TW201905210A - LIQUID BIOPSY FOR cfRNA - Google Patents

Abstract

cfRNA is used to identify and quantitate expression levels of disease related genes and further allows for non-invasive monitoring of changes in such genes. Moreover, quantitative analysis of disease related genes will enable prediction of treatment response where the treatment is dependent on the presence of the disease related gene.

Description

Liquid biopsy for cfRNA

The field of the invention is systems and methods for detecting and quantifying circulating free RNA (cfRNA), particularly in relation to cfRNA from tumor cells.

The background description includes information that can be used to understand the invention. It is not an admission that any of the information provided herein is prior art or related to the invention as claimed herein, or any publication that is explicitly or implicitly referred to is prior art.

All publications and patent applications herein are hereby incorporated by reference in their entirety in the extent of the extent of the disclosures The definition of a term provided herein is applied when the definition or use of a term in the incorporated reference is inconsistent or contrary to the definition of the term provided herein, and the definition of the term in the reference does not apply.

In the past decade, cancer treatment has changed from general chemotherapy-based therapies combined with surgery and radiation therapy to more personalized therapies that take into account the genetic variability of tumors among different patients. Therefore, treatment planning now often requires identification of molecular markers that provide more targeted treatment. In many cases, this information is obtained by analyzing various nucleic acid molecules from living tissue sections of cancer tissues. However, biopsies of tissue are usually limited to initial diagnosis or surgery, and subsequent biopsy is likely to present significant risks and discomfort to the patient. In addition, tumor tissue biopsies are prone to problems with sampling bias and have limited ability to monitor nucleic acid molecules as patient tumor markers during treatment.

Although it is known to obtain nucleic acid molecules from tumors and non-tumor cells from blood (see, for example, Clin Canc. Res . (1999) Vol 5, 1961-1965; Canc Res . (1977) 37:646-650), it is not clear Whether these nucleic acids are associated or associated with any vector or other structure. Indeed, it has recently been discovered that RNA can be derived from a variety of sources, including circulating tumor cells (see, for example, WO 2017/180499), exosomes (see, for example, WO 2015/082372), and carrier proteins (see, for example, WO 2010/079118, or Proc). Natl. Acad. Sci. (1985) 82, 3455).

Unfortunately, and possibly due to the different positions/associations of RNA with various vectors or other structures, accurate quantification of circulating nucleic acids is often problematic. For example, the detection of disease states of neuroblastoma using cell-free RNA does not reliably replace whole-cell RNA analysis (see, for example, Pediatr Blood Cancer . 2010 Jul 1; 54(7): 897-903). Similarly, as described in WO 2016/077709, although a relatively small amount of cfRNA can be detected from a mutant or inappropriately fused gene in the blood, regardless of its specific binding, the amount of detection of these RNA varies significantly. Furthermore, whether any detected amount reflects the actual physiological condition in a cell or the stability function of the particular RNA in question is still unknown. For example, the data in the '709 patent publication shows that the amount of cfRNA encoding PD-1/PD-L1 is typically highly variable and may depend on the sample, patient condition, and other factors. Therefore, there has been no report so far on the degree of PD-L1 cfRNA expression as a predictive agent and/or indicator to determine the eligibility of cancer patients for anti-PD-1/PD-L1 therapy.

Thus, while various nucleic acid analysis methods from biological fluids are known in the art, they all or almost all have various disadvantages. Therefore, there is still a need to improve systems and methods for cfRNA analysis.

The present subject matter relates to various compositions and methods for predicting a therapeutic response, tracking therapy, and/or diagnosing a cancer using the degree of cfRNA of one or more cfRNAs. In a particularly preferred aspect, the inventors have discovered that the performance threshold of certain cfRNAs, particularly PD-L1 and HER2, can be confirmed to predict the therapeutic response of certain cancers.

In one aspect of the subject matter of the present invention, a method of predicting a therapeutic response to an individual having cancer to a treatment with a checkpoint inhibitor, comprising: a step of obtaining blood from the individual and isolating cfRNA from the blood, wherein The cfRNA encodes a checkpoint suppressor gene, and a step of further quantifying the cfRNA using a quantitative PCR method. A positive therapeutic response is predicted when the amount of cfRNA is above a threshold.

In a preferred embodiment, the checkpoint inhibitor is an antibody to PD1 or PD-L1 and the cfRNA is PD-L1 cfRNA. Further, usually, the step of isolating the cfRNA is preferably at least one of RNA stabilization and cell preservation. Most typically, the quantitative PCR method involves real-time PCR, preferably with β-actin as an internal standard. In the case of quantitative PD-L1, the threshold of PD-L1 relative to β-actin may be ΔΔCT>10. In addition, at least one second cfRNA can be quantified using a quantitative PCR method if desired. Although not limited to the subject matter of the present invention, the second cfRNA contemplated may encode TIM3 or LAG3, a gene having a tumor and patient-specific mutation, a tumor associated gene, or a cancer specific gene.

In another aspect of the inventive subject matter, the inventors of the present invention also contemplate a method of monitoring treatment of a subject having cancer comprising: the step of obtaining blood from the individual and isolating cfRNA from the blood, wherein the cfRNA encoding a checkpoint suppressor gene, or wherein the cfRNA encodes a tumor-associated or cancer-specific gene, or wherein the cfRNA encodes a gene having a tumor and a patient-specific mutation; a step of quantifying the cfRNA using a quantitative PCR method; The step of updating a patient record using the amount of cfRNA.

For example, suitable checkpoint suppression genes include PD-L1, TIM3, or LAG3, tumor-associated or cancer-specific genes, including CEA, MUC1, brachyury, HER2, PCA3, or AR-V7, and have a tumor and patient specificity The appropriate gene for the mutation preferably encodes a new epitope. As noted above, it is generally preferred that the step of isolating the cfRNA uses RNA stabilization and cell preservation, and the quantitative PCR method includes real-time PCR (e.g., using β-actin as an internal standard). The patient record is updated when the amount of HER2 cfRNA is ΔΔCT>5 relative to β-actin, or the amount of PCA3 cfRNA is ΔΔCT>10 relative to β-actin.

In another aspect of the inventive subject matter, the inventors contemplate a method of detecting prostate cancer comprising the steps of: obtaining blood from a body and isolating cfRNA from the blood, wherein the cfRNA encodes PCA3 or an androgen receptor Splice variant 7; a further step of quantifying the cfRNA using a quantitative PCR method; and a further step of diagnosing the individual as having cancer when the amount of the cfRNA is above a threshold. Most typically, when the amount of the PCA3 cfRNA is ΔΔCT > 10 relative to β-actin, the individual is diagnosed as having cancer.

If desired, at least a second cfRNA encoding a gene having a tumor and patient-specific mutation, a tumor associated gene, a cancer specific gene, or a checkpoint suppressor gene can be quantified. Thus, such second genes include PD-L1, LAG3, TIM3, AR-V7, PSA, and PSMA.

In yet another aspect of the inventive subject matter, the inventors of the present invention also contemplate a method of treating a cancer comprising the steps of administering a drug to an individual diagnosed with a PD-L1 negative cancer; by isolating cfRNA from blood To monitor the treatment of the individual, wherein the cfRNA encodes PD-L1; quantify the cfRNA using a quantitative PCR method; and add a checkpoint inhibitor to the treatment after detecting the cfRNA.

In such a method, it is generally considered that the PD-L1 negative cancer is a substantially solid cancer (for example, breast cancer), and/or the drug is cancer afinitor. Most typically, the step of quantifying the cfRNA uses real-time PCR, and when the cfRNA is detected and increases over time, the checkpoint inhibitor is added. In a further preferred aspect of the method, the checkpoint inhibitor is added when the cfRNA is detected and the cfRNA content is ΔΔCT > 10 relative to β-actin.

Furthermore, the inventors of the present invention also contemplate a method of determining an immune profile in a patient comprising: the step of determining the amount of different cfRNA molecules in the blood of a subject, wherein the cfRNA molecule encodes a different checkpoint suppressor gene (eg, PD-L1, LAG3, or TIM3). In general, the determining step is performed before or during treatment with at least one of a checkpoint inhibitor, a chemotherapeutic drug, an immunotherapeutic drug, and radiation therapy.

The various features, aspects, aspects and advantages of the present invention will become more apparent from the detailed description of the preferred embodiments of the invention.

The inventors of the present invention have discovered that cfRNA can be used as a sensitive, selective, and quantitative marker for use as a diagnostic, monitoring treatment, and even as a discovery tool that allows for repeated and non-invasive sampling of a patient. In the best aspect, cfRNA is isolated from whole blood treated under conditions that maintain cellular integrity and stabilize cfRNA and/or ctDNA. Notably, under such cell preservation conditions, the ratio of cfRNA to RNA released by non-tumor cells damaged during whole blood processing is high enough to allow for quantitative analysis that can provide clinically meaningful results. Once the non-nucleic acid component is separated and then the circulating nucleic acid is quantified, it is preferred to use real-time quantitative PCR. Accordingly, the inventors of the present invention also consider kits, reagents, and instructions for isolating, monitoring, and quantifying cfRNA in blood, and specifically considering oligonucleotides suitable for quantitatively determining the presence of cfRNA of a particular gene, as follows Further discussion in more detail.

Of course, and as discussed in more detail below, it should be understood that one or more desired ones may be selected for a particular disease, stage of the disease, a particular mutation, or even the presence of a new antigenic epitope based on an individual mutational map or expression. Nucleic acid. Alternatively, in case a new mutation or a change in the performance of a particular gene needs to be discovered or scanned, the real-time quantitative PCR can be replaced or supplemented by RNA sequencing (RNAseq) to encompass at least a portion of a patient cfRNA transcriptome. In addition, it should be recognized that the analysis can be performed statically, or oversampled over time to obtain dynamic images without the need to biopsied tumors or metastases.

From a different perspective, the inventors of the present invention have generally found various methods and compositions for blood-based RNA expression testing for circulating tumor RNA (cfRNA) that recognize and quantify performance and allow for non-invasive changes to indicators. Surveillance, and/or disease drivers that have so far only been obtained by protein-based analysis of biopsied tissue. For example, contemplated systems and methods allow for monitoring changes in indicators and/or drivers of disease, and/or identifying changes in drug targets that may be associated with resistance to chemotherapy. Advantageously, the contemplated systems and methods are integrated with other omics analysis platforms, particularly GPS cancers (providing whole genome or exon sequencing, RNA sequence and performance analysis, and quantitative protein analysis) to establish powerful primary analysis/monitoring A combination tool in which changes identified by a set of learning platforms are non-invasive, and molecular monitoring is performed by the systems and methods presented herein.

In some embodiments, the inventors contemplate a method of determining a state of cancer (eg, a substantially solid state) in a patient, comprising the step of selecting a cancer associated gene based on a known association of a gene with the cancer and/or At least one of the previous omics analysis of cancerous tissue in the patient. In another step, the cfRNA of the cancer associated gene is quantified in the patient's body fluid (eg, whole blood, serum, or plasma), and in a further step, the amount of cfRNA is associated with the cancer state. Alternatively, or in addition to cancer-related genes, other cfRNAs can be monitored. For example, the state of cancer may be the sensitivity of the cancer to treatment with a drug, or the presence or absence of cancer in the patient. Most typically, the cancer associated gene is a cancer associated gene, a cancer specific gene, or a gene encoding a patient and a tumor specific new epitope (which can be determined using GPS cancer histology analysis). In a further contemplated aspect, as described in more detail below, the step of quantifying will comprise isolating cfRNA under RNA stabilization and cell preservation, and/or the quantifying step comprises performing real-time quantitative PCR on cDNA prepared from cfRNA.

In other specific embodiments, the inventors of the present invention also contemplate a method of screening a patient treated with a checkpoint inhibitor, which can include the steps of: obtaining a unitary fluid from the patient, and quantifying at least one of the body fluids associated with checkpoint inhibition Gene cfRNA. Among other suitable cfRNAs, specifically contemplated cfRNAs include those encoding PD-L1 and HER2. Of course, it should be recognized that cfRNA does not need to encode a complete gene, but may be a gene fragment under investigation. The number of cfRNAs is then compared to a threshold that correlates the number with possible treatment outcomes. Thus, and in other options, the therapeutic outcome may be associated with one or more checkpoint inhibitors (eg, antibodies or antibody fragments directed against PD-1, PD-L1, TIM3, and/or LAG3) and/or Receptors (eg, EGFR, ERCC1, IGF1H, ER2, etc.) are involved in the treatment of antibodies to the target.

Accordingly, the inventors of the present invention also contemplate various methods of treating cancer comprising the steps of determining the amount of cfRNA of the first and second markers in a blood sample of a patient, wherein the first marker is a checkpoint inhibition-related gene, and wherein the first The second marker is a cancer-associated gene, a cancer-specific gene, or a gene encoding a patient and a tumor-specific new epitope. It is further contemplated that the amounts of the first and second markers in the method are (e.g., positively) associated. The amount of the second marker can then be used to determine treatment with a checkpoint inhibitor. For example, the first marker is PD-1 or PD-L1 (or other checkpoint suppression related marker) and the second marker is HER2. Similarly, the first marker is PD-1 or PD-L1 (or other checkpoint inhibition related marker), which is a cfRNA encoding a novel epitope.

In other embodiments, the inventors of the present invention also contemplate a method of determining an immunological profile in a patient, comprising the step of determining the amount of a plurality of labeled cfRNAs in the blood sample of the patient, wherein the plurality of markers comprises a checkpoint inhibition-related gene . Most typically, the determining step is performed prior to or during treatment with at least one of a checkpoint inhibitor, a chemotherapeutic drug, an immunotherapeutic drug, and radiation therapy. Moreover, the method contemplated may further comprise the steps of determining the amount of cfRNA of at least one co-stimulatory marker, and/or the step of generating or updating a treatment plan based on the determined amount.

In general, consider using any body fluid containing cfRNA for cfRNA analysis. Thus, suitable body fluids include whole blood, plasma, serum, lymph, saliva, ascites, spinal fluid, urine, and the like, each of which may be fresh or preserved/frozen. However, it is particularly preferred that cfRNA analysis uses whole blood as a biological sample. Whole blood can be easily obtained without significant patient discomfort and can be handled in a simple and effective manner. As described in more detail below, the inventors have found that protocols for removing cells from whole blood have a significant impact on RNA stability and yield. It is worth noting that the inventors of the present invention found that quantitative cfRNA analysis was significantly improved when cells were removed from whole blood under conditions that maintained cell integrity. While not wishing to be bound by any theory or hypothesis, the inventors of the present invention have considered that cell lysis of non-tumor cells in the blood is a substantial contributor to the release of non-cfRNA. In addition, certain RNA stabilizers may also adversely affect white blood cells and red blood cells, and thus help release non-cfRNA into the plasma.

For example, for the analysis presented herein, the specimen was accepted as 10 ml of whole blood, which was drawn into a cell-free RNA BCT tube or a cell-free DNA BCT tube containing RNA or DNA stabilizers (both available from Streck, 7002 S). 109th St., La Vista NE 68128, commercially available). Advantageously, cfRNA in cell-free RNA BCT® tubes can be stable in whole blood for 7 days, while ctDNA in cell-free DNA BCT® tubes can be stable in whole blood for 14 days, so there is time to ship from different locations Patient samples without degradation of cfRNA or ctDNA. However, it should be noted that as long as the RNA stabilizer does not cause a large amount of cell lysis (for example, equal to or less than 3%, equal to or less than 1%, or equal to or less than 0.1%, or equal to or less than 0.01%, or equal to or Less than 0.001%) lysed white blood cells and/or red blood cells. From a different perspective, a suitable RNA stabilizing agent does not result in a substantial increase in the amount of RNA in the serum or plasma after binding of the blood to the agent (eg, the total RNA increase does not exceed 10%, or does not exceed 5%, or does not More than 2%, or no more than 1%). Of course, it should be recognized that many other or additional collection formats are also considered suitable, and that cfRNA and/or ctDNA can be at least partially purified or temporarily adsorbed onto a solid phase to increase stability prior to further processing.

As will be readily appreciated, plasma stratification and extraction of ctDNA and cfRNA can be accomplished in a number of ways. In an exemplary preferred aspect, whole blood in a 10 mL tube is centrifuged and plasma stratified at 1600 rcf for 20 minutes. The appropriate centrifugation speed can be calculated according to known conversions (for example, RCF = 1.1118 x 10 ^-5 x rpm ² , where r is the rotor radius calculated in cm). The plasma thus obtained was further centrifuged at 16,000 rcf for 10 minutes to remove cell debris. Of course, various alternative centrifugation protocols are also considered suitable, as long as centrifugation does not result in substantial cell lysis/maintainment of blood cell integrity (eg, lysis no more than 3%, or no more than 1%, or no more than 0.1%, Or no more than 0.01%, or no more than 0.001% of all cells). The cfDNA and cfRNA can then be extracted from the desired volume (eg, 2 mL) of plasma using Qiagen or other commercially available reagents. All isolated ctDNA and/or cfRNA are then preferably stored in a barcode matrix storage tube (eg, the DNA is stored at -4 ° C, the RNA is stored at 80 ° C or reverse transcribed into cDNA, and then stored in - 4 ° C).

Quantification of cfRNA can be performed in a variety of ways, and contemplated methods include quantification by digital PCR methods, absolute quantification using external standards, and most typically relative quantification using internal standards (eg, expressed as 2 ^ΔΔCt ). For example, real-time qPCR amplification can be performed using a 10 μL reaction mixture assay containing 2 μL of cDNA, primers, and probes. --actin can be used as an internal standard for the degree of ct-cDNA input. A standard curve for each analyte of known concentration can be included in each PCR dish, as well as positive and negative controls for each gene. The test sample is then identified by scanning a 2D barcode on a substrate tube containing the nucleic acid. The Ct value of the β-actin from each individual patient's blood sample was subtracted from the Ct value obtained by quantitative PCR (qPCR) amplification of each analyte, and ΔCt (dCT) was calculated. The relative performance of the patient specimens was calculated using a standard curve of ΔCts of serial dilutions of universal human reference RNA set to a gene expression value of 10 (when ΔCT is plotted against the logarithmic concentration of each analyte). The ctDNA can be analyzed in a similar manner.

Regarding ctDNA, it should be noted that the accuracy of ctDNA in diagnostic tests has been questioned since it was adopted as a diagnostic tool for cancer. When relying on ctDNA to monitor disease progression, especially considering the use of ctDNA to predict the presence of disease, the problem of abnormally high false positive rates must be addressed. As shown in Figure 1, healthy individuals produce a similar amount of total ctDNA as cancer patients, however, the total cfRNA content of healthy individuals (e.g., as determined by the use of beta actin quantification) is significantly lower. Furthermore, when the cfRNA isolation protocol is carried out under conditions that do not result in substantial cell lysis, the extent of total cfRNA between cancer patients and healthy individuals is significantly different. In fact, there is no overlap between healthy individuals, allowing cancer patients to be differentiated by their total cfRNA content. Conversely, there is an overlap between the degree of ctDNA in cancer patients and healthy individuals. Therefore ctDNA cannot distinguish between the two groups. In a further contemplated method, it will be appreciated that where the total cfRNA is isolated, a suitable DNase (eg, on-column digestion using DNA) can be used to remove and/or degrade cfDNA. Similarly, in the case of isolation of ctDNA, cfRNA can be removed and/or degraded using an appropriate RNase. Furthermore, the linear detection range of cfRNA (herein: PD-L1) is significant when the isolation protocol is carried out without causing a large amount of cell lysis, as shown in more detail below.

It should be noted that the term cfRNA includes full length RNA as well as fragments of full length RNA (which may have 50-150 bases, 15-500 bases, or 500-1,000 bases, or longer lengths). Thus, cfRNA can represent a portion of RNA that can be between 100-80% of full-length RNA (usually mRNA), or between 80-60%, or between 60-40%, or at 40-20 Between %, or even less. Furthermore, it should be understood that the term cfRNA generally refers to tumor-derived RNA (as opposed to RNA from non-tumor cells), and thus cfRNA can be derived from tumor cells of solid solid tumors, blood-borne cancers, circulating tumor cells, and cells. Exo body. Most typically, however, the cfRNA will not be surrounded by a membrane (and thus from circulating tumor cells or extracellular bodies). Furthermore, it is to be understood that cfRNA can be uniquely expressed in a tumor (eg, as a drug resistance or a response to a therapeutic regimen, as a splice variant, etc.) or as a mutant form of a gene (eg, as a fusion transcript, as having Transcripts of genes with single or multiple base mutations, etc.). Thus, and from a different perspective, the cfRNA contemplated includes, inter alia, a unique transcript relative to the corresponding non-tumor cell for the tumor cell, or a significant overexpression relative to the corresponding non-tumor cell (eg, at least 3 fold, Or at least 5 fold, or at least 10 fold), or with mutations relative to the corresponding non-tumor cells (e.g., missense or nonsense mutations result in a new epitope).

Thus, with regard to suitable target nucleic acids, it should be understood that suitable targets include, inter alia, genes associated with the treatment of disease and/or disease. For example, disease targets include one or more cancer-related genes, cancer-specific genes, genes with patient and tumor-specific mutations (especially those that lead to the formation of new epitopes), cancer-driven genes, and known in cancer. Over-performing genes. Thus, suitable targets include those encoding "functional" proteins (eg, enzymes, receptors, transcription factors, etc.), as well as those encoding "non-functional" proteins (eg, structural proteins, tubulin, etc.). From a different perspective, suitable targets may also include targets specific to diseased cells or organs (eg, PCA3, PSA, for prostate, etc.) or targets that are more common in different cancers, such as various mutant KRAS ( For example, G12V, G12D, G12C, etc.) or BRAF (for example, V600E). Exemplary targets validated by the inventors of the present invention include: AKT1, BRAF, CDK6, CYP3A4, ERBB3, FGFR1, JAK1, MAP2K1, AR-ALK, BRCA1, CDKN2A, DDR2, ERBB4, FGFR2, JAK2, MET, AR, ARAF, BRCA2 , CTNNB1, OPYD, FGF19, FGFR3, KOR, MTOR, PD-U, ATM, CCND1, CYP2C19, EGFR, FGF3, FLT3, KIT, NRAS, PD-1, BIM, CDK4, CYP2D6, HER2, FGF4, HRAS, KRAS , NRG1, TIM3, NTRK1, PTCH1, SMO, NTRK2, PTEN, STK11, NTRK3, RAF1, LAG3, TP53, PDGFRA, RET, TSC1, PIK3CA, RO-S1, TSC2, and UGT1A1.

Thus, it should be understood that a suitable therapeutic target includes one or more markers that can indicate the susceptibility of a diseased cell to treatment of a particular drug that is targeted to a particular molecular entity. For example, the present systems and methods can be used to identify the presence and extent of a particular kinase that is targeted by a kinase inhibitor, or the extent and presence, or presence and performance of a particular signaling receptor that is targeted by a synthetic ligand. The degree of specific checkpoint receptors to which the synthetic antagonist or antibody is targeted, and the like, and suitable targets can also be grouped by the instructions shown in Table 1 below. Table 1

In addition to known markers such as tumor associated antigens and tumor specific antigens, it should also be understood that previous histological analysis of a patient's tumor may reveal the presence of one or more new epitopes. For example, the tumor can be compared to the whole genome or exome pairings normally, preferably using the incremental synchronization alignment described in US Pat. No. 9721 062 and/or using RNAseq for prior analysis. In addition, proteomic analysis can be performed, and quantitative mass spectrometry is best used. Thus, it should be understood that cfRNA can also be used to detect tumor RNA in a patient- and tumor-specific manner, wherein cfRNA contains such patients and tumor-specific mutations (eg, new epitopes). For example, such detection can be used to monitor therapeutic effects, particularly where the treatment is directed to a patient and a tumor-specific mutation (eg, a new epitope) immunotherapy. In another embodiment, detection of a patient with a tumor-specific mutation can also reveal a (newly generated) therapeutic target that can be treated with immunization or chemotherapy.

Thus, it will be appreciated that contemplated compositions and methods can be used to discover disease-associated markers, and more generally to quantify suitable targets to obtain information about the presence of mechanism-targeted therapies and/or to obtain cancer cell population acceptance. Quantitative agent baseline for treatment or predictive response development. For example, contemplated compositions and methods are particularly useful in immunotherapy where a new epitope is targeted, as the performance and amount of a new epitope can be used to validate a new epitope as a therapeutic target, and to use a new epitope. Performance and quantity serve as markers for the progression of alternative therapies. Therefore, it should be noted that cfRNA can be used to determine the presence of new epitopes of expression before, during, and after treatment, and thus allow for the prediction and/or quantification of therapeutic efficacy on an individual basis.

Alternatively, and in other preferred uses, the cfRNA can be quantified to identify patients (e.g., targets PD-1 and PD-L1) that are suitable for treatment with a checkpoint inhibitor. This is particularly useful because there is currently no convenient and non-invasive way to determine the levels of PD-1 and PD-L1, which will inform clinicians whether patients will benefit from checkpoint inhibitors (eg, Navuzumab) , pemrolizumab, atezolizumab, etc.). In fact, immune checkpoints, such as programmed death ligand 1 (PD-L1) or its receptor, programmed death 1 (PD-1), appear to be the Achilles heel of multiple tumor types. PD-L1 not only provides immune escape to tumor cells, but also opens apoptotic switches on activated T cells. Therapies that block this interaction show promising clinical activity in several tumor types. It has been shown that tumor PD-L1 expression status includes a variety of melanoma (MEL), renal cell carcinoma (RCC), and non-small-cell lung cancer (NSCLC). Prognosis is in the type of tumor. Furthermore, tumor PD-L1 expression appears to be closely related to the response of anti-PD-1 antibodies. However, none of the tests were accepted as the standard for quantitative PD-L1 performance. In addition, some anti-PD-L1 antibodies are in clinical trials and two antibodies have been approved by the FDA for the treatment of non-small cell lung cancer (NSCLC). Therefore, it is important to measure PD-L1 performance before administering anti-PD-L1 immunotherapy to patients. The inventors have now discovered that by analyzing the frequency and extent of PD-L1 (and other markers) expression in cfRNA isolated from various cancer types, cfRNA can be used to quantify the performance of PD-L1 as well as other immunotherapeutic-related cancer markers, as in The following is a more detailed description. Example

Separation of crRNA from whole blood : Whole blood was obtained by venipuncture, and 10 ml of whole blood was collected into cell-free RNA BCT® tubes or cell-free DNA BCT® tubes containing RNA or DNA stabilizers respectively (Streck, 7002 S. 109th St., La Vista NE 68128). The sample tube was then centrifuged at 1,600 rcf for 20 minutes, the plasma was removed and further centrifuged at 16,000 rcf for 10 minutes to remove cell debris. Plasma was separated and slightly modified using a commercially available RNA isolation kit according to the manufacturer's protocol. Specifically, DNA is removed from the sample during DNase digestion on the column.

In another method, cfRNA was obtained in an automated manner using a robotic extraction method on a QiaSymphony instrument (Qiagen, 19300 Germantown Road, Germantown, MD 20874), slightly modified to accommodate the desired DNA removal. The robot extract contains approximately 12% of the DNA contamination in the cfRNA sample. We measured the relative performance of the Excision Repair Cross-Complementing enzyme (ERCC1) relative to β-actin in the same 21 NSCLC samples to determine if there were significant differences between the two extraction procedures. It is worth noting that the relative performance of the automated process and the manual process is not statistically different, as shown in the table below. p = 0.4111 (paired t-test; for this test, the statistical difference would be p < 0.05).

The custom kit from Qiagen (QiaSymphony Circulating NA kit #1074536) contains two virus extraction kits in a custom kit (this virus kit is called QiaSymphony DSP Virus/Pathogen Midi Kit Version 1#937055) . Analysis was performed in a single proprietary program on the Qiagen instrument (custom program protocol CF 2000S_CR21040_ID993; from Qiagen).

Quantification of cfRNA: Relative quantitation was quantified by rtPCT with gene-specific primer pairs and primer pairs for β-actin as an internal control, unless otherwise stated. For example, amplification is carried out using a 10 μL reaction mixture containing 2 μL of cDNA, primers, and probes. --actin can be used as an internal standard for ct-cDNA input content. A standard curve of each known sample of each analyte filler was included in each PCR dish along with positive and negative controls for each gene. The test sample is identified by scanning a two-dimensional barcode on a substrate tube containing the nucleic acid. The Ct value of the β-actin in the blood sample of each individual patient was subtracted from the Ct value obtained by quantitative PCR (qPCR) amplification of each analyte, and ΔCt (dCT) was calculated. The relative performance of the patient samples was calculated using a standard curve of ΔCts of serial dilutions of universal human reference RNA set to a gene expression value set to 10 (when ΔCT is plotted against the logarithmic concentration of each analyte). The ctDNA was analyzed in a similar manner.

The ΔCts versus log10 relative gene expression (standard curve) for each gene test was captured in hundreds of PCR reaction plates (historical reaction). Linear regression analysis was performed for each assay and used to calculate gene expression from a single point of the original standard curve.

It is worth noting that, as shown in Figure 1, ctDNA was quantified from healthy donors and cancer (non-small cell lung cancer (NSCLC)) patients, including 10 cancers and 9 healthy individuals. There was no statistically significant difference in total ctDNA between the two populations. In contrast, the total amount of cfRNA between the two populations (measured by β-actin) was significantly different, indicating that the measurement of total cfRNA may be a useful indicator of the presence of cancer.

The inventors of the present invention also investigated whether the above results can be confirmed across various other cancer types and selected genes (for example, PD-L1), and analyzed from diseases diagnosed with breast cancer, colon cancer, stomach cancer, lung cancer, and prostate cancer. Suffering. In this series of tests, the relative performance of PD-L1 cfRNA was quantified and the results are shown in Figure 2A. Interestingly, as shown in Figure 2A, not all cancers exhibited PD-L1, and the frequency of positives in various cancers was consistent with the PD-L1 expression disclosed in IHC in parenchymal solid tissue. PD-L1 cfRNA was not detected in healthy patients, as shown in Figure 2B.

Analytical Validation - Accuracy: Analysis of results from current PD-L1 analysis ("LiquidGeneDx") from 61 clinical samples and digital PCR PD-L1 (laboratory reference method, an alternative PD-L1 assay) Comparisons were made to determine the accuracy of the exemplary PD-L1 performance analysis. This result was used to determine the clinical sensitivity and clinical specificity of the analysis. Table 2 summarizes the accuracy results of the current PD-L1 analysis and the digital PCR PD-L1 assay. Table 2

Analysis verification - the detection limit (Limit of Detection, LOD): analytical sensitivity through repeating 20 times the current measured PD-L1 Analysis ( "LiquidGeneDx") in a detection rate of 95%. The cfRNA was extracted from the patient's plasma and reverse transcribed into cDNA using a random hexamer primer with Thermo Fisher's preamplification product Taqman® Preamp Master Mix and PD-L1 and β-actin primers according to the manufacturer's instructions. Explain that 10 cycles are pre-amplified. The resulting pre-amplified cDNA was diluted 2-fold in increments of cDNA from PD-L1 negative patients. The minimum amount of PD-L1 cDNA required for amplification and successful PCR in all diluted samples was determined by LiquidGeneDx. The estimated LOD content was then repeated 20 times to confirm the final LOD. The limit of detection (LOD) acceptance criteria in this study was determined to be the lowest concentration of 95% higher for all 20 replicates. If 20 replicate samples were unable to produce a detection rate greater than 95%, the next higher concentration of diluted sample was used as the assumed LOD to repeat 20 replicates. Table 3 shows a summary of the results of the LOD study, where * represents the final LOD. table 3

Assay Validation - Detection Limit (LOD) : The accuracy group included a low positive PD-L1 sample, a medium positive PD-L1 sample, a high negative PD-L1 sample, a positive control group, and a no template control group. All positive samples were from PD-L1 positive cancer cell lines. Operated by 3 different operators (Op), each precision group is run four times for each run, two instruments per instrument, two instruments per day for a total of 3 days (continuous or non-continuous). Each sample of the accuracy group generated a total of 48 data points in 3 days. The study design is shown in Table 4 . Table 4

The accuracy of the measurement was measured using two instruments, two operators, one day, and four replicates per sample. The consistency of all replicated results was 96% or more. Table 5 is an exemplary summary of the intra-assay accuracy. table 5

Two instruments, two operators, three runs in three days, and four repeat runs tested for inter-batch accuracy. For all replicates of stand-alone operation, the result consistency was 96% or more. Table 6 lists the results. Table 6

Assay Validation - Linear Range: Determine the quantitative linear range of the current PD-L1 assay ("LiquidGeneDx") by diluting the cDNA from PD-L1 positive patients from cfRNA into pooled negative matrices (PD-L1 negative cDNA from cfRNA) . ctRNA was extracted from patient plasma, reverse transcribed into cDNA using random hexamer primers, and Thermo Fisher's preamplified product Taqman® Preamp Master Mix and PD-L1 and β-actin primers were used according to the manufacturer's instructions. 10 cycles of preamplification. The resulting pre-amplified cDNA was diluted 2-fold in increments of cDNA from PD-L1 negative patients. LiquidGeneDx PD-L1 detects all diluted samples to determine their quantitative linear range. Figure 2C shows the final linear range. The linear portion of the line extends to a Ct of approximately 32.5. Beta actin is also consistent with the PD-L1 slope.

Assay Validation - Specificity: Test samples were prepared by serial dilution of human PD-L1 cell line cDNA in TE buffer matrix. The medium analyte sample has a target analyte concentration that is four times the LOD concentration. A medium positive sample (one analyte and each interferent) with each interferent and a baseline sample were repeatedly examined by the current PD-L1 assay ("LiquidGeneDx"). Table 7 lists the interferents and their test concentrations. All samples with different interference concentration tests were still positive by the LiquidGeneDx PD-L1 test. Table 7

It is worth noting that all samples with different interferor test concentrations were still positive by the LiquidGeneDx PD-L1 assay.

The current PD-L1 assay ("LiquidGeneDx") is designed as an instant PCR assay to detect the expression of the PD-L1 gene and other genes in the blood of cancer patients. Among other benefits, such measurements may inform the clinician prior to and during drug treatment that a particular drug (eg, an anti-PD-1 antibody) may successfully treat the patient.

Based on the above findings, cfRNA can be accurately quantified, and the inventors of the present invention have attempted to determine whether the quantified cfRNA content will also correlate with known analyte levels measured by conventional methods such as FISH, mass spectrometry, and the like. More specifically, the frequency and intensity of PD-L1 expression was measured using LiquidGenomicsDx from cfRNA in plasma from 320 consecutive NSCLC patients and using tissue IHC testing, and pemizumab in the Keynote test (registered trial) The frequency of positive patients (pembrolizumab, Keytruda) was compared. It is worth noting that 66% of NSCLC patients (1,475/2,222) in the Keynote trial had IHC (>1% cell positive), while 64% of NSCLC patients (204/320) had blood-based cfRNA. PD-L1 detection, as can be seen from Figures 3A and 3B, the test results were positive. It is worth noting that there is no significant difference in PD-L1 status between the two assays, but cfRNA assays provide quantitative data.

It is worth noting that the difference in PD-L1 status (ie, PD-L1 positive or PD-L1 negative) between the two selected patients (Pt#1 and Pt#2) is also related to the use of nivolumab (nivolumab). IHC analysis and treatment response are well correlated and the results can be seen in Figure 4. Here, two patients with squamous cell lung cancer were treated with the anti-PD-1 antibody nivolumab. Patient 1 did not exhibit PD-L1 in tissues or blood as measured by cfRNA. Patient 1 did not respond to nivolumab. CT scans recorded tumor growth and the patient quickly failed. In contrast, patient 2 has a high level of PD-L1 in tissues and blood as measured by cfRNA. Patient 2 responded to nivolumab for several cycles of the drug. The CT scan confirmed that the reaction had a significant tumor shrinkage. Interestingly, a high degree of gene expression in the patient's blood (measured by cfRNA) disappeared after three and a half weeks of sustained response in the patient's body.

Based on the correlations observed above, the inventors of the present invention set out to study whether the degree of expression of PD-L1 cfRNA can provide a therapeutic agent suitable for the treatment of nivolumab or other interfering PD1/PD-L1 signaling. The threshold level of the response prediction. To this end, PD-L1 expression was measured in the plasma of NSCLC patients using cfRNA and compared to the IHC status. Figure 5A shows the correlation between the therapeutic response status determined by IHC and the anti-PD-L1 therapeutic and PD-L1 status, and PD-L1 performance above cfRNA above the response threshold. Patients identified as responding to treatment were also identified as PD-L1 positive by IHC, and all patients identified as not responding to treatment were determined to be PD-L1 negative by IHC. It is worth noting that when the reaction threshold is applied to the data, the same separation between responders and non-responders can be achieved using the PD-L1 cfRNA content. In the present embodiment, the relative performance threshold with a threshold of 10 accurately separates responders from non-responders. Figure 5B shows that the cfRNA response threshold of PD-L1 is predicted to be positive for PD1/PD-L1 checkpoint inhibitor (here: nivolumab) relative to β-actin ΔΔCT>10. All patients who responded to nivolumab showed PD-L1 above the threshold before treatment.

The inventors of the present invention further studied whether the degree of PD-L1 cfRNA expression can be used as an indicator of progressive disease (PD), stable disease (SD) and/or partial response (PR) in other cancer treatments. For this purpose, dynamic changes in PD-L1 as measured by cfRNA were found during the course of treatment of various treatment regimens, as exemplarily shown in Figures 6A-6D. Panel A shows the relative degree of PD-L1 expression in the treatment of breast cancer with abraxane in patients with progressive disease. The lack of therapeutic response was reflected in the elevation of PD-L1 cfRNA, and abraxane treatment discontinued treatment with CDX-011 (glembatumumab vedotin). As can be seen from Figure 6A, treatment with CDX-011 resulted in stable disease, which is also reflected in the reduction of PD-L1 cfRNA. Similarly, as can be seen from Figure 6B, lung cancer patients were treated with carboplatin/alimta combination therapy in stable disease, and the initial high degree of PD-L1 cfRNA was significant as the patient showed partial response. reduce. In the case of colon cancer, patients with progressive disease are treated with capecitabine and bevacizumab. There was a significant increase in PD-L1 cfRNA performance during treatment. After treatment of cancer with 5-FU and bevacizumab, the patient had a partial response with a significant decrease in the extent of PD-L1 cfRNA, as shown in Figure 6C. Therefore, the inventors of the present invention considered that the quantitative degree of PD-L1 cfRNA can also be accurately used to monitor the therapeutic response.

In another embodiment, the inventors observed a rapid increase in PD-L1 cfRNA in stable disease breast cancer patients treated with exemestane/exemestane/afinitor, as shown in Figure 6D. It is worth noting that patients did not receive PD-L1 cfRNA before treatment. Based on this observation, the inventors of the present invention further tested samples of breast cancer patients undergoing cancer afinitor treatment, and exemplary results are shown in FIG. It is apparent that the PD-L1 cfRNA is treated with a significant increase after the second blood draw to the extent that it is suitable for treatment with the PD1/PD-L1 checkpoint inhibitor. Therefore, the inventors of the present invention also considered that at least PD-L1 cfRNA can be monitored to identify the appearance of PD-L1 cfRNA after cancer treatment (especially those treated with drugs other than PD1/PD-L1 checkpoint inhibitors), It can then be used as an indicator of treatment with a PD1/PD-L1 checkpoint inhibitor. From a different perspective, the detection and quantification of previously undetected PD-L1 cfRNA expression during cancer treatment can be used as an (additional) indicator for treating patients with a PD1/PD-L1 checkpoint inhibitor.

Interestingly, the disease state of cancer is also at least somewhat parallel to the β-actin cfRNA, as shown in Figure 8. Blood was drawn from the patient under various therapies every 6-8 weeks while completing a CT scan. cfRNA was extracted from the plasma of 45 patients with metastatic breast cancer, and 30 patients completed the first two courses of treatment: 6/6 patients with PR showed no change in β-actin cfRNA content (NC) or Reduction (DEC), cfRNA content in 13/16 SD patients showed NC or DEC, and 6/8 PD patients had elevated cfRNA levels (INC). The cfRNA was reverse transcribed into cDNA using a random hexamer primer. The content of cfRNA was quantified by RT-qPCR and the correlation between patient response (PR/SD/PD) and cfRNA content was determined by CT scan. The degree of gene expression in cfRNA (including PD-L1 and HER2) was monitored in blood drawn patients. It is worth noting that the beta actin cfRNA content of patients with advanced breast cancer is higher than that of patients with stable disease and/or partial remission. Therefore, it should be recognized that an increase in the β-actin cfRNA content can serve as a leading indicator of disease status, particularly as a leading indicator of progressive disease in patients who have been diagnosed with cancer.

In further study of breast cancer samples, the inventors of the present invention also found that HER2 cfRNA in tumors appears to co-exhibit or co-regulate with PD-L1, as shown in Figure 9A. On this basis, the inventors then used the HER2 status classification by immunohistochemical analysis (IHC) using anti-HER2 antibodies to correlate the IHC-HER2 status with the quantitative relative performance of HER2 measured by cfRNA content. Notably, there was a significant correlation (82% identity) between the extent of HER2 cfRNA expression and the IHC HER2 status, where ΔΔCT>5 of HER2 relative to β-actin was applied, as exemplarily shown in Figure 9B. . Therefore, it is expected that the detection and quantification of HER2 cfRNA can also be used to determine the status of HER2 using the performance thresholds provided above.

In further experiments, the inventors have also discovered that HER2 cfRNA in at least some gastric tumors appears to co-exhibit or co-regulate with PD-L1, as shown in FIG. This finding is particularly noteworthy because it is known that about 15% of gastric cancer does exhibit HER2. Therefore, the inventors of the present invention contemplate a method of detecting or quantifying HER2 cfRNA in a gastric cancer patient. In addition, the inventors have also considered that one or more immunological checkpoint genes (eg, PD-L1, TIM3, LAG3) measured by cfRNA can be used as other cancer-specific markers or tumor-associated markers (eg, CEA, PSA, MUC1). Brachyury, etc.).

As will be readily understood, quantification of HER2 cfRNA content can also be used after treatment, particularly to assess whether treatment with anti-HER2 drugs has a therapeutic effect. For example, two anti-HER2 drugs (pertuzumab and trostuzumab) in two exemplary patients (patients 25 and 12, respectively) of patients with metastatic breast cancer of the same age group Part of the treatment response showed a positive response directly related to the reduction in cfRNA, as shown in Figure 11. In fact, there was no detectable amount of HER2 cfRNA in the treatment of the past three months.

Based on the observed co-expression or co-regulation, the inventors of the present invention then investigated whether other cfRNA content of the immunological checkpoint-related gene is related to the PD-L1 cfRNA content, and exemplary results are shown in FIG. Here, the contents of PD-L1, TIM3, and LAG3 cfRNA were measured from blood samples of prostate cancer patients. It is worth noting that all checkpoint related genes are strongly expressed except for one sample. Interestingly and importantly, the levels of TIM3 and LAG3 (the former has been shown to be an escape or resistance factor for PD-1 or PD-L1 inhibition) generally reflect the performance of PD-L1, emphasizing that in addition to PD-1 and PD- In addition to L1, all checkpoint proteins need to be processed. Therefore, it should be recognized that cancer patients can be analyzed for cfRNA content of their immune checkpoint-related genes to obtain immunological characteristics or the patient, and then more than one checkpoint inhibitory drug can be recommended for appropriate treatment. As will be appreciated, suitable gene thresholds can be established according to the methods described above for PD-L1 and HER2.

In a still further aspect of the inventive subject matter, it is demonstrated that various alternative cfRNA species are quantitatively distinguished from healthy individuals and individuals afflicted with cancer and/or predictive therapeutic response. For example, the detection of androgen receptor splice variant 7 (AR-V7) has been an important consideration in the treatment of prostate cancer with hormone therapy. Therefore, the inventors of the present invention investigated whether hormone therapy resistance is associated with prostate cancer tumor growth, and detects AR-V7 by detecting and quantifying AR-V7 cfRNA. Figure 13 shows exemplary results of AR and AR-V7 gene expression using plasma from the cfRNA method from prostate cancer patients. AR-V7 was also measured using the IHC technique of CTCs from the same patient. It is worth noting that the CTC of AR-V7 is consistent with the cfRNA results (data not shown).

In addition, PCA3 was identified as a marker of prostate cancer in a test for detecting and quantifying PCA3 cfRNA in the plasma of a prostate cancer patient, and in which the non-prostate cancer patient sample has a relatively low undetectable content. Non-prostate cancer patients are patients with NSCLC and CRC. As can be seen in Figure 14, PCA3 showed a differential expression between the two groups via cfRNA (non-overlapping median between prostate cancer and non-prostate cancer patients), indicating that non-invasive blood-based cfRNA assays are available. For the detection of prostate cancer. Again, based on a priori knowledge of the test population, a threshold for performance (here: ΔΔCT > 10 of PCA3 relative to β-actin) can be established, as exemplarily shown in FIG.

In a further study, the inventors used the analysis of total cell-free circulating tumor RNA (cfRNA) extracted from plasma (pts) of cancer patients as a measure of the dynamic changes in gene expression and the total amount of nucleic acid including cfRNA. These analyses again provide insight into disease status and allow prediction of the outcome of anti-tumor treatment.

More specifically, blood is drawn from the patient at various treatments (tx) every 6-8 weeks while the CT scan is completed. As described above, cfRNA was extracted from the obtained plasma and reverse transcribed into cDNA with a random hexamer primer. The total cfRNA content was quantified by RT-qPCR as determined by CT scan and correlated with pt reaction (PR/SD/PD). In this study, a total of 30 lung cancer patients participated in a 2-year clinical study. Races include: 73% (22/30) Caucasus, 20% (6/30) Hispanic, and 7% (2/30) other races. Non-SQCCs accounted for 87% (26/30). Twenty-three cancer patients completed the first two cycles of treatment (tx). Among them, 6/8 patients with advanced disease (PD) had elevated total cfRNA content (INC), 8/12 patients had stable disease (SD) and showed no change in total cfRNA content (NC) or decrease (DEC). The total cfRNA of patients with partial remission (PR) at 3/3 was reduced (DEC), which corresponds to a 74% agreement between total cfRNA and pt response. PD-L1 performance measured in plasma cfRNA was consistent with tissue in 7/10 cancer patients (pts). In a cancer patient (pt) in which PD-L1 is negative in the blood and PD-L1 is positive in the tissue, the cancer patient (pt) has progress using pemizumab. Of the 7 patients treated with immunotherapy (nivolumab, pembrolizumab, and atezolizumab), 3/3 of patients with advanced disease (PD) PD-L1 cfRNA showed increased degree of expression (INC), 3/3 patients with stable disease (SD) had no change in PD-L1 cfRNA (NC), and 1 patient with partial response (PR) showed PD-L1 cfRNA Reduced content (DEC) corresponds to a 100% correlation between PD-L1 performance and cancer patient response. After treatment, significant agreement was observed between clinical response and changes in plasma cfRNA levels (74%) in NSCLC patients. Detection of PD-L1 expression in plasma of cancer patients was also associated with results obtained from the same patient tissue (70%). At the target treatment, the degree of PD-L1 expression was correlated with the response in 7/7 patients. It can therefore be concluded that the cfRNA content can indicate a response to various treatments (tx) and that PD-L1 in plasma can be used to monitor response to immunotherapy.

It will be apparent to those skilled in the art that many modifications may be made in addition to those already described, without departing from the inventive concept. Therefore, the subject matter of the present invention is not limited by the scope of the appended claims. Moreover, in interpreting the specification and the scope of the patent application, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprises" or "comprises" or "an" Exist, or be utilized, or combined. In the case where the specification claims that the patent scope relates to at least one selected from the group consisting of A, B, C, ..., and N, the content should be interpreted as requiring only one element from the group, rather than A plus N, or B plus N and so on.

Figure 1 is a graph showing plasma concentrations of cfDNA and cfRNA in healthy subjects versus subjects diagnosed with cancer.

Figure 2A shows a graph comparing plasma concentrations of PD-L1 cfRNA across various cancer types.

Figure 2B is a graph showing plasma concentrations of PD-L1 cfRNA in healthy subjects.

Figure 2C is a graph showing the linear range of plasma concentrations of PD-L1 cfRNA.

Figure 3A is a graph showing the relative expression of PD-L1 cfRNA in lung cancer patients in a clinical trial.

Figure 3B shows data showing PD-L1 expression in lung cancer patients measured by IHC in a clinical trial.

Figure 4 shows PD-L1 cfRNA content showing no response to nivolumab and responders, corresponding IHC staining with lung tumor samples, and PD-L1 cfRNA content during treatment.

Figure 5A is a graph showing the association of PD-L1 cfRNA content and PD-L1 status determined by PD-L1 IHC.

Figure 5B is a graph showing the association of PD-L1 cfRNA content with the reaction status of nivolumab, showing the clinically relevant performance threshold for PD-L1 cfRNA content.

Figures 6A-6D are graphs showing plasma concentrations of PD-L1 cfRNA levels in subjects who are diagnosed with cancer and are being treated.

Figure 7 is a graph showing the PD-L1 cfRNA content as a function of treatment with an afinitor and an anti-PD1/PD-L1 composition.

Figure 8 is a graph showing the association of cancer treatment response status with total cfRNA/β-actin cfRNA.

Figure 9A is a graph showing the relative co-expression of PD-L1 and HER2 measured by cfRNA content.

Figure 9B is a graph showing the association of HER2 cfRNA content and HER2 status as determined by HER2 IHC/FISH, showing the clinically relevant performance threshold for HER2 cfRNA content.

Figure 10 is a graph showing the relative co-expression of PD-L1 and HER2 in gastric cancer measured by cfRNA content.

Figure 11 is a graph showing the association of pertuzumab/trustuzumab treatment response with HER2 cfRNA content.

Figure 12 shows the cfRNA characteristics of the selected checkpoint related genes.

Figure 13 shows exemplary results for AR-V7 cfRNA content and AR cfRNA content in prostate cancer patients, indicating that AR-V7 cfRNA is a suitable marker.

Figure 14 shows exemplary results for PCA3 cfRNA content in non-prostate and prostate cancer patients, indicating that PCA3 cfRNA is a suitable marker.

Claims (41)

A method of using a primer pair to amplify a nucleic acid encoding at least one checkpoint suppressor gene, comprising: obtaining blood from the individual and isolating cfRNA from the blood, wherein the cfRNA encodes a checkpoint suppressor gene; using the primer Quantifying the cfRNA by a quantitative PCR method; and correlating a predicted effect of a checkpoint inhibitor with the amount of the cfRNA when the amount of the cfRNA is above a threshold. The method of claim 1, wherein the checkpoint inhibitor is an antibody against PD1 or PD-L1, and wherein the cfRNA is PD-L1 cfRNA. The method of claim 1, wherein the step of isolating the cfRNA uses RNA stabilization and cell preservation. The method of claim 1, wherein the quantitative PCR method comprises real-time PCR. The method of claim 1, wherein the quantifying step uses a β-actin as an internal standard. The method of claim 1, wherein the threshold of the PD-L1 relative to β-actin is ΔΔCT>10. The method of claim 1, further comprising the step of quantifying at least one second cfRNA using the quantitative PCR method. The method of claim 7, wherein the at least one second cfRNA encodes TIM3 or LAG3. The method of claim 1, further comprising the step of at least one second cfRNA encoding a gene having a tumor and a patient-specific mutation, a tumor-associated gene, or a cancer specific Sex gene. A method of using a primer pair to amplify a nucleic acid encoding at least one of a checkpoint inhibition gene, a tumor associated or cancer specific gene, and a gene having a tumor and a patient-specific mutation, including: The individual obtains blood and isolates cfRNA from the blood, wherein the cfRNA encodes a checkpoint suppressor gene, or wherein the cfRNA encodes a tumor associated or cancer specific gene, or wherein the cfRNA encodes a tumor and patient specific mutation Gene; quantify the cfRNA using a quantitative PCR method; and update a patient record using the amount of the cfRNA. The method of claim 10, wherein the checkpoint inhibition gene is PD-L1, TIM3, or LAG3. The method of claim 10, wherein the tumor-associated or cancer-specific gene is CEA, MUC1, brachyury, HER2, PCA3, or AR-V7. The method of claim 10, wherein the gene having a tumor and a patient-specific mutation encodes a novel epitope. The method of claim 10, wherein the step of isolating the cfRNA uses RNA stabilization and cell preservation. The method of claim 10, wherein the quantitative PCR method comprises real-time PCR. The method of claim 10, wherein the quantifying step uses a β-actin as an internal standard. The method of claim 10, wherein when the amount of the HER2 cfRNA is ΔΔCT>5 relative to β-actin, or the amount of the PCA3 cfRNA is ΔΔCT>10 relative to β-actin, the patient The record is updated. A method of using a primer pair to amplify at least a portion of a nucleic acid encoding a splice variant 7 of PCA3 or an androgen receptor, comprising: obtaining blood from a body and isolating cfRNA from the blood, wherein the cfRNA encodes PCA3 or a male Splice variant 7 of a hormone receptor; quantify the cfRNA using a quantitative PCR method; and correlating a predicted effect of a treatment with the amount of the cfRNA when the amount of the cfRNA is above a threshold. The method of claim 18, wherein the predictive effect is correlated when the amount of the PCA3 cfRNA is ΔΔCT &gt; 10 relative to β-actin. The method of claim 18, further comprising the step of quantifying at least one second cfRNA, wherein the at least one second cfRNA encodes a gene having a tumor and a patient-specific mutation, a tumor-associated gene, a cancer specific Sex genes, or a checkpoint suppressor gene. The method of claim 20, wherein the second cfRNA encodes PD-L1, LAG3, TIM3, AR-V7, PSA, and PSMA. A method of using a primer pair to amplify at least a portion of a nucleic acid encoding PD-L1, comprising: isolating cfRNA from blood of an individual diagnosed with a PD-L1 negative cancer, wherein the cfRNA encodes PD-L1; A quantitative PCR method quantifies the cfRNA; and correlates a predicted effect of a checkpoint inhibitor with the amount of the cfRNA upon detection of the cfRNA. The method of claim 22, wherein the PD-L1 negative cancer is a substantially solid cancer. The method of claim 23, wherein the substantially solid cancer is breast cancer. The method of claim 22, wherein the drug is afinitor. The method of claim 22, wherein the step of quantifying the cfRNA uses real-time PCR. The method of claim 22, wherein the checkpoint inhibitor is added when the cfRNA is detected and increases over time. The method of claim 22, wherein the checkpoint inhibitor is added when the cfRNA is detected and the cfRNA content is ΔΔCT>10 relative to β-actin. A method of using a primer pair to amplify a nucleic acid encoding at least one checkpoint suppressor gene, comprising: determining an amount of a different cfRNA molecule in a blood of a body, wherein the cfRNA molecule encodes a different checkpoint suppressor gene; The step of determining is performed in the blood of one body before or during treatment with at least one of a checkpoint inhibitor, a chemotherapeutic drug, an immunotherapeutic drug, and radiation therapy. The method of claim 29, wherein at least one of the different cfRNA molecules encodes PD-L1, LAG3, or TIM3. The method of claim 29, wherein the step of determining the amount comprises real-time PCR. The use of cfRNA to predict the response of a subject having cancer to a therapeutic response to a checkpoint inhibitor, wherein the cfRNA encodes a checkpoint suppressor gene, and wherein the cfRNA is above a threshold. The use of claim 32, wherein the checkpoint inhibitor is an antibody against PD1 or PD-L1, and wherein the cfRNA is PD-L1 cfRNA. The use of the scope of claim 32, wherein the threshold of the PD-L1 relative to the β-actin is ΔΔCT &gt; The use of cfRNA to monitor the treatment of a subject having cancer, wherein the cfRNA encodes a checkpoint suppressor gene, or wherein the cfRNA encodes a tumor associated or cancer specific gene, or wherein the cfRNA encodes a tumor and patient specific Gene of sexual mutation. The use of claim 35, wherein the checkpoint suppressor gene is PD-L1, TIM3, or LAG3. The use of claim 35, wherein the tumor-associated or cancer-specific gene is CEA, MUC1, brachyury, HER2, PCA3 or AR-V7. For example, in the application of claim 35, wherein the gene having a tumor and a patient-specific mutation encodes a novel epitope. The use of a cfRNA encoding a splice variant 7 of PCA3 or androgen receptor for detecting an in vivo prostate cancer. The use of claim 39, wherein the individual is diagnosed with prostate cancer when the amount of the cfRNA is above a threshold. The use of claim 40, wherein the threshold of the PCA3 cfRNA is ΔΔCT &gt; 10 relative to β-actin.