many biofluids contain circulating nucleic acids and/or nucleic acid-containing EVs. Examples of these biofluids include blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or a combination thereof.
- In some embodiments, the biofluid sample is obtained from a subject who has been diagnosed with prostate cancer based on tissue or liquid biopsy and/or surgery or clinical grounds. In some embodiments, the biofluid sample is obtained from a subject showing a clinical manifestation of prostate cancer advancing to CRPC, including without limitation, rising PSA levels prior to diagnosis, after initial surgery or radiation, or despite hormone therapy. In some embodiments, the biofluid sample is obtained from a subject who has been on hormone therapy or who has had a bilateral orchiectomy and whose testosterone levels have dropped to less than 50 ng/dl, and who shows evidence of disease progression in the form of rising PSA levels or bone or soft tissue metastases. In some instances, the biofluid sample is obtained from a subject who has been undergoing primary hormone therapies, which are the LHRH agonists, for example, leuprolide (Lupron) or goserelin (Zoladex). In other embodiments, the biofluid sample is obtained from a healthy subject or a subject deemed to be at high risk for prostate cancer and/or metastasis of existing prostate cancer based on known clinically established criteria including, for example, age, race, family and history.
- In some embodiments, the AR splicing variant detected in the present disclosure has a functional DNA binding domain, but not a functional ligand binding domain. In some instances, the AR splicing variant includes the entire or at least a functional portion of
exon 1 encoding the N-terminal domain and the entire or at least a functional portion ofexons exon 4 encoding the short hinge region or at least a functional portion of exons 4-8 encoding the ligand binding domain. In other instances, the AR splicing variant that substantially consists of exons 1-3 may also include a non-functional portion of the nucleic acid sequence from a genomic region or exons 4-8. It is contemplated that the splicing process may give rise to such AR splicing variants that retain DNA binding function but not ligand binding function, indicative of a castrate-resistant prostate cancer. - In some embodiments, the androgen receptor gene splice variant is a truncated AR with C-terminal loss and therefore lacking LBD. In some instance, the LBD-lacking truncated AR variant is AR-V7, AR-V9, ARv567es or other known or novel splicing variants. In other instance, the AR genetic alteration is AR-T878A or other related mutation, copy number gain, RNA over expression, among others.
- In some embodiments, the present disclosure provides a method of detecting genetic alterations related to resistance to androgen receptor (AR) targeted therapy in a prostate cancer patient comprising assaying the presence or absence of one or more androgen receptor genetic alterations in a biofluid sample from the patient; wherein the presence of the one or more AR-Vs indicates the presence of resistance to AR-targeted therapy in the prostate cancer patient. In some embodiments, the castration resistance is de novo resistance. In other embodiments, the castration resistance preexists. In some particular embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).
- In some embodiments, the prediction of resistance to AR-targeted therapy informs a subsequent treatment decision. Androgens in the form of testosterone or the more potent dihydrotestosterone (DHT) have been well-defined drivers of progression of prostate cancer and differentiation of the prostate gland. As such, the backbone of treatment for advanced prostate cancers was established decades ago when castration in the form of surgical orchiectomy achieved significant prostate tumor regression. Since then, substitution to chemical castration has been employed mostly due to patient preference. Androgen Deprivation Therapy (ADT) has therefore become the standard systemic treatment for locally advanced or metastatic prostate cancer. While ADT is almost always effective in most patients, disease progression to castration resistance inevitably occurs. It is now recognized that the androgen receptor (AR) remains overexpressed despite seemingly castrate levels of testosterone since alternative receptors may activate the AR or other target genes may help perpetuate the castrate-resistant phenotype, hence the term “castration-resistance” has become widely adopted in the literature.
- The androgen receptor axis is a validated target for the treatment of castration-resistant prostate cancer. Several perturbations in this pathway are postulated to lead to androgen-independent growth, including androgen receptor mutation and amplification as well as the autocrine production of testosterone. Two drugs targeting this pathway in castration-resistant prostate cancer— abiraterone acetate (Zytiga®) and enzalutamide (Xtandi®)— are approved for use in prostate cancer patients. Mechanistically, abiraterone exerts antitumor activity by inhibition of the 17,20-lyase pathway, crucial to testosterone synthesis. Enzalutamide binds to the androgen receptor and prevents its translocation into the nucleus. Abiraterone is also approved for patients in the prechemotherapy setting based on results of recent clinical trials.
- With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. Chemotherapy is often indicated in the treatment of castration-resistant prostate cancer, and taxane-based agents, such as docetaxel, are often the first choice. Abiraterone (Zytiga), an anti-androgen drug, was initially only approved for the treatment of castration-resistant prostate cancer after chemotherapy. However, due to successful treatment results, many physicians currently prescribe abiraterone early during the course of treatment. Combining abiraterone with enzalutamide appears safe, and the efficacy of this combination over enzalutamide alone is being presently being evaluated to detect a survival advantage for the combination approach as upfront treatment as compared to the expected standard sequential use of these agents. Recent studies have suggested that since taxane-based chemotherapy and abiraterone are effective partially due to similar mechanisms, prior abiraterone treatment may contribute to taxane resistance and decreased chemotherapy effectiveness, suggesting clinical cross-resistance. The present methods enable optimal sequencing of care between hormone agents (abiraterone and enzalutamide) and cytotoxic chemotherapy (docetaxel and cabazitaxel).
- In one embodiment, barcode or barcoding with random oligonucleotide sequences such as 5, 10 12, 14, 15 nucleotides to uniquely tag individual target DNA molecules can be used. Such application increases the accuracy and reduce sequencing. For example, it can be used for PCR or NGS analysis to identify individual molecules (DNA or RNA fragments) in samples.
- It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a biomarker” includes a mixture of two or more biomarkers, and the like.
- The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.
- As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
- As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
- The term “patient,” as used herein preferably refers to a human, but also encompasses other mammals. It is noted that, as used herein, the terms “organism,” “individual,” “subject,” or “patient” are used as synonyms and interchangeably.
- The term “genetic alteration” comprise gene splice variants, SNV, InDel, CNV, fusion and combination thereof.
- The term “barcoding” or “barcode” means using one or more oligonucleotides as tags/markers to incorporate into a dsDNA. Such tagged dsDNA is used for future analysis.
- The term “androgen receptor targeted therapy” or “AR-targeted therapy” in the context of the methods described herein encompasses any therapy that directly or indirectly inhibits the AR signaling pathway, including, for example, through inactivation of androgen production, inactivation of androgen binding to the AR or inactivation of the AR directly. Any therapy that disrupts the AR signaling axis and inhibits the signaling pathway is therefore included in the term AR-targeted therapy. An example of direct inhibition, enzalutamide, also known as MDV3100, is one of the most frequently studied AR antagonists. Enzalutamide targets several steps in the AR-signaling pathway. Due to its increased binding affinity for the AR, it is able to block androgens from binding to the receptor, preventing nuclear translocation of the AR, DNA binding, and co-activator recruitment of the ligand-receptor complex. One of the more notable characteristics of enzalutamide is that it is able to bind and inhibit not only wild type but also mutant AR, which means point mutations of the AR that commonly occur after the progression of PCa which causes castration resistance. Another emerging agent in the treatment of CRPC is abiraterone acetate. While enzalutamide targets the AR directly, abiraterone acts by indirectly inhibiting the AR signaling pathway. CYP17, an enzyme of the cytochrome P450 family, is inhibited by abiraterone. This inhibition is significant because CYP17 plays a critical role in testosterone synthesis. Accordingly, inhibition of causes inhibition of testosterone synthesis, limiting the amount of androgens circulating in the body, thus also limiting the action of the AR. While castration is able to decrease testosterone and DHT synthesis, it does not remove all possible sources of androgens within the body, such as intratumoral or adrenal androgens. As will be understood by those skilled in the art, any mechanism of androgen inhibition is a potential avenue for AR-targeted therapy.
- As used herein, the term “resistance” in the context of AR-targeted therapy or chemotherapy, including taxanes, means that the subject does not show a response to the therapy based on an underlying ability of tumor cells to escape the effect of the therapeutic agent. Resistance includes de novo resistance and acquired resistance. Cancer patients that exhibit de novo resistance do not respond to chemotherapy from the start. However, in acquired resistance, the cancer cells initially respond to a chemotherapeutic drug but eventually acquire resistance to it. The cells might also show cross-resistance to other structurally and mechanistically unrelated drugs—a phenomenon commonly known as multidrug resistance (MDR). Owing to the acquisition of MDR, treatment regimens that combine multiple agents with different targets are no longer effective.
- The example describes the detailed steps of assaying the presence or absence of one or more androgen receptor gene splice variants (AR-Vs) in a biofluid sample with spiked in AR-Vs and AR-FL (WT) oligos in the cfDNA extracted from the biofluid sample.
- In this example, we used the Gene RADAR (RNA and DNA single molecular digital Reading) workflow, as shown in
FIG. 1 , to process about 5 ml plasma of healthy donor. We used optimized nucleic acid extraction procedure to extract RNA and DNA from plasma. Then cfDNA was size selected using Agencourt AMPure XP beads (Beckman Coulter) and subsequently quantified using Qubit 3.0 fluorometer. To mimic the condition that cDNAs, reverse transcription of targeted AR region, is mixed with cfDNA samples, AR-FL DNA fragment of 139 bp and AR-V7 DNA fragment of 125 bp were synthesized and pre-annealed, and then a mixer of AR-FL DNA fragment and AR-V7 DNA fragment equivalent to the copy number of cfDNAs were spiked into cfDNA samples. We performed a titration experiment of AR-V7 vs AR-FL spike into plasma. The titration experiment was performed in 6 levels of AR-V7 percent: 1.56, 3.13, 6.25, 12.5, 25, and 50. - cfDNA fragments were subsequently converted to digital sequence libraries according to the kit manufacturer's instructions with modification. Briefly, DNA was processed to end repaired, 5′-phosphorylated, and 3′-dA-tailed dsDNA fragments. Then DNA fragments were ligated to dsDNA adapters with 3′-dTMP overhangs. The libraries were amplified and then subsequently enriched for target genes using biotinylated custom baits of DNA probes and IDT xGen lockdown reagents. Enriched libraries were sequenced on NextSeq 500 (Illumina). As shown in
FIG. 2 , the measured percentage of AR-V7 reads is highly correlated with the expected percentage of AR-V7 reads. - This example partly illustrates one embodiment of the disclosed method of detecting AR-V7 and AR-FL in castration-resistant prostate cancer patients.
- Similar to Example 1, circulating nucleic acid is extracted from blood plasma samples using optimized nucleic acid extraction procedure. RNA in the above extraction is then reverse transcribed into cDNA.
- In order to quantify the individual RNA molecules, we used two-layer RNA molecular barcoding scheme, as shown in
FIG. 1 . The first layer RNA molecular barcoding scheme includes adding RNA specific tags plus random molecular barcodes. After cDNA is converted as dsDNA (double-stranded DNA), it was the same as regular ds cfDNA. We then added the DNA level barcodes by ligation of adaptors. - For reverse transcription step, cDNA Synthesis Mix is prepared by adding sequentially 10×RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then heat inactivated. Then RNA was removed. The cDNA synthesis reaction can be stored at −30° C. to −10° C.
- To make the second strand from the cDNA, the following reagents are added to a 0.2-mL tube with 1st strand cDNA from above, DNA polymerase and AR specific primers (contain molecular barcode information). The second strand synthesis reaction is performed as follows: 37° C. for 60 min. The reaction is then cleaned up using a 2×AMPure XP beads according to the manufacturer's instructions. Quantification of DNA sample is performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution is analyzed using Agilent High Sensitivity DNA chips.
- cfDNA fragments were subsequently used in PCR test or converted to NGS digital sequence libraries according to the commercial kit manufacturer's instructions with modification. Briefly, DNA was processed to end repaired, 5′-phosphorylated, and 3′-dA-tailed dsDNA fragments. Then DNA fragments were ligated to dsDNA adapters with 3′-dTMP overhangs. The adapters contain sample/molecular barcode information. The libraries were amplified, quantified, and then pooled together. The pooled libraries then subsequently enriched for target genes using biotinylated custom baits of DNA probes and IDT xGen lockdown reagents (IDT) according to the manufacturer's instructions with modification. Quantification of enriched libraries was performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution was analyzed using Agilent High Sensitivity DNA chips. Certain number of enriched libraries were denatured, diluted and loaded on NextSeq 500 (Illumina) for sequencing.
-
FIG. 3 shows the analysis result, the AR-V7 and AR-FL specific junction read count. The result shows that AR-V7 junction reads were detected in 6 out of 7 samples. And no AR-V7 junction reads were detected in an AR-V7 negative control sample, and high level of AR-V7 junction reads was detected in the AR-V7 positive control sample (Vcap cell line). - Similar to Example 1, circulating nucleic acid was extracted from blood plasma samples using optimized nucleic acid extraction procedure.
- RNA in the above extraction is then reverse transcribed into cDNA. Specifically, in a 0.2 mL PCR tube with total nucleic acid extraction, primer, dNTP mix are mixed. The following cDNA Synthesis Mix was prepared by adding sequentially 10×RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then heat inactivated. Then RNA was removed. The cDNA synthesis reaction can be stored at −30° C. to −10° C.
- Second strand cDNA synthesis, and following library preparation, Enrichment, Sequencing on illumine NextSeq 500 procedure were same as Example 2.
-
FIG. 4 shows one example of detecting multiple AR splicing variants (AR-V1/V2, AR-V7, AR-V9 and AR-FL) from one clinical plasma sample. InFIG. 4 , the read arches represented the junction reads across two exons. - This example partly illustrates one embodiment of the disclosed method of detecting gene fusion or other gene splicing isoforms in prostate cancer patient biofluid samples.
- Similar to Example 1, circulating nucleic acid was extracted from blood plasma samples using optimized nucleic acid extraction procedure.
- RNA in the above extraction was then reverse transcribed into cDNA. Specifically, in a 0.2 mL PCR tube with total nucleic acid extraction random hexamer, all Predicine-designed fusion gene specific primers with a molecular barcode, dNTP mix are mixed. The following cDNA Synthesis Mix was prepared by adding sequentially 10×RT buffer, MgCl2, DTT, RNaseOUT, and Superscript® III Reverse Transcriptase. The reverse transcription reaction was performed on thermocycler, then was heat inactivated. Then RNA was removed. The cDNA synthesis reaction can be stored at −30° C. to −10° C.
- To make the second strand from the cDNA, the following reagents were added to a 0.2-mL tube with 1st Strand cDNA from above, DNA polymerase and Predicine-designed splicing/fusion gene specific primers (contain molecular barcode information). The second strand synthesis reaction was performed as follows: 37° C. for 60 min. The reaction was then cleaned up using a 2×AMPure XP beads according to the manufacturer's instructions. Quantification of DNA sample is performed using Qubit dsDNA High Sensitivity Assay kit and the DNA size distribution was analyzed using Agilent High Sensitivity DNA chips.
- The following library preparation, Enrichment, Sequencing on Illumina sequencing platform were same as Example 2.
-
FIGS. 5A-5B show an example of TMPRSS2-ERG gene fusion was identified in a prostate cancer patient plasma.FIG. 5A shows the gene model of the TMPRSS2-ERG fusion.FIG. 5B shows the IGV screen shot of sequencing data from a patient plasma sample. The plot shows two types of fusion junction reads were identified. - This example partly illustrates one embodiment of the disclosed method of detecting DNA copy number gains while measuring other RNA level variants at the same time from the patient biofluid sample.
- DNA copy number change is one of the important genomic abnormalities causing cancers. It is important to detect DNA copy number changes while detecting other RNA level variations in cancer patients. We used the Gene RADAR (RNA and DNA single molecular digital Reading) bioinformatics analysis workflow, as shown in
FIG. 6 , to analyze both DNA and RNA reads. The sequencing reads were first separated as RNA reads (with RNA specific tags arcodes) and DNA reads (without RNA specific tags/barcodes). For DNA reads, we aligned the reads to human reference genome, and then calculate the consensus sequences based on molecular barcodes and alignment start and end location of the fragments using in-house developed algorithm. SNV, Indel variant calls, and CNV analysis were performed based on the bam file of aligned consensus sequences using in-house developed algorithms. For RNA reads, we first aligned the reads to human reference genome and transcriptome, calculated the consensus sequences based on molecular barcodes and alignment start and end location of the fragments using in-house developed algorithm. We counted the junction reads for splicing junctions of interest based on aligned consensus sequences. We detected SNV and Indels from RNA data. Finally, we integrated the DNA and RNA analysis results and received comprehensive variant analysis results. -
FIG. 7 shows an example of detecting copy number changes following the analysis workflow shown inFIG. 6 .FIG. 7 shows Case-3 has significant AR copy number gain while Case-4 has less significant AR copy number gains, and other two patients had their calculated AR copy numbers close to the normal control samples (shown in grey color). - This case is about a prostate cancer patient, who took Abiraterone for a period of time and got drug resistance. At
time 1, before taking a new drug, the measured AR-V7 and DNA mutation using our Gene RADAR technology. The NGS data was analyzed by following the workflow shown inFIG. 6 . The NGS result shows AR-V7 positive and AR T878A point mutation with 7% mutation allele frequency attime point 1. The new drug aims to suppress the AR expression (including AR-V7 expression). Attime point 2, after taking the new drug for about two weeks, the took another measurement. The NGS analysis result shows that AR-V7 was negative and the mutation allele frequency of AR T878A point mutation was increased to 21% (FIG. 10 ). This result provided very good guidance to the drug treatment.
Claims (16)
1. A method for detecting castration resistant prostate cancer in a subject, comprising:
(a) assaying a biofluid sample obtained or derived from the subject to determine a presence or an absence of an androgen receptor gene splice variant (AR-V) or AR gene mutation in the biofluid sample; and
(b) detecting castration resistant prostate cancer in the subject, based at least in part on the presence or the absence of the AR-V or AR gene mutation in the biofluid sample.
2. The method of claim 1 , wherein the biofluid sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, a urine sample, a sputum sample, a spinal fluid sample, a cerebrospinal fluid sample, a pleural fluid sample, a nipple aspirate sample, a lymph fluid sample, a fluid sample of the respiratory, intestinal, or genitourinary tracts, a tear sample, a saliva sample, a breast milk sample, a fluid sample from a lymphatic system, a semen sample, an intra-organ system fluid sample, an ascitic fluid sample, a tumor cyst fluid sample, an amniotic fluid sample, and a combination thereof.
3. The method of claim 2 , wherein the biofluid sample is the plasma sample.
4. The method of claim 2 , wherein the biofluid sample is the urine sample.
5. The method of claim 1 , wherein the biofluid sample is a cell-free sample.
6. The method of claim 5 , wherein (a) further comprises assaying cell-free DNA molecules or cell-free RNA molecules, and wherein (b) further comprises detecting the castration resistant prostate cancer in the subject, based at least in part on the presence or the absence of the AR-V or the AR gene mutation in the cell-free DNA molecules or cell-free RNA molecules.
7. The method of claim 6 , wherein (a) further comprises assaying cell-free DNA molecules and cell-free RNA molecules, and wherein (b) further comprises detecting the castration resistant prostate cancer in the subject, based at least in part on the presence or the absence of the AR-V or the AR gene mutation in the cell-free DNA molecules and the cell-free RNA molecules.
8. The method of claim 1 , wherein (a) further comprises determining the presence or the absence of the AR-V in the biofluid sample, and wherein (b) further comprises detecting the castration resistant prostate cancer in the subject, based at least in part on the presence or the absence of the AR-V in the biofluid sample.
9. The method of claim 8 , wherein the AR-V is selected from the group consisting of AR-V1, AR-V2, AR-V7, AR-V9, and AR-V567es.
10. The method of claim 1 , wherein (a) further comprises determining the presence or the absence of the AR gene mutation in the biofluid sample, and wherein (b) further comprises detecting the castration resistant prostate cancer in the subject, based at least in part on the presence or the absence of the AR gene mutation in the biofluid sample.
11. The method of claim 10 , wherein the AR gene mutation is selected from the group consisting of AR-FL, AR-T878A, and AR-F876L.
12. The method of claim 1 , wherein (a) further comprises extracting ribonucleic acid (RNA) molecules from the biofluid sample, and reverse transcribing the extracted RNA molecules to produce complementary deoxyribonucleic acid (DNA) molecules.
13. The method of claim 1 , wherein (a) further comprises extracting both DNA molecules and RNA molecules from the biofluid sample simultaneously, and reverse transcribing the extracted RNA molecules to produce complementary DNA molecules.
14. The method of claim 13 , further comprising sequencing the complementary DNA molecules to produce sequence reads, and analyzing the sequence reads to determine the presence or the absence of the AR-V or the AR gene mutation in the biofluid sample.
15. The method of claim 13 , further comprising analyzing the complementary DNA molecules using polymerase chain reaction.
16. The method of claim 13 , further comprising analyzing the complementary DNA molecules using an array-based technology.
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WO2018057820A1 (en) | 2016-09-21 | 2018-03-29 | Predicine, Inc. | Systems and methods for combined detection of genetic alterations |
WO2017181161A1 (en) | 2016-04-15 | 2017-10-19 | Predicine, Inc. | Systems and methods for detecting genetic alterations |
IL302912A (en) * | 2016-12-22 | 2023-07-01 | Guardant Health Inc | Methods and systems for analyzing nucleic acid molecules |
EP3592863A1 (en) * | 2017-03-08 | 2020-01-15 | H. Hoffnabb-La Roche Ag | Primer extension target enrichment and improvements thereto including simultaneous enrichment of dna and rna |
SG11201903333SA (en) | 2017-12-29 | 2019-08-27 | Clear Labs Inc | Automated priming and library loading services |
EP3755814A4 (en) * | 2018-02-23 | 2021-12-01 | Cornell University | Assay for detection of androgen receptor variants |
EP3620538A1 (en) * | 2018-09-04 | 2020-03-11 | Jessenius Faculty of Medicine of Comenius University in Martin | Means and methods for stratifying prostate cancer patients |
CN109321569B (en) * | 2018-10-29 | 2022-04-12 | 迈杰转化医学研究(苏州)有限公司 | Primer probe composition and application thereof |
CN110438199A (en) * | 2019-08-15 | 2019-11-12 | 深圳谱元科技有限公司 | A kind of method of novel the pathogenic microorganism examination |
CN114616343A (en) | 2019-09-30 | 2022-06-10 | 夸登特健康公司 | Compositions and methods for analyzing cell-free DNA in methylation partition assays |
EP4073247A4 (en) * | 2019-12-10 | 2024-01-03 | Washington University St Louis | Method of cell-free dna analysis to identify high-risk metastatic prostate cancer |
WO2022212590A1 (en) * | 2021-03-31 | 2022-10-06 | Predicine, Inc. | Systems and methods for multi-analyte detection of cancer |
WO2024077080A1 (en) * | 2022-10-05 | 2024-04-11 | Predicine, Inc. | Systems and methods for multi-analyte detection of cancer |
Family Cites Families (32)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4661450A (en) | 1983-05-03 | 1987-04-28 | Molecular Genetics Research And Development Limited Partnership | Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides |
FR2788780B1 (en) | 1999-01-27 | 2001-03-30 | Ap Cells Inc | PROCESS FOR THE PREPARATION OF MEMBRANE VESICLES |
GB9927320D0 (en) | 1999-11-18 | 2000-01-12 | Chiron Spa | Exosome separation |
US6812023B1 (en) | 2000-04-27 | 2004-11-02 | Anosys, Inc. | Methods of producing membrane vesicles |
WO2004092338A2 (en) | 2003-04-11 | 2004-10-28 | Diadexus, Inc. | Compositions, splice variants and methods relating to cancer specific genes and proteins |
FI20040723A0 (en) * | 2004-05-26 | 2004-05-26 | Orpana Aarne | Method for a quantitative and / or relative measurement of mRNA expression levels in small biological samples |
US20100227771A1 (en) * | 2006-05-11 | 2010-09-09 | Universite Laval | Methods for detection of target on responsive polymeric biochips |
ES2703363T3 (en) | 2008-02-01 | 2019-03-08 | Massachusetts Gen Hospital | Use of microvesicles in the diagnosis and prognosis of brain tumors |
EP3181705A1 (en) | 2008-11-12 | 2017-06-21 | Caris Life Sciences Switzerland Holdings GmbH | Methods and systems of using exosomes for determining phenotypes |
ES2547715T3 (en) | 2009-07-16 | 2015-10-08 | The General Hospital Corporation | Nucleic acid analysis |
WO2011031892A1 (en) | 2009-09-09 | 2011-03-17 | The General Hospital Corporation | Use of microvesicles in analyzing kras mutations |
WO2011031877A1 (en) | 2009-09-09 | 2011-03-17 | The General Hospital Corporation | Use of microvesicles in analyzing nucleic acid profiles |
CA2791244A1 (en) | 2010-03-08 | 2011-09-15 | Regents Of The University Of Minnesota | Androgen receptor isoforms and methods |
WO2012129363A2 (en) * | 2011-03-24 | 2012-09-27 | President And Fellows Of Harvard College | Single cell nucleic acid detection and analysis |
US20160040229A1 (en) * | 2013-08-16 | 2016-02-11 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US9671405B2 (en) | 2012-09-19 | 2017-06-06 | Cornell University | Identifying taxane sensitivity in prostate cancer patients |
WO2014089536A1 (en) * | 2012-12-07 | 2014-06-12 | Invitae Corporation | Multiplex nucleic acid detection methods |
EP2774997A1 (en) * | 2013-03-05 | 2014-09-10 | Bayer Pharma Aktiengesellschaft | Detection of Androgen Receptor (AR) mutations in circulating tumor DNA from plasma samples of castration-resistant prostate cancer patients using locked nucleic acid-clamp PCR |
US10612088B2 (en) * | 2013-03-14 | 2020-04-07 | The Broad Institute, Inc. | Massively multiplexed RNA sequencing |
BR112016002970A2 (en) * | 2013-08-12 | 2017-09-12 | Tokai Pharmaceuticals Inc | biomarkers for the treatment of neoplastic disorders using androgen-directed therapies |
RU2698125C2 (en) | 2013-08-19 | 2019-08-22 | Эбботт Молекьюлар Инк. | Libraries for next generation sequencing |
ES2857908T3 (en) | 2013-08-28 | 2021-09-29 | Becton Dickinson Co | Massively parallel single cell analysis |
EP4094834A1 (en) * | 2013-12-30 | 2022-11-30 | Atreca, Inc. | Analysis of nucleic acids associated with single cells using nucleic acid barcodes |
US10774387B2 (en) * | 2014-05-19 | 2020-09-15 | The Johns Hopkins University | Methods for identifying androgen receptor splice variants in subjects having castration resistant prostate cancer |
EP3253479B1 (en) | 2015-02-04 | 2022-09-21 | The Regents of The University of California | Sequencing of nucleic acids via barcoding in discrete entities |
US9727810B2 (en) | 2015-02-27 | 2017-08-08 | Cellular Research, Inc. | Spatially addressable molecular barcoding |
US20180045727A1 (en) | 2015-03-03 | 2018-02-15 | Caris Mpi, Inc. | Molecular profiling for cancer |
JP7189401B2 (en) | 2015-08-12 | 2022-12-14 | サークロジーン・セラノスティクス・インコーポレイテッド | Methods for preparing cell-free nucleic acid molecules by in situ amplification |
WO2017181161A1 (en) | 2016-04-15 | 2017-10-19 | Predicine, Inc. | Systems and methods for detecting genetic alterations |
US20210102256A1 (en) | 2016-04-15 | 2021-04-08 | Predicine, Inc. | Systems and methods for predicting and monitoring cancer therapy |
WO2018057820A1 (en) | 2016-09-21 | 2018-03-29 | Predicine, Inc. | Systems and methods for combined detection of genetic alterations |
WO2018183796A1 (en) | 2017-03-31 | 2018-10-04 | Predicine, Inc. | Systems and methods for predicting and monitoring cancer therapy |
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