Predicting peptide receptor radionuclide therapy using gene expression assay

Abstract

To provide a method for prediction of response of a subject having a neuroendocrine tumor (NET) to peptide receptor radionuclide therapy (PRRT) treatment.SOLUTION: The method comprises: determining the expression level of each of at least 12 biomarkers including ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2 and ALG9.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 592,647, filed November 30, 2017, the entire contents of which are incorporated herein by reference.

[0002] [Sequence Listing] This application contains a Sequence Listing that has been submitted in ASCII format via EFS Web, and is incorporated herein by reference in its entirety. The ASCII copy, created on November 15, 2018, has the file name "LBIO-003_001WO_ST25.txt" and is 52,601 bytes in size. FIELD OF THE INVENTION

[0003] The present invention relates to predicting response to peptide receptor radionuclide therapy (PRRT) using gene expression assays. [Background technology]

[0004] The most commonly used form of radionuclide therapy in neuroendocrine tumors is peptide receptor radionuclide therapy (PRRT), which exploits the overexpression of somatostatin receptors, a major feature of NETs. PRRT uses octreotide, an analog of somatostatin, as a peptide to target the somatostatin receptor. Radiolabeled derivatives of this analog include: 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide, so-called 177 This therapeutic strategy is widely used in Europe and has recently been introduced in the United States.

[0005] A wide range of non-comparative studies in pancreatic and bronchopulmonary NETs have 177Lu-octreotate has proven effective with objective responses and beneficial effects on survival parameters. Most recently, the ongoing Phase III randomized controlled clinical trial of midgut NETs (NETTER-1) compared standard octreotide LAR treatment with 177 Lu-octreotate proved to be more effective than the high dose octreotide somatostatin analog.

[0006] The use of PRRT is currently based on somatostatin receptor (SSR) expression levels, information obtained by tissue biopsy and immunohistochemistry, or 111 In-pentetreotide scan or 68 or by somatostatin-based scans such as Ga-DOTATATE / DOTATOC PET / CT.

[0007] However, immunohistochemistry is limited by the heterogeneity of somatostatin receptor expression in tumors, and because individual antibodies have different binding affinities, evaluation of staining by a pathologist cannot provide the desired results. Further limitations include the inability to define receptor functionality and the inability to distinguish expression in other tumors not examined by biopsy.

[0008] Assessment of somatostatin expression using imaging involves comparing the uptake of radionuclides in target lesions with non-tumor organs such as the spleen. The degree of uptake is graded according to the Krenning grade from low to strongly positive. However, this approach has poor predictive power. For example, 111 Strongly positive tumors, with a Krenning grade of 4 on in-pentetreotide scans, have an accuracy of response of only 60%. Various semiquantitative tools have been attempted, but all have failed. Somatostatin receptor expression can be useful in determining whether a tumor is targetable and amenable to isotope delivery, but it does not provide an accurate assessment of the likelihood of radiation sensitivity (and treatment efficacy).

[0009] Other clinical parameters (e.g., extent of disease), tumor grading, and biomarkers (e.g., chromogranin A) are being investigated as useful predictive tools. However, while grading using morphological criteria or KI67 index assessment has shown some clinical utility, none have proven to be robust predictors of treatment response. The accuracy of grading is approximately 70% for predictive PRRT. Typically, low-grade tumors (well-differentiated grade 1 or 2, i.e., KI67 detectable in ≤20% of tumor cells) respond more frequently to PRRT than high-grade tumors (KI67 >20%). However, grading is limited by tumor heterogeneity, subjective observer variability, and low kappa values. Furthermore, tissue biopsies are rarely obtained from multiple affected areas, and metastases are often significantly different from the primary lesion biopsied for diagnosis.

[0010] Due to the complexity of the molecular drivers of tumor cells that determine therapeutic response during cancer or disease progression, there is a clear need for more sophisticated assessment tools. The development of technologies based on molecular biology characterization of various cancers has led to the evolution of strategies for assessing circulating molecular information arising from tumorigenesis. One such strategy, "liquid biopsy," has proven remarkably effective in lung tumorigenesis, for example, for monitoring therapeutic response to EFGR inhibitors through the identification of the T790M mutation in circulating tumor DNA. The opportunity to limit biopsies, define potential therapeutic targets, and provide a real-time monitoring tool to assess disease progression has considerable clinical benefits. Summary of the Invention

[0011] The present disclosure relates to a method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations for a subject having a neuroendocrine tumor (NET), the method comprising: determining an expression level of each of at least nine biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of the at least nine biomarkers, wherein the nine biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining a normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; summing the normalized expression levels of each of ATP6V1H, OAZ2, PANK2, and PLD3, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 when the NET is designated as high grade, or the second score is 0 when the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and providing a recommendation that the NET will respond to PRRT if the third score is less than the second predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the third score is greater than the second predetermined cutoff value.

[0012] In the aforementioned methods of the present disclosure, the first predetermined cutoff value may be 5.9. The second predetermined cutoff value may be 0.

[0013] The present disclosure relates to a method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a neuroendocrine tumor (NET), the method comprising: determining an expression level of each of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression level of at least 12 biomarkers, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. obtaining a normalized expression level for each of PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and providing a recommendation that the NET will respond to PRRT if the third score is less than the second predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the third score is greater than the second predetermined cutoff value.

[0014] In the aforementioned methods of the present disclosure, the first predetermined cutoff value may be 10.9. The second predetermined cutoff value may be 0.

[0015] The present disclosure relates to a method for providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a neuroendocrine tumor (NET), the method comprising: determining the expression levels of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of at least 12 biomarkers, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression levels of at least 12 biomarkers from the test sample. obtaining normalized expression levels of PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; and providing a recommendation that the NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the total expression level is less than the predetermined cutoff value.

[0016] In the aforementioned methods of the present disclosure, the predetermined cutoff value may be 10.9.

[0017] The present disclosure relates to a method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: measuring the expression levels of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. obtaining normalized expression levels of PANK2, PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; and providing a recommendation that the low-grade or high-grade NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value, or providing a recommendation that the low-grade or high-grade NET will not respond to PRRT if the total expression level is less than the predetermined cutoff value.

[0018] In the aforementioned methods of the present disclosure, the predetermined cutoff value may be 10.9.

[0019] The present disclosure relates to a method for providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: determining the expression levels of at least nine biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; The normalized expression levels of ATP6V1H, OAZ2, PANK2, and PLD3 are summed to thereby obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, a recommendation is provided that the low-grade or high-grade NET will respond to PRRT, or if the total expression level is less than the predetermined cutoff value, a recommendation is provided that the low-grade or high-grade NET will not respond to PRRT.

[0020] In the method of the present disclosure, at least one of the at least nine biomarkers can be RNA, cDNA or protein.In an embodiment where the biomarker is RNA, the RNA can be reverse transcribed to produce cDNA, and the expression level of the produced cDNA can be detected.In an embodiment where the biomarker is protein, the protein can be detected by forming a complex between the biomarker and a labeled probe or primer.

[0021] In the methods of the present disclosure, the expression level of a biomarker can be detected by forming a complex between the biomarker and a labeled probe or primer.

[0022] In the disclosed method, when the biomarker is RNA or cDNA, the RNA or cDNA can be detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer. The complex between the RNA or cDNA and the labeled nucleic acid probe or primer can be a hybridization complex.

[0023] In the disclosed methods, the test sample can be blood, serum, plasma, or tumor tissue. In the disclosed methods, the test sample can be blood.

[0024] In the methods of the present disclosure, NETs can be designated as high grade when they are poorly differentiated.

[0025] In the methods of the present disclosure, a NET can be designated as low grade if it is a well-differentiated bronchial typical carcinoid or bronchial atypical (atypical) carcinoid.

[0026] The methods of the present disclosure can further include administering PRRT to the subject if the third score is less than or equal to a second predetermined cutoff value.

[0027] The methods of the present disclosure can further include administering PRRT to the subject if the total expression level is equal to or greater than a predetermined cutoff value.

[0028] The disclosed methods can have a sensitivity of greater than 90%. The disclosed methods can have a specificity of greater than 90%.

[0029] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), the method comprising: determining an expression level of each of at least nine biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression level of each of at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining a normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; summing the normalized expression levels of ATP6V1H, OAZ2, PANK2, and PLD3, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and if the third score is equal to or greater than a predetermined cutoff value, the subject undergoes PRRT.

[0030] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting the expression levels of at least 12 biomarkers, thereby measuring the expression levels of each of the at least 12 biomarkers from the test sample, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby detecting the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, obtaining a normalized expression level for each of PANK2, PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and if the third score is equal to or greater than a predetermined cutoff value, the subject undergoes PRRT.

[0031] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting the expression levels of at least 12 biomarkers, thereby measuring the expression levels of each of the at least 12 biomarkers from the test sample, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby detecting the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. A normalized expression level of each of PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed to thereby obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT.

[0032] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: measuring expression levels of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of each of the at least 12 biomarkers, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, A normalized expression level of each of OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed to thereby obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT.

[0033] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: measuring the expression levels of each of at least nine biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of the at least nine biomarkers, wherein the nine biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to ALG9, thereby obtaining normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; The normalized expression levels of each of ATP6V1H, OAZ2, PANK2 and PLD3 are summed to obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject undergoes PRRT.

[0034] In the methods of the present disclosure, administering PRRT to a subject comprises: 177 This may include administering Lu-based PRRT. 177 Lu-based PRRT 177 Lu-DOTA-Tyr 3 -Thr 8 -Can be octreotide.

[0035] In the method of the present disclosure, 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of about 7.4 GBq (200 mCi) approximately once every 8 weeks for a total of about 4 doses. 177 Lu-DOTA-Tyr 3 -Thr 8-Octreotide can be administered at a dose of about 6.5 GBq approximately once every 8 weeks for a total of about 4 doses. 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of approximately 4.6 GBq approximately once every 8 weeks for a total of approximately 4 doses.

[0036] In the method of the present disclosure, 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of about 3.2 GBq (100 mCi) approximately once every 8 weeks for a total of about 4 doses. 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of about 3.7 GBq approximately once every 8 weeks for a total of about 4 doses.

[0037] In the method of the present disclosure, 177 Lu-based PRRT can be administered intravenously. 177 Lu-based PRRT can be administered intra-arterially.

[0038] In some embodiments of any one of the above aspects, the method further includes administering PRRT to the subject when the NET is predicted to be responsive to PRRT.

[0039] Any of the above aspects can be combined with any other aspect.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. As used herein, the singular includes the plural unless the context clearly dictates otherwise. By way of example, the terms "a," "an," and "the" are interpreted as singular or plural, and the term "or" is interpreted as inclusive. By way of example, "an element" means one or more elements. Throughout this specification, the word "comprising," or variations thereof (comprises or comprising) is understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. "About" should be interpreted as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0041] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0042] [Figure 1]Figure 1 shows the utility of the PRRT quotient for predicting PFS in the study cohort. Study cohort (n=72): Patients predicted by the PPQ to be "responders" before treatment (biomarker positive) did not achieve mPFS. Patients predicted to be "non-responders" (biomarker negative) achieved mPFS of 8 months. This difference was significant (HR 36.4, p<0.0001). [Figure 2] Figure 2 shows the utility of the PPRT predictive index for predicting PFS in validation cohort I. Responders did not achieve mPFS. Non-responders achieved mPFS of 14 months (HR 17.7, p<0.0001). [Figure 3] Figure 3 shows the utility of the PPRT predictive index for predicting PFS in validation cohort II. The predicted responders did not achieve mPFS. The predicted non-responders had a mPFS of 9.7 months, a significant difference (HR 92, p<0.0001). [Figure 4] Figure 4 shows the utility of the PRRT predictive index for predicting PFS in patients treated with SSAs. For predicted responders, mPFS was 10 months. For predicted non-responders, mPFS was not reached. This difference was not significant (HR 0.8, p=NS). [Figure 5] Figure 5 shows the utility of the PRRT predictive index for predicting PFS in registry patients. For predicted responders, mPFS was 10 months. For predicted non-responders, mPFS was 15 months. This difference was significant (HR 0.9, p=NS). [Figure 6A]Figures 6A-6D are graphs demonstrating the utility of PPQ as a predictive marker. Figure 6A shows PPQ in the PRRT cohort and comparison cohort in biomarker-positive cases. In the predicted responder, i.e., PPQ "positive," group, PRRT-treated patients did not achieve mPFS compared with those treated with SSA or those in the registry [Validation Cohort I (n=44) and Validation Cohort II (n=42)]. [Figure 6B] Figures 6A-6D are graphs demonstrating the utility of PPQ as a predictive marker. Figure 6B shows PPQ in the PRRT cohort and comparison cohort in biomarker-negative cases: in predicted non-responders, i.e., the PPQ "negative" group, mPFS was similar regardless of whether or not they were treated with PRRT. [Figure 6C] Figures 6A-6D are graphs demonstrating the utility of PPQ as a predictive marker. Figure 6C shows the ideal predictive biomarker "Positive"; in this ideal case, there is a "treatment effect," i.e., a quantitative difference in mPFS between those receiving treatment (mPFS undetermined) and those not receiving treatment (17 months). [Figure 6D] Figures 6A-6D are graphs demonstrating the utility of PPQ as a predictive marker. Figure 6D shows the ideal predictive biomarker "Negative." In this ideal case, mPFS is the same (18 months) regardless of treatment. [Figure 7] FIG. 7 shows progression-free survival (PFS) of PPQ-negative subjects after treatment with PRRT or a combination of PRRT and chemotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description of the Invention Details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein. Other features, objects, and advantages of the invention will become apparent from the detailed description and claims. In this specification and the appended claims, the singular forms include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited herein are incorporated by reference in their entirety.

[0044] The present invention is based, in part, on the discovery that the expression levels of circulating neuroendocrine tumor (NET) transcripts can predict whether patients with NETs will respond to peptide receptor radionuclide therapy (PRRT). Circulating neuroendocrine tumor (NET) transcripts include: (a) growth factor (GF)-related genes (ARAF1, BRAF, KRAS, and RAF-1); and (b) genes involved in metabolism (M) (ATP6V1H, OAZ2, PANK2, and PLD3). The expression levels of these genes can be normalized to ALG9, which serves as a housekeeping gene. It was found that if the combined expression level (after normalization) of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 is equal to or greater than a predetermined cutoff value, the NET will respond to PRRT, regardless of the histological grade of the NET. Additionally, it has been discovered that if the combined expression levels (after normalization) of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 are below a predetermined cutoff value, the NET will not respond to PRRT, regardless of the histological grade of the NET. In some embodiments, circulating NET transcripts may further include genes involved in proliferation (P) (NAP1L1, NOL3, and TECPR2). Expression levels of NAP1L1, NOL3, and TECPR2 can also be measured and normalized to the expression level of ALG9.

[0045] In some embodiments, the total expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3 can be obtained by including the following steps: (a1) determining the expression level of each of at least nine biomarkers from the subject by contacting the test sample with a plurality of agents specific for detecting the expression of at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; (b1) normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby determining the total expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3. (c1) obtain the normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3; and (c2) sum the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3, thereby obtaining the total expression level.

[0046] Alternatively, the total expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 can be obtained by, after determining the expression level of each of the at least nine biomarkers, including the following steps: (a2) summing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3, thereby obtaining a total value; and (b2) normalizing the summed value to the expression level of ALG9, thereby obtaining a total expression level.

[0047] One aspect of the present disclosure relates to a method for providing a PRRT treatment recommendation to a subject with low-grade or high-grade NETs, ​​the method providing a recommendation that the low-grade or high-grade NET will respond to PRRT if the combined expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 is equal to or greater than a predetermined cutoff value, or providing a recommendation that the low-grade or high-grade NET will not respond to PRRT if the combined expression level is less than a predetermined cutoff value. In some embodiments, a NET is designated as high-grade if it is poorly differentiated. In some embodiments, a NET is designated as low-grade if it is well-differentiated with bronchial typical carotenoid or bronchial atypical carotenoid.

[0048] In a similar aspect, the disclosure relates to a method of providing a PRRT treatment recommendation to a subject having a NET, the method providing a recommendation that the NET will respond to PRRT if the combined expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3 is equal to or greater than a predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the combined expression level is less than a predetermined cutoff value.

[0049] In some embodiments, the predetermined cutoff value is 5.9, which is derived from the assumption that the combined expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 is 5.9 times higher than the expression level of ALG9.

[0050] In another embodiment, the histological grade of the NET can be used in combination with the expression level of circulating neuroendocrine tumor transcripts. Accordingly, the present disclosure provides a method for providing a PRRT treatment recommendation to a subject with a NET, the method comprising: (a3) ​​determining a first score, wherein the first score is 1 if the total expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 is equal to or greater than a predetermined cutoff value, or the first score is 0 if the total expression level is less than a predetermined cutoff value; (b3) determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; (c3) calculating a third score based on the following equation: third score=39.22787−40.80341×(first score)−18.441×(second score); and (d3) if the third score is less than or equal to the second predetermined cutoff value, provide a recommendation that the NET will respond to PRRT, or if the third score is greater than the second predetermined cutoff value, provide a recommendation that the NET will not respond to PRRT.

[0051] In some cases, the first predetermined cutoff value is 5.9, which is derived from the assumption that the combined expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 is 5.9 times higher than the expression level of ALG9.

[0052] In some embodiments, the second predetermined cutoff value is zero.

[0053] In one aspect, the present disclosure relates to a method of providing a PRRT treatment recommendation to a subject with a low-grade or high-grade NET, the method comprising: (a) measuring the expression level of each of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression level of each of the at least 12 biomarkers, wherein the 12 biomarkers are ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; (b) normalizing the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby normalizing the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9. (c) obtaining a normalized expression level for each of PLD3, NAP1L1, NOL3, and TECPR2; (c) summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; and (d) providing a recommendation that the low-grade or high-grade NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value, or providing a recommendation that the low-grade or high-grade NET will not respond to PRRT if the total expression level is less than a predetermined cutoff value. In some embodiments, the predetermined cutoff value is 10.9.

[0054] In another embodiment, the disclosure provides a method of providing a PRRT treatment recommendation to a subject having a NET, the method comprising: (a) measuring the expression level of each of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression level of the at least 12 biomarkers, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and (b) normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby normalizing the expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, (c) obtaining a normalized expression level for each of NAP1L1, NOL3, and TECPR2; (c) summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; and (d) providing a recommendation that the NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the total expression level is less than the predetermined cutoff value. In some embodiments, the predetermined cutoff value is 10.9.

[0055] In another aspect, the present disclosure relates to a method of providing a PRRT treatment recommendation to a subject having a NET, the method comprising: (a) measuring the expression level of each of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting the expression level of the at least 12 biomarkers, wherein the 12 biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and (b) normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, (c) obtaining a normalized expression level for each of NAP1L1, NOL3, and TECPR2; (c) summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; (d) determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than the first cutoff value; (e) determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; (f) calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and (f) providing a recommendation that the NET will respond to PRRT if the third score is less than a second predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the third score is greater than the second predetermined cutoff value. In some embodiments, the first predetermined cutoff value is 10.9. In some embodiments, the second predetermined cutoff value is 0.

[0056] A responder (i.e., one whose NET responds to PRRT) refers to an individual who is predicted to achieve disease stabilization or show a partial response according to the methods described herein. A non-responder (i.e., one whose NET does not respond to PRRT) refers to an individual who shows progressive disease.

[0057] The test sample can be any biological fluid obtained from a subject. Preferably, the test sample is blood, serum, plasma, or neoplastic tissue. In some embodiments, the test sample is blood. In some embodiments, the test sample is serum. In some embodiments, the test sample is plasma.

[0058] Expression levels can be measured in a variety of ways, including, but not limited to, measuring the mRNA encoded by a particular gene; measuring the amount of a protein encoded by a particular gene; and measuring the activity of a protein encoded by a particular gene.

[0059] Biomarkers can be RNA, cDNA, or proteins. When the biomarker is RNA, the RNA can be reverse transcribed to generate cDNA (e.g., by RT-PCR), and the expression level of the generated cDNA is detected. The expression level of the biomarker can be detected by forming a complex between the biomarker and a labeled probe or primer. When the biomarker is RNA or cDNA, the RNA or cDNA can be detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer. The complex between the RNA or cDNA and the labeled nucleic acid probe or primer can be a hybridization complex.

[0060] Gene expression can also be detected by microarray analysis. Variable gene expression can be identified or confirmed using microarray technology. Thus, biomarker expression profiles can be measured in either fresh or fixed tissues using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated or arrayed on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. The source of mRNA is typically total RNA isolated from a biological sample, and variable expression can be detected using corresponding normal tissues or cell lines.

[0061] In some embodiments of microarray technology, PCR-amplified inserts of cDNA clones are applied to a substrate in the form of a high-density array. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. Microarray genes immobilized on a microchip, each with 10,000 elements, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated through the incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes smeared on the chip specifically hybridize to each DNA spot on the array. After stringent washing to remove nonspecifically bound probes, the microarray chip is scanned using a device such as a confocal laser microscope or another detection method, such as a CCD camera. Quantifying hybridization of each arrayed element allows assessment of the abundance of the corresponding mRNA. Using dual-color fluorescence, differentially labeled cDNA probes generated from RNA from two sources hybridize to the array in pairs. Thus, the relative abundance of transcripts from the two sources corresponding to each specified gene is determined simultaneously. Microarray analysis can be performed using commercially available equipment according to the manufacturer's protocols.

[0062] In some embodiments, biomarkers can be detected in biological samples using qRT-PCR. The first step in gene expression profiling by RT-PCR is to extract RNA from the biological sample, followed by reverse transcription of the RNA template into cDNA and amplification by PCR. The reverse transcription step is generally primed using specific primers, random hexamers, or oligo-dT primers, depending on the goal of expression profiling. Two commonly used reverse transcriptases are avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MLV-RT).

[0063] When a biomarker is a protein, the protein can be detected by forming a complex between the protein and a labeled antibody. The label can be any label, such as a fluorescent label, a chemiluminescent label, or a radioactive label. Typical methods for protein detection include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). For example, biomarkers can be detected by ELISA, in which a biomarker antibody is bound to a solid phase and an enzyme-antibody conjugate is used to detect and / or quantify the biomarker present in a sample. Alternatively, Western blot analysis can be used, in which solubilized and separated biomarkers are bound to nitrocellulose paper. The combination of a liquid conjugate with a highly specific, stable, and sensitive chromogenic substrate allows for rapid and accurate sample identification.

[0064] In some embodiments, the methods described herein further include administering PRRT to the subject if the NET is predicted to respond to PRRT. For example, according to some aspects of the present disclosure, the method further includes administering PRRT to the subject if the total expression level is equal to or greater than a predetermined cutoff value. According to another aspect of the present disclosure, the method further includes administering PRRT to the subject if the third score is equal to or less than a second predetermined cutoff value. In PRRT, a cell-targeting protein (or peptide) called octreotide is combined with a small amount of radioactive material or radionuclide to create a special form of radiopharmaceutical called a radiopeptide. When injected into a patient's bloodstream, this radiopeptide is transported to and binds to neuroendocrine tumor cells, delivering a high dose of radiation to the cancer.

[0065] When it is predicted that the NET will not respond to PRRT, the methods described herein further include monitoring the subject over a period of time, for example, 1 to 6 months.

[0066] In some embodiments, the methods described herein can have a specificity, sensitivity, and / or accuracy of at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0067] The present disclosure provides a method of treating a patient with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least nine biomarkers, thereby determining an expression level of each of the at least nine biomarkers from a test sample from the subject, wherein the at least nine biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining a normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; summing the normalized expression levels of each of RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and if the third score is equal to or greater than the predetermined cutoff value, the subject will undergo PRRT.

[0068] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the patient has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining the expression level of each of said at least 9 biomarkers from the test sample, wherein said 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to ALG9, thereby obtaining a normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; summing the normalized expression levels of each of OAZ2, PANK2, and PLD3, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is equal to or greater than a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: third score=39.22787−40.80341×(first score)−18.441×(second score); and if the third score is equal to or greater than the predetermined cutoff value, the subject is administered PRRT, or if the third score is less than the predetermined cutoff value, the subject is administered an alternative form of treatment.

[0069] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the patient has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining the expression level of each of said at least 12 biomarkers from the test sample, wherein said 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. obtaining a normalized expression level for each of PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: Third score = 39.22787 - 40.80341 x (first score) - 18.441 x (second score); and if the third score is equal to or greater than the predetermined cutoff value, the subject will undergo PRRT.

[0070] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the patient has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining the expression level of each of said at least 12 biomarkers from the test sample, wherein said 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. obtaining a normalized expression level for each of PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histological grade of the NET, wherein the second score is 1 if the NET is designated as high grade, or the second score is 0 if the NET is designated as low grade; and calculating a third score based on the following equation: third score=39.22787−40.80341×(first score)−18.441×(second score); and if the third score is equal to or greater than the predetermined cutoff value, the subject is administered PRRT, or if the third score is less than the predetermined cutoff value, the subject is administered an alternative form of treatment.

[0071] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the patient has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining an expression level of each of said at least 12 biomarkers from the test sample, wherein said 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. A normalized expression level of each of PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed to thereby obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT.

[0072] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the patient has a neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting the expression of each of at least 12 biomarkers, thereby determining expression levels of said at least 12 biomarkers from the test sample, wherein said 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. A normalized expression level for each of PANK2, PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed, thereby obtaining a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT, or if the total expression level is less than the predetermined cutoff value, the subject is administered an alternative form of treatment.

[0073] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), the patient having a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining the expression level of each of at least 12 biomarkers from the test sample, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. A normalized expression level for each of PANK2, PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed, thereby obtaining a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT, or if the total expression level is less than a predetermined cutoff value, the subject is administered an alternative form of treatment.

[0074] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), the patient having a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least 12 biomarkers, thereby determining the expression level of each of at least 12 biomarkers from the test sample, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; and normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby determining the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2. A normalized expression level for each of PANK2, PLD3, NAP1L1, NOL3, and TECPR2 is obtained; the normalized expression levels for each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 are summed, thereby obtaining a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT, or if the total expression level is less than a predetermined cutoff value, the subject is administered an alternative form of treatment.

[0075] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least nine biomarkers, thereby determining the expression levels of each of the at least nine biomarkers from a test sample from the subject, wherein the nine biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; The normalized expression levels of each of RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 are summed to obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject undergoes PRRT.

[0076] The present disclosure provides a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), the method comprising: contacting a test sample from the subject with a plurality of agents specific for detecting expression of at least nine biomarkers, thereby determining the expression levels of each of at least nine biomarkers from the test sample, wherein the nine biomarkers comprise ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; normalizing the expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 to the expression level of ALG9, thereby obtaining normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; The normalized expression levels of ATP6V1H, OAZ2, PANK2 and PLD3 are summed to obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT, or if the total expression level is less than a predetermined cutoff value, the subject is administered an alternative form of therapy.

[0077] In the methods of the present disclosure, administering PRRT to a subject comprises: 177 This involves administering Lu-based PRRT. 177 Lu-based PRRT 177 Lu-DOTA-Tyr 3 -Thr 8 - Can be octreotide (Lutathera).

[0078] In the method of the present disclosure, 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of approximately 7.4 GBq (200 mCi) approximately once every 8 weeks for a total of approximately 4 doses. 177 Lu-DOTA-Tyr 3-Thr 8 -Octreotide can be administered at a dose of approximately 6.5 GBq, approximately once every 8 weeks for a total of four doses. 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of approximately 4.6 GBq, approximately once every 8 weeks for a total of approximately 4 doses. 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of approximately 3.2 GBq (100 mCi) approximately once every 8 weeks for a total of approximately 4 doses. 177 Lu-DOTA-Tyr 3 -Thr 8 -Octreotide can be administered at a dose of about 3.7 GBq, about once every 8 weeks for a total of about 4 doses.

[0079] In the methods of the present disclosure, PRRT can be administered intravenously. Alternatively, PRRT can be administered intra-arterially.

[0080] In the method of the present disclosure, 177 Lu-based PRRT can be administered intravenously. 177 Lu-based PRRT can be administered intra-arterially.

[0081] In the methods of the present disclosure, an alternative form of treatment may include administering chemotherapy to the subject. An alternative form of treatment may include administering immunotherapy to the subject. An alternative form of treatment may include administering radiation therapy to the subject. An alternative form of treatment may include administering a combination of PRRT and chemotherapy to the subject. An alternative form of treatment may include administering a combination of PRRT and immunotherapy to the subject. An alternative form of treatment may include administering a combination of PRRT and radiation therapy to the subject. An alternative form of treatment may include administering a combination of PRRT, immunotherapy, and radiation therapy to the subject. An alternative form of treatment may include administering a combination of PRRT, immunotherapy, chemotherapy, and radiation therapy to the subject. An alternative form of treatment may include administering a combination of immunotherapy and chemotherapy to the subject.

[0082] The immunotherapy may include administering a checkpoint inhibitor. The checkpoint inhibitor may include an antibody. Checkpoint inhibitors include, but are not limited to, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-A2AR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-BTLA antibody, anti-IDO antibody, anti-KIR antibody, anti-LAG3 antibody, anti-TIM3 antibody, and anti-VISTA (V domain Ig suppressor of T cell activation) antibody.

[0083] Anti-CTLA4 antibodies include, but are not limited to, ipilimumab, tremelimumab, and AGEN-1884. Anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab / pidilizumab, cemiplimab, REGN2810, AMP-224, MEDI0680, PDR001, and CT-001. Anti-PD-L1 antibodies include, but are not limited to, atezolizumab, avelumab, and durvalumab. Anti-CD137 antibodies include, but are not limited to, urelumab. Anti-B7-H3 antibodies include, but are not limited to, MGA271. Anti-KIR antibodies include, but are not limited to, lirilumab. Anti-LAG3 antibodies include, but are not limited to, BMS-986016.

[0084] The term "immunotherapy" can refer to activating immunotherapy or suppressive immunotherapy. As will be understood by those skilled in the art, activating immunotherapy refers to the use of a therapeutic agent that induces, enhances, or promotes an immune response, such as a T cell response, while suppressive immune responses interfere with, suppress, or inhibit an immune response, such as a T cell response. Activating immunotherapy can include the use of a checkpoint inhibitor. Activating immunotherapy can include administering to a subject a therapeutic agent that activates a stimulatory checkpoint molecule. Stimulatory checkpoint molecules include, but are not limited to, CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. Therapeutic agents that activate stimulatory checkpoint molecules include, but are not limited to, MEDI0562, TGN1412, CDX-1127, and lipocalin.

[0085] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An antibody that binds to a target refers to an antibody that can bind to a target with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting the target. In one embodiment, the extent to which an anti-target antibody binds to unrelated non-target proteins is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA) or Biacore assay. In certain embodiments, an antibody that binds to a target has an affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 Less than M, e.g., 10 8 M ~10 13 M, e.g. 10 9 M~10 13 M) In certain embodiments, the anti-target antibody binds to an epitope of the target that is conserved across different species.

[0086] A "blocking antibody" or "antagonist antibody" is one that partially or completely blocks, inhibits, interferes with, or neutralizes the normal biological activity of the antigen to which it binds. For example, an antagonist antibody can block signaling through an immune cell receptor (e.g., a T cell receptor) and restore a functional response (e.g., enhancement, cytokine production, target cell killing) by T cells from a dysfunctional state to an antigen-stimulated state.

[0087] An "agonist antibody" or "activating antibody" is one that mimics, promotes, stimulates, or enhances the normal biological activity of the antigen to which it binds. An agonist antibody can also enhance or initiate signaling by the antigen to which it binds. In some embodiments, an agonist antibody causes or activates signaling in the absence of a natural ligand. For example, an agonist antibody increases memory T cell proliferation, increases cytokine production by memory T cells, inhibits regulatory T cell function, and / or inhibits regulatory T cell suppression of effector T cell function, such as effector T cell proliferation and / or cytokine production.

[0088] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0089] Administering a chemotherapeutic agent to the subject can include administering a therapeutically effective amount of at least one chemotherapeutic agent, including, but not limited to, 13-cis-retinoic acid, 2-CdA, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 5-FU, 6-mercaptopurine, 6-MP, 6-TG, 6-thioguanine, abemaciclib, abiraterone acetate, Abraxane, Accutane, actinomycin-D, Adcetris, ado-trastuzumab emtansine, Adriamycin, Adrsil, and Afatin. Afinitor, Agrylin, Ala-Coat, Aldesleukin, Alemtuzumab, Alecensa, Alectinib, Alimta, Alitretinoin, Alkaban-AQ, Alkeran, All-trans retinoic acid, alpha interferon, altretamine, Alunbrig, amethopterin, amifostine, aminoglutethimide, anagrelide, anandrone, anastrozole, apalutamide, arabinosylcytosine, Ara-C, arane Sp, Aredia, Arimidex, Aromasin, Alano, Arsenic trioxide, Alzera, Asparaginase, Atezolizumab, Atra, Avastin, Avelumab, Axicaptagen-ciloleucel, Axitinib, Azacitidine, Bavencio, Bcg, Beleodac, Belinostat, Bendamustine, Bendeca, Besponsa, Bevacizumab, Bexarotene, Bexar, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Blinaz Momab, Blincyto, Bortezomib, Bosurif, Bosutinib, Brentuximab vedotin, Brigatinib, Busulfan, Busulfex, C225, Cabazitaxel, Cabozantinib, Leucovorin calcium, Campas, Camptosar, Camptothecin-11, Capecitabine, Caprelsa, Carac, Carboplatin, Carfilzomib, Carmustine, Carmustine wafer, Casodex, CCI-779, CcNU, Cddp Ceenu, cetirinib, cerbidine, cetuximab, chlorambucil, cisplatin, citrovorum factor, cladribine, clofarabine, chlorar, cobimetinib, cometrik, cortisone, cosmegen, cotellic, Cpt-11, crizotinib, cyclophosphamide, ciramza, citadren, cytarabine, cytarabine liposomal formulation, Cytosar U,Cytoxan, dabrafenib, dacarbazine, Dacogen, dactinomycin, daratumumab, darbepoetin alfa, Darzalex, dasatinib, daunomycin, daunorubicin, daunorubicin-cytarabine (liposomal formulation), daunorubicin hydrochloride, daunorubicin liposomal formulation, daunoxom, Decadron, decitabine, degarelix, δ-Cortef, Deltasone, denileukin-diftitox, denosumab, DepoCyt, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexasone, dexamethasone Surazoxane, Dhad, Dic, Diodex, docetaxel, Doxil, doxorubicin, liposomal doxorubicin, Droxia, DTIC, Dtic-Dome, Duralon, durvalumab, eculizumab, Efudex, Elence, elotuzumab, Eloxatin, Elspar, Eltrombopag, Emcyt, Empliciti, enasidenib, enzalutamide, epirubicin, epoetin alfa, Erbitux, eribulin, Elivege, Erleda, erlotinib, Erwinia L-asparaginase, estalamustine, ethyolol , etopophos, etoposide, etoposide phosphate, eurexin, everolimus, Evista, exemestane, Fareston, Faridak, Faslodex, Femara, filgrastin, Farmagon, floxuridine, Fludara, fludarabine, Fluoroplex, fluorouracil, fluorouracil (cream), fluoxymesterone, flutamide, folinic acid, Folotin, FUdR, fulvestrant, G-Csf, Gazyva, gefitinib, gemcitabine, gemtuzumab ozogamicin, Gemzar, Dilotrif, Glebe K, Gloostin, Gliadel Uefa, Gm-Csf, Goserelin, Granix, Granulocyte colony-stimulating factor, Granulocyte-macrophage colony-stimulating factor, Halaven, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, Hmm, Hycamtin, Hydrea, Hydrocort acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibrance, Ibritumomab, Ibritumomab tiuxetan,Ibrutinib, Iclusig, idamycin, idarubicin, idelalisib, Idhifa, Ifex, IFN-α, ifosfamide, IL-11, IL-2, Imbruvica, imatinib mesylate, Imfinzi, imidazole carboxamide, Imlygic, Inlyta, inotuzumab ozogamicin, interferon α, interferon α-2b (PEG conjugate), interleukin-2, interleukin-11, Intron A (Interferon α-2b), ipilimumab, Iressa, irinotecan, irinotecan (liposomal formulation), isotretinoin, Istodax, ixabepilone, ixazomib, Ixempra, Jakafi, Jevtana, Kadcyla, Keytruda, Quidrolase, Kisqali, Kymriah, Cyprolis, lanacort, lanreotide, lapatinib, Latorvo, L-asparaginase, Lubrance, Lcr, lenalidomide, lenvatinib , Lenvima, Letrozole, Leucovorin, Leukeran, Leukin, Leuprolide, Leulocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, Lonsurf, L-PAM, L-Sarcolysin, Lupron, Lupron Depot, Lynparza, Markivo, Matulan, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralon, Medrol, Megas, Megestrol, Megestrol Acetate, Mequi Nist, mercaptopurine, mesna, mesnex, methotrexate, methotrexate sodium, methylprednisolone, methycortene, midostaurin, mitomycin, mitomycin C, mitoxantrone, M-prednisolone, MTC, MTX, Mustagen, Mustine, Mutamycin, Myleran, Mylocel, Mylotarg, navelbine, necitumumab, nelarabine, Neosar, neratinib, Nerlinx, Neulasta , Neumega, Neupogen, Nexavar, Nilandrone, Nilotinib, Nilutamide, Ninlaro, Nipent, Niraparib, Nitrogen Mustard, Nivolumab, Nolvadex, Novantrone, Nplate, Obinutuzumab, Octreotide, Octreotide acetate, Odomzo, Ofatumumab, Olaparib, Olaratumab, Omacetaxine, Oncospar, Oncovin, Onivyde, Ontac, Onxar, Opdivo,Oprelvekin, Olapred, Olazone, Osimertinib, Otrexup, Oxaliplatin, Paclitaxel, Paclitaxel protein-bound, Palbociclib, Pamidronate, Panitumumab, Panobinostat, Panretin, Paraplatin, Pazopanib, Pediapred, Peginterferon, Pegaspargase, Pegfilgrastim, PegIntron, PEG-L-asparaginase, Pembrolizumab, Pemetrexed, Pentostatin, Pergenta, Pertuzumab, Phenylanine mustard, Platinol, Platinol-AQ, Pomalidomide, Pomalyst, Ponatinib, Portraza, Pralatrexate, Prednisolone, Prednisone, Prelon, Procarbazine, Procrit, Proleukin, Prolia, Carmustine implant Prolifeprospan 20, Promacta, Provenge, Purinetol, Radium-223 dichloride, Raloxifene, Ramucirumab, Lasbo, Regorafenib, Revlimid, Rheumatrex, Ribociclib, Rituxan, Rituxan-Hycera, Rituximab, Rituximab-Hyaluronase, Roferon-A (Interferon α-2a), romidepsin, romiplostim, Rivex, Rubraca, rucaparib, ruxolitinib, Ridapt, Sandostatin, Sandostatin LAR, sargramostimab, siltuximab, sipuleucel-T, Soliris, Sol-Cortef, Sol-Medrol, somatulin, sonidegib, sorafenib, Splicel, Sti-571, Stivarga, streptozocin, SU11248, sunitinib, Sutent, Silvant, Synribo, Tafinlar, Tagrisso, talimogene laherparepvec, tamoxifen, Tarceva, Targretin, Tasigna, Taxol, Taxotere, Tecentriq, Temodar, temozolomide, temsirolimus, teniposide, Tespa, thalidomide, talomide, Terasis, thioguanine, thioguanine tabloid, thiophosphamide, thioplex, thiotepa, Tice, tisagenlecleucel, toposal, topotecan, toremifene, Tolicel, tositumomab, trabectedin, trametinib, trastuzumab, Treanda, Trelstar, tretinoin, Trexal, trifluridine / tipiricil, triptorelin pamoate, Trisenox,Tspa, T-VEC, Tykerb, valrubicin, Valstar, vandetanib, VCR, Vectibix, Velban, Velcade, vemurafenib, Venclexta, venetoclax, Bepcid, Verzenio, Vesanoid, Viazul, Vidaza, vinblastine, vinblastine sulfate, Vincasar Pfs, vincristine, vincristine liposomal formulation, vinorelbine, vinorelbine tartrate, vismodegib, Vlb, VM-26, bornostat, Votrient, VP-16, Vumon, Vyxeos These include Xalkori capsules, Xeloda, Xigeva, Xofigo, Xitandi, Yervoy, Yescata, Yondelis, Zaltrap, Zanostar, Zalxio, Zejula, Zelboraf, Zevalin, Zinecard, Ziv-Aflibercept, Zoladex, zoledronic acid, Zolinza, Zometa, Zydelig, Zykadia, Zytiga, or any combination thereof.

[0090] Table 1 presents the biomarker / housekeeper sequence information. The amplicon position identified for each biomarker is underlined.

[0091] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0092] definition

[0093] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0094] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0095] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to a polymeric form of nucleotides (either ribonucleotides or deoxynucleotides) at least 10 bases or base pairs in length, or modified forms of either type of nucleotide, and are intended to encompass single- and double-stranded DNA. As used herein, nucleic acid molecules or nucleic acid sequences that function as probes in microarray analysis preferably comprise a chain of nucleotides, more preferably DNA and / or RNA. In alternative embodiments, nucleic acid molecules or nucleic acid sequences comprise other types of nucleic acid structures, such as DNA / RNA helices, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and / or ribozymes. Thus, the term "nucleic acid molecule" as used herein also encompasses chains comprising non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks that perform the same function as natural nucleotides.

[0096] The terms "hybridize," "hybridizing," "hybridizes," and the like, as used herein with respect to polynucleotides, refer to conventional hybridization conditions, preferably hybridization in 50% formamide / 6xSSC / 0.1% SDS / 100 μg / mL ssDNA, with a hybridization temperature above 37°C and a wash temperature above 55°C in 0.1xSSC / 0.1% SDS, most preferably stringent hybridization conditions.

[0097] As used herein, the term "normalization" or "normalizer" refers to the expression of a differential value relative to a reference value to adjust for effects resulting from technical variations in sample processing, sample preparation, and assay methodology, rather than biological variations in biomarker concentration in a sample. For example, when measuring the expression of a differentially expressed protein, the absolute value for the expression of that protein is expressed relative to the absolute value of expression of a standard protein whose expression is substantially constant.

[0098] The terms "diagnosis" and "diagnostic method" encompass the terms "prognosis" and "prognostic method," respectively, and also encompass the application of such procedures to diagnose across two or more time points and / or monitor prognosis over time, and statistical modeling. Furthermore, the term diagnosis encompasses (a) predictive prediction (determining whether a patient is likely to develop aggressive disease (hyperproliferative / invasive)), (b) prognosis (predicting whether a patient is likely to have a more benign or malignant outcome at a preselected time point in the future), (c) therapy selection, (d) therapeutic drug monitoring, and (e) relapse monitoring.

[0099] The term "provide" used herein in relation to biological sample refers to directly or indirectly obtaining a biological sample from a subject.For example, "provide" can refer to the act of directly obtaining a biological sample from a subject (for example, by blood sampling, tissue biopsy, irrigation, etc.).Similarly, "provide" can also refer to the act of indirectly obtaining a biological sample.For example, "provide" can refer to the act of a laboratory receiving a sample from the party who directly obtained the sample, or the act of obtaining a sample from a repository.

[0100] "Accuracy" refers to the degree to which a measured or calculated quantity (test-reported value) corresponds to its actual (i.e., true) value. Clinical accuracy is related to the ratio of true outcomes (true positives (TP) or true negatives (TN)) to misclassified outcomes (false positives (FP) or false negatives (FN)), and may be referred to as sensitivity, specificity, positive predictive value (PPV) or negative predictive value (NPV), or likelihood, odds ratio, among other measures.

[0101] As used herein, the term "biological sample" refers to any sample of biological origin that potentially contains one or more biomarkers. Examples of biological samples include tissues, organs, or bodily fluids, such as whole blood, plasma, serum, tissue, lavage fluid, or any other specimen used in the detection of disease.

[0102] As used herein, the term "subject" refers to a mammal, preferably a human.

[0103] As used herein, "treating" or "treatment" with respect to a condition can refer to preventing the condition, delaying the onset or rate of occurrence of the condition, reducing the risk of the condition occurring, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, or any combination thereof.

[0104] Biomarker levels can change due to disease treatment. Changes in biomarker levels can be measured by the present invention. Changes in biomarker levels can be used to monitor the progression of a disease or treatment.

[0105] The term "stable disease" refers to a diagnosis of the presence of a NET, but the NET has been treated and remains stable, i.e., is not progressing, as determined by imaging data and / or best clinical judgment.

[0106] The term "progressive disease" refers to a diagnosis of the presence of a highly active state of NETs, ​​i.e., untreated and not stable, treated but not responding to therapy, or treated but with persistently active disease, as determined by imaging data and / or best clinical judgment.

[0107] Use of an Agent or Compound The terms "effective amount" and "therapeutically effective amount" are used in the broadest sense to refer to a nontoxic but sufficient amount of an active agent or compound to provide a desired effect or benefit.

[0108] The term "benefit" is used in the broadest sense and refers to any desired effect, specifically including clinical benefit as defined herein. Clinical benefit can be measured by various endpoints, such as some degree of inhibition of disease progression, for example, slowing and complete cessation; a reduction in the number of disease histories and / or symptoms; a reduction in lesion size; inhibition (i.e., reduction, delay, or complete cessation) of pathological cell infiltration in nearby peripheral organs and / or tissues; inhibition (i.e., reduction, delay, or complete cessation) of disease progression; a reduction in autoimmune response, which may, but does not necessarily, result in regression or alleviation of lesions; some degree of alleviation of one or more symptoms associated with the disorder; an increase in the length of time that disease-free status remains after treatment, for example, an increase in progression-free survival; an increase in survival rate; a high response rate; and / or a decrease in mortality at a given time point after treatment.

[0109] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by upregulated cell growth. Included within this definition are benign and malignant cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include adrenocortical carcinoma, urothelial carcinoma of the bladder, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumor large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, chromophobe kidney carcinoma, clear cell renal carcinoma, papillary cell renal carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, uterine cancer, and uveal melanoma. Further examples include breast cancer, lung cancer, lymphoma, melanoma, liver cancer, colorectal cancer, ovarian cancer, bladder cancer, kidney cancer, and gastric cancer. Further examples of cancer include neuroendocrine cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, thyroid cancer, endometrial cancer, bile duct cancer, esophageal cancer, anal cancer, salivary gland cancer, vulvar cancer or cervical cancer.

[0110] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0111] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly dictates otherwise.

[0112] Unless otherwise specified or clear from context, the term "or" as used herein is to be interpreted as including both "or" and "and." [Example]

[0113] The present disclosure is illustrated by the following examples, which should not be construed as limiting the scope or spirit of the disclosure to the specific procedures described therein. It should further be understood that various alternative embodiments, modifications, and equivalents must be resorted to and may suggest themselves to those skilled in the art without departing from the spirit of the disclosure and / or the scope of the appended claims.

[0114] Example 1

[0115] Derivation of RRT predictive quotient (PPQ): an eight-marker gene panel combined with grade

[0116] The PRRT predictive predictive value (PPQ) includes expression of genes involved in growth factor expression / metabolism (Table 2) and histological grade (malignancy). It provides two biomarker outputs: "positive" (or predicted responder) and "negative" (or predicted non-responder). The model was developed from an initial cohort of 54 patients and then validated in four separate cohorts (n=214).

[0117] [Table 2]

[0118] A two-step protocol (RNA isolation, cDNA production, and PCR) was used to measure the expression of growth factor (GF)-related genes (ARAF1, BRAF, KRAS, and RAF-1), genes involved in metabolism (M) (ATP6V1H, OAZ2, PANK2, and PLD3), and, occasionally, genes involved in replication (P) (NAP1L1, NOL3, and TECPR2). Expression levels were normalized to ALG9. In some embodiments, a total GF+M value of ≥ 5.9 was scored as "1," and a value of < 5.9 was scored as "0." In some embodiments, a total GF+M+P value of ≥ 10.9 was scored as "1," and a value of < 10.9 was scored as "0." From histology, low grade (G1 / G2, well-differentiated, or bronchial typical or atypical carcinoid) was scored as "0"; high grade (G3, poorly differentiated) was scored as "1." Using a logistic regression classification method, these data were integrated into a predictive model by generating a score for each sample. The PPQ for a sample was derived as follows: PPQ = 39.22787 - 40.80341 × (total GF+M gene expression) - 18.441 × (grade) or PPQ = 39.22787 - 40.80341 × (total GF+M+P gene expression) - 18.441 × (grade)

[0119] From this model, two-phase forces can be derived.

[0120] (1) Responders refer to individuals predicted by PPQ values ​​as having stabilized disease or showing a partial response. They are scored as biomarker "positive" and exhibit a p-value <0.5.

[0121] (2) Non-responders were defined as individuals who exhibited progressive disease during the follow-up period (PRRT failure). They were considered biomarker "negative" and exhibited a p-value ≥ 0.5.

[0122] Five example outputs are given in Table 3.

[0123] Table 3 shows an example of output from the algorithm. [Table 3] *Normalized gene expression of ARAF1, BRAF, KRAS, and RAF-1; **Normalized gene expression of APT61VH, OAZ2, PANK2, and PLD3; ***Normalized gene expression of NAP1L1, NOL3, and TECPR2; & low-grade (G1 / G2, well-differentiated, or bronchial typical or atypical carcinoid); high-grade (G3, poorly differentiated); $ A value of >0.5 is classified as a non-responder; # R = responder (PPQ-positive); NR = non-responder (PPQ-negative).

[0124] This model has the following criteria: Chi-square (χ 2 )=41.6, DF=2, p<0.00001, Cox & Snell regression R 2 =0.537, Nagelkerke regression R 2 =0.722.

[0125] The accuracy of the classifier was 94% in the study population, which included 97% responders and 91% non-responders.

[0126] This cohort was expanded to 72 patients. The PPQ accurately predicted responders at the beginning (100%) and end (100%) of follow-up (Table 4). Non-responders were predicted in 65% (beginning) and 84% (end) (Fisher's test, p=NS). Overall, at the end of follow-up, 67 / 72 (93%) cases were correctly predicted. PRRT responders were predicted in 100% of cases, and non-responders in 84% (Table 4). Assessment of progression-free survival identified that mPFS was not achieved in PPQ-predicted responders. In predicted non-responders, mPFS was 8 months. This difference was significant (HR 36.4, p<0.0001) (Figure 1). The sensitivity of this study was 100%, and the NPV was 100%.

[0127] This model has the following metrics: Chi-square (χ2) = 41.6, DF = 2, p < 0.00001, Cox & Snell regression R 2 =0.722.

[0128] The accuracy of the classifier was 94% in the study population, which included 97% responders and 91% non-responders.

[0129] This cohort was expanded to 72 patients. The PPQ accurately predicted responders at the beginning (100%) and end (100%) of follow-up (Table 4). Non-responders were predicted in 65% (beginning) and 84% (end) (Fisher's test, p=NS). Overall, at the end of follow-up, 67 / 72 (93%) were correctly predicted. PRRT responders were predicted in 100% of cases, and non-responders in 84% (Table 4). Assessment of progression-free survival identified that mPFS was not confirmed in PPQ-predicted responders. In predicted non-responders, mPFS was 8 months. This difference was significant (HR 36.4, p<0.0001) (Figure 1). The sensitivity of this test was 100%, and the NPV was 100%.

[0130] Table 4. Predictive accuracy of PPQ in the PRRT-treated cohort [Table 4]

[0131] In Table 4, *Follow-up was approximately 6 to 9 months after the end of the last PRRT cycle; **Se = sensitivity, Sp = specificity, PPV = positive predictive value, NPV = negative predictive value.

[0132] Predicted response to PRRT

[0133] Validation I (n=44): The PPQ accurately predicted responders at follow-up with 97% accuracy. Non-responders were predicted with 93% accuracy (final). Overall, 42 / 44 (95%) were correctly predicted (Table 4). Survival assessment did not extend to mPFS in those predicted to respond. For "non-responders," mPFS was 14 months (HR 17.7, p<0.0001) (Figure 2). The sensitivity of this test was 97% and NPV 93%.

[0134] Validation II (n=42): The PPQ accurately predicted responders at follow-up with 94% accuracy. Non-responders were predicted with 100% accuracy. Overall, 40 / 42 (95%) were correctly predicted at final follow-up. PRRT responders were correctly predicted with 95% accuracy, and non-responders with 100% accuracy (Table 4). Survival assessment did not include mPFS in predicted responders. For "non-responders," mPFS was 9.7 months (HR 92, p<0.0001) (Figure 3). The sensitivity of this test was 94%, and the NPV was 95%.

[0135] Specificity of PPQ - predicting response to non-radioactive somatostatin

[0136] The PPQ was retrospectively determined in 28 patients treated with SSAs alone. At follow-up, 15 patients (54%) had stable disease and 13 patients (46%) developed progressive disease. The PPQ correctly predicted disease stabilization in 8 patients (53%) and progressive disease in 6 patients (47%, p = NS). Survival analysis identified no impact on PFS (Figure 4). Sensitivity and NPV were 53% and 46%, respectively. The PPQ did not predict response to SSAs.

[0137] Specificity of PPQ - its function as a predictive marker

[0138] PPQ was retrospectively determined in 100 patients included in the registry. Analysis was by intent-to-treat group. At follow-up, 48 patients (48%) had stable disease and 52 patients (52%) developed progressive disease. PPQ correctly predicted disease stabilization in 32 patients (67%) and progressive disease in 19 patients (37%, p = NS). Survival analysis identified no impact on PFS (Figure 5). Sensitivity and NPV were 67% and 50%, respectively. PPQ did not function as a predictive biomarker over the follow-up period.

[0139] Demonstration of the predictive utility of PRRT

[0140] To prove that a biomarker is predictive of treatment, studies should be conducted to assess biomarker levels in those expected to benefit from treatment as well as in those not treated with the agent. Because biomarkers have both predictive and prognostic features, the association between biomarkers and outcomes should be evaluated with and without treatment.

[0141] Comparison of Kaplan-Meier curves (PFS) between these cohorts is shown in Figure 6A (predicted "responders") and Figure 6B (predicted "non-responders"). A "treatment effect" was observed only in biomarker-positive, i.e., predicted "responders," individuals receiving PRRT treatment (Validation I and Validation II cohorts). Specifically, a quantitative difference (statistically significant, p<0.0001) was observed in median PFS (mPFS) between the PRRT-treated and non-PRRT-treated groups. This effect was observed regardless of whether they were all biomarker-positive. In contrast, no difference in PFS was observed in the biomarker-negative group. This effect was observed regardless of treatment. These data demonstrate that PPQ functions as a predictive biomarker.

[0142] The ideal biomarker metrics are included in Figures 6C-D. Treatment benefit was observed only in those who were treated and PPQ biomarker "positive" (Figure 6C - two validation cohorts). This particular case is important to emphasize that the ideal biomarker is not a predictor. This is underscored by the similar survival curves obtained for the biomarker-positive and -negative groups in its absence. Comparison of Figure 6A (biomarker-positive) confirms that the survival curves for the SSA-treated and registry cohorts (both not treated with PRRT - 10-month period) are not different from the survival curve for the PRRT-treated cohort in Figure 6B (biomarker-negative - 10-15-month survival), confirming that PPQ is not a predictor.

[0143] Evaluation of PPQ-negative patients

[0144] We further analyzed the clinical outcomes of patients identified by the PPQ as predicted non-responders to PRRT therapy. As shown in Figure 7, PPQ-predicted non-responders treated with four cycles of standard PRRT using Lutathera demonstrated a median PFS (mPFS) of 9 months. In contrast, PPQ-predicted non-responders treated with the same protocol and a personalized approach that added chemotherapy demonstrated a longer PFS of 14 months, as shown in Figure 7. These data indicate that PPQ-negative patients (predicted non-responders) and those who received additional therapy responded better than those who received standard therapy. Thus, the PPQ can be used to identify patients who should receive additional agents (e.g., immunotherapeutic or chemotherapeutic agents) in conjunction with PRRT, thereby optimizing outcomes.

[0145] References :

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[0179] While the present invention has been described in terms of the specific embodiments set forth above, various alternatives, modifications and other variations thereof will be apparent to those skilled in the art. Such alternatives, modifications and variations are intended to be within the spirit and scope of the invention. [1] 1. A method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a neuroendocrine tumor (NET), comprising: determining the expression levels of each of at least nine biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 were summed, thereby obtaining the total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histologic grade of the NET, wherein the second score is 1 when the NET is designated as high grade or the second score is 0 when the NET is designated as low grade; Calculate a third score based on the following equation: Third score = 39.22787 - 40.80341 × (First score) - 18.441 × (Second score); and providing a recommendation that the NET will respond to PRRT if the third score is less than a second predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the third score is greater than the second predetermined cutoff value. The method includes: [2] Item 10. The method of item 1, wherein the first predetermined cutoff value is 5.9. [3] 3. The method of item 1 or 2, wherein the second predetermined cutoff value is 0. [4] 4. The method of any one of items 1 to 3, having a sensitivity of greater than 90%. [5] 5. The method of any one of items 1 to 4, having a specificity greater than 90%. [6] 6. The method of any one of items 1 to 5, wherein at least one of the at least nine biomarkers is RNA, cDNA, or protein. [7] 7. The method according to item 6, wherein when the biomarker is RNA, the RNA is reverse transcribed to generate cDNA, and the expression level of the generated cDNA is detected. [8] 7. The method of claim 6, wherein the expression level of the biomarker is detected by forming a complex between the biomarker and a labeled probe or primer. [9] 7. The method according to item 6, wherein when the biomarker is a protein, the protein is detected by forming a complex between the protein and a labeled antibody.

[10] 7. The method according to item 6, wherein when the biomarker is RNA or cDNA, the RNA or cDNA is detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer.

[11] 11. The method of claim 10, wherein the complex of RNA or cDNA and labeled nucleic acid probe or primer is a hybridization complex.

[12] 12. The method of any one of items 1 to 11, wherein the test sample is blood, serum, plasma or tumor tissue.

[13] 13. The method of claim 12, wherein the test sample is blood.

[14] 13. The method of any one of items 1 to 12, wherein if the NET is poorly differentiated, the NET is designated as high grade.

[15] 15. The method of any one of items 1 to 14, wherein the NET is designated as low grade if the NET is well-differentiated, a bronchial typical carcinoid or a bronchial atypical carcinoid.

[16] 16. The method of any one of items 1 to 15, further comprising administering PRRT to the subject if the third score is less than a second predetermined cutoff value.

[17] 1. A method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a neuroendocrine tumor (NET), comprising: determining the expression level of each of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression level of at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3 and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3 and TECPR2; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 PLD3, NAP1L1, NOL3 and TECPR2 were summed, thereby obtaining the total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; Determine a second score based on the histological grade of the NET, where the second score is 1 if the NET is designated as high grade or the second score is 0 if the NET is designated as low grade; and calculate a third score based on the following equation: Third score = 39.22787 - 40.80341 × (First score) - 18.441 × (Second score); and providing a recommendation that the NET will respond to PRRT if the third score is less than a second predetermined cutoff value, or providing a recommendation that the NET will not respond to PRRT if the third score is greater than the second predetermined cutoff value. The method includes:

[18] Item 18. The method of item 17, wherein the first predetermined cutoff value is 10.9.

[19] 19. The method of item 17 or 18, wherein the second predetermined cutoff value is 0.

[20] 1. A method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a neuroendocrine tumor (NET), comprising: determining the expression level of each of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression level of at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3 and TECPR2 were summed, thereby obtaining the total expression level; providing a recommendation that the NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value; or If the total expression level is below a predetermined cutoff value, a recommendation is provided that the NET will not respond to PRRT. The method includes: [twenty one] 21. The method of item 20, wherein the predetermined cutoff value is 10.9. [twenty two] 1. A method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a low-grade or high-grade neuroendocrine tumor (NET), comprising: measuring the expression level of each of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression level of at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining a total expression level; and providing a recommendation that a low-grade or high-grade NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value; or If the total expression level is below a predetermined cutoff value, a recommendation is provided that the low-grade or high-grade NET will not respond to PRRT. The method includes: [twenty three] 23. The method of item 22, wherein the predetermined cutoff value is 10.9. [twenty four] 1. A method of providing peptide receptor radionuclide therapy (PRRT) treatment recommendations to a subject having a low-grade or high-grade neuroendocrine tumor (NET), comprising: determining the expression levels of each of at least nine biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3; summing the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3, thereby obtaining a total expression level; and providing a recommendation that a low-grade or high-grade NET will respond to PRRT if the total expression level is equal to or greater than a predetermined cutoff value; or If the total expression level is below a predetermined cutoff value, a recommendation is provided that the low-grade or high-grade NET will not respond to PRRT. The method includes: [twenty five] 25. The method of item 24, wherein the NET is designated as high grade if the NET is poorly differentiated.

[26] 25. The method of item 24, wherein the NET is designated as low grade if the NET is a well-differentiated bronchial typical carcinoid or bronchial atypical carcinoid.

[27] 27. The method according to any one of items 24 to 26, wherein the predetermined cutoff value is 5.9.

[28] 28. The method of any one of items 24 to 27, having a sensitivity of greater than 90%.

[29] 29. The method of any one of items 24 to 28, having a specificity greater than 90%.

[30] 30. The method of any one of items 24 to 29, wherein at least one of the at least nine biomarkers is RNA, cDNA, or protein.

[31] 31. The method of item 30, wherein when the biomarker is RNA, the RNA is reverse transcribed to generate cDNA, and the expression level of the generated cDNA is detected.

[32] 32. The method according to any one of items 24 to 31, wherein the expression level of the biomarker is detected by forming a complex between the biomarker and a labeled probe or primer.

[33] 31. The method of claim 30, wherein when the biomarker is a protein, the protein is detected by forming a complex between the protein and a labeled antibody.

[34] 31. The method of claim 30, wherein when the biomarker is RNA or cDNA, the RNA or cDNA is detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer.

[35] 35. The method of claim 34, wherein the complex of RNA or cDNA and labeled nucleic acid probe or primer is a hybridization complex.

[36] 36. The method of any one of items 24 to 35, wherein the test sample is blood, serum, plasma or tumor tissue.

[37] 37. The method of claim 36, wherein the test sample is blood.

[38] 38. The method of any one of items 24 to 37, further comprising administering PRRT to the subject if the total expression level is equal to or greater than a predetermined cutoff value.

[39] Provided is a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), determining the expression level of each of at least nine biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression level of each of the at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 were summed, thereby obtaining the total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; Determine a second score based on the histological grade of the NET, where the second score is 1 if the NET is designated as high grade or the second score is 0 if the NET is designated as low grade; and calculate a third score based on the following equation: Third score = 39.22787 - 40.80341 × (First score) - 18.441 × (Second score); and Subjects will undergo PRRT if the third score is equal to or greater than the predetermined cutoff value. The method includes:

[40] Provided is a method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), measuring the expression levels of each of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of the at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 PLD3, NAP1L1, NOL3 and TECPR2 were summed, thereby obtaining the total expression level; determining a first score, wherein the first score is 1 if the total expression level is greater than or equal to a first predetermined cutoff value, or the first score is 0 if the total expression level is less than a first predetermined cutoff value; determining a second score based on the histologic grade of the NET, wherein the second score is 1 if the NET is designated as high grade or the second score is 0 if the NET is designated as low grade; Calculate a third score based on the following equation: Third score = 39.22787 - 40.80341 × (First score) - 18.441 × (Second score); and Subjects will undergo PRRT if the third score is equal to or greater than the predetermined cutoff value. The method includes:

[41] 1. A method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a neuroendocrine tumor (NET), measuring the expression levels of each of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of the at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; The normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were summed, thereby obtaining the total expression level; and administering PRRT to the subject if the total expression level is equal to or greater than a predetermined cutoff value. The method includes:

[42] 1. A method of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), measuring expression levels of at least 12 biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of each of the at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; The expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 were normalized to the expression level of ALG9, thereby obtaining the normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; summing the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2, thereby obtaining the total expression level; and If the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT. The method includes:

[43] Provided are methods of treating a subject with peptide receptor radionuclide therapy (PRRT), wherein the subject has a low-grade or high-grade neuroendocrine tumor (NET), measuring the expression levels of each of at least nine biomarkers from a test sample from a subject by contacting the test sample with a plurality of agents specific for detecting the expression levels of the at least nine biomarkers, wherein the nine biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, and ALG9; The expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3 were normalized to ALG9, thereby obtaining the normalized expression levels of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2 and PLD3; The normalized expression levels of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, and PLD3 are summed to obtain a total expression level; and if the total expression level is equal to or greater than a predetermined cutoff value, the subject is administered PRRT. The method includes:

[44] PRRT is administered to the target 177 44. The method of any one of items 39 to 43, comprising administering Lu-based PRRT.

[45] The aforementioned 177 Lu-based PRRT 177 Lu-DOTA-Tyr 3 -Thr 8 - The method according to item 44, wherein the compound is octreotide.

[46] The aforementioned 177 Lu-DOTA-Tyr 3 -Thr 8 - The method of item 45, wherein the octreotide is administered at a dose of about 7.4 GBq (200 mCi) about once every 8 weeks for a total of about 4 doses.

[47] The aforementioned 177 Lu-DOTA-Tyr 3 -Thr 8 46. ​​The method of item 45, wherein octreotide is administered at a dose of about 6.5 GBq about once every 8 weeks for a total of about 4 doses.

[48] The aforementioned 177 Lu-DOTA-Tyr 3 -Thr 8 46. ​​The method of item 45, wherein octreotide is administered at a dose of about 4.6 GBq about once every 8 weeks for a total of about 4 doses.

[49] The aforementioned 177 Lu-DOTA-Tyr 3 -Thr 8 - The method of item 45, wherein the octreotide is administered at a dose of about 3.2 GBq (100 mCi) about once every 8 weeks for a total of about 4 doses.

[50] The aforementioned 177 Lu-DOTA-Tyr 3 -Thr 8 46. ​​The method of item 45, wherein the octreotide is administered at a dose of about 3.7 GBq about once every 8 weeks for a total of about 4 doses.

[51] The aforementioned 177 45. The method of item 44, wherein Lu-based PRRT is administered intravenously.

[52] The aforementioned 177 45. The method of item 44, wherein Lu-based PRRT is administered intra-arterially.

Claims

1. 1. A method for predicting the response of a subject having a neuroendocrine tumor (NET) to peptide receptor radionuclide therapy (PRRT) treatment, comprising: determining expression levels of at least 12 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific for detecting expression of at least 12 biomarkers, wherein the 12 biomarkers include ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, TECPR2, and ALG9; Normalizing the expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2 to the expression level of ALG9, thereby obtaining a normalized expression level of each of ARAF1, BRAF, KRAS, RAF-1, ATP6V1H, OAZ2, PANK2, PLD3, NAP1L1, NOL3, and TECPR2; determining a score based on the normalized expression level; Determine the histological grade of NETs To predict whether subjects with NETs will respond to PRRT based on the score and histological grade of the NETs The method comprising:

2. 10. The method of claim 1, wherein a NET is designated as low grade if it is a G1 or G2 NET and a NET is designated as high grade if it is a G3 NET.

3. 10. The method of claim 1, wherein at least one of the biomarkers is RNA, cDNA, or protein.

4. The method according to any one of claims 1 to 3, wherein when at least one of the biomarkers is RNA, the RNA is reverse transcribed to generate cDNA, and the expression level of the generated cDNA is detected.

5. The method of claim 3, wherein the expression level of the biomarker is detected by forming a complex between the biomarker and a labeled probe or primer.

6. 4. The method of claim 3, wherein when at least one of the biomarkers is a protein, the protein is detected by forming a complex between the protein and a labeled antibody.

7. 4. The method of claim 3, wherein when at least one of the biomarkers is RNA or cDNA, the RNA or cDNA is detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer.

8. The method of claim 7, wherein the complex of RNA or cDNA and labeled nucleic acid probe or primer is a hybridization complex.

9. The method of any one of claims 1 to 3, wherein the test sample is blood, serum, plasma, or tumor tissue.

10. The method of claim 9 , wherein the test sample is blood.

11. The PRRT 177 The method according to any one of claims 1 to 3, which is a Lu-based PRRT.

12. 177 Lu-based PRT 177 12. The method of claim 11, wherein the compound is Lu-DOTA-Tyr3-Thr8-octreotide.

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