Compositions and methods for treating cancer

Abstract

The present disclosure relates to compositions, systems, and methods for treating cancer. In particular, the present disclosure relates to compositions, systems, and methods for stratifying and treating adrenocortical carcinoma using gene expression and methylation profiles, as well as drugs that have utility in patients stratified by these means.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 859,933, filed June 11, 2019, and U.S. Provisional Application No. 62 / 882,147, filed August 2, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] [Technical field to which the disclosure pertains] The present disclosure relates to compositions, systems, and methods for treating cancer. In particular, the present disclosure relates to compositions, systems, and methods for stratifying and treating adrenocortical carcinoma using gene expression and methylation profiles, as well as drugs that have utility in patients stratified by these means.

[0003] BACKGROUND OF THE INVENTION Adrenocortical carcinoma (ACC) is a rare malignant tumor with an overall poor prognosis. Treatment options for ACC are limited, with surgery being the only treatment that can result in long-term remission and cure. Despite surgery, many patients with early-stage disease develop metastases after surgery, necessitating systemic treatment. For this reason, adjuvant therapy with the adrenaline-degrading compound mitotane after margin-free surgical resection is now part of the standard of care for most patients with ACC; however, current pharmacological treatment options are very limited, leaving a large unmet medical need for additional options. Recent studies have confirmed that mitotane is modestly effective while highly toxic. Therapeutic serum concentrations of mitotane typically require several months of drug administration to achieve, and up to 90% of patients inevitably relapse during adjuvant mitotane therapy after surgery or progress during mitotane therapy for unresectable disease (either during this dose-escalation period or thereafter). Furthermore, the efficacy of cytotoxic chemotherapy for unresectable disease is similarly limited, with significant side effects. As a result, there is a critical unmet medical need for new treatments that are safer, more effective, or both, than current options for patients.

[0004] Current approaches to patient stratification rely on histologic assessment of cell proliferation. The gold standard, KI67 index or mitotic count, has several limitations and cannot reliably identify patients who are likely to respond to a particular treatment. Improved methods for identifying patients who are likely to respond to a particular treatment are needed.

[0005] Summary of the Invention The present disclosure relates to compositions, systems, and methods for treating cancer. In particular, the present disclosure relates to compositions, systems, and methods for stratifying and treating adrenocortical carcinoma using gene expression and methylation profiles, as well as drugs that have utility in patients stratified by these means.

[0006] During the development of embodiments of the present invention, compositions and methods were developed to identify patient populations amenable to treatment with IGF1R inhibitors (e.g., linsitinib). In particular, Mohan & Lerario et al. (2019) observed that G0S2 methylation was prevalent in the primary tumors of 54% of patients with metastatic disease. High BUB1B-PINK1 expression predicts a slower disease course, even in patients with metastatic disease (Table 4). In conjunction with G0S2 methylation, BUB1B-PINK1 expression scores are additionally used, particularly to exclude patients with "low" BUB1B-PINK1 expression, with the goal of identifying non-obvious and previously unidentified subpopulations of patients who would typically be predicted not to respond to IGF1R inhibitor treatment. To clarify this unique population and further define this subpopulation of patients with unusual responses, given the 25% survival split observed in the placebo / treatment survival curves of a previous linsitinib clinical trial (Figure 9 and Example 2), a BUB1B-PINK1 cutoff at the 44th percentile for non-CIMP-elevated patients with a history of metastatic disease was used. This resulted in the inclusion of 56% of patients with non-CIMP-elevated metastatic disease. After applying these collective steps, a subpopulation was obtained with (0.56)(1-0.54)=25.8% of all patients with metastatic disease, reflecting the patient population at the 25% survival rate identified in Figure 9. A valuable feature of this cutoff is that it optimizes the identification of patients with chromosomal tumors that harbor recurrent alterations leading to IGF2 upregulation and are unlikely to harbor other recurrent somatic alterations from ACC-TCGA (COC1; Figures 1, 2, 3), thereby aligning with the biological motivation for the use of the targeted therapies described herein.

[0007] Thus, the compositions and methods described herein provide improved patient care by customizing ACC treatment to specific subtypes of ACC and improving the efficacy of IGF1R inhibitor treatment by providing such treatment only to patients identified as likely to respond.

[0008] For example, in some embodiments, provided herein are methods for treating adrenocortical carcinoma (ACC), comprising administering an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor such as linsitinib) to a subject identified as having cluster 1 (COC1) ACC. In some embodiments, the subject is identified as having COC1 ACC by measuring G0S2 methylation levels and BUB1B and PINK1 expression levels. For example, in some embodiments, a G0S2 methylation level below a threshold level (e.g., less than 4.696% methylation, as determined by methylation-sensitive restriction digestion / amplification) and a BUB1B-PINK1 expression score above a threshold level cutoff for BUB1B-PINK1 indicate COC1 ACC. In some embodiments, the threshold level of G0S2 methylation is determined using unsupervised full hierarchical clustering using Euclidean distance on logit-transformed methylation β values. In some embodiments, linsitinib is administered as monotherapy.

[0009] The present disclosure is not limited to a specific threshold level for the BUB1B-PINK1 expression score. In some embodiments, the threshold level is the 44th percentile (e.g., ±1, 2, 3, 4, 5, 6, 7, 8, 9, or 10th percentile) of ACC samples with a history of metastatic disease and a G0S2 methylation level below the threshold level (e.g., less than 4.696% methylation, as determined by methylation-sensitive restriction). In some specific embodiments, the BUB1B-PINK1 expression threshold is, for example, 1.4, 1.5, 1.6, 1.7, or greater, as determined, for example, by subtracting the copy thresholds of BUB1B and PINK1 in samples assessed using quantitative polymerase chain reaction. In some embodiments, subjects with a G0S2 methylation level above the threshold level are excluded from treatment with an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor). In some embodiments, subjects with G0S2 methylation below a threshold level and a BUB1B-PINK1 expression score below a threshold level are excluded from treatment with an agent that blocks IGF1R signaling (eg, an IGF1R inhibitor).

[0010] A further embodiment is a method for treating ACC in a subject, comprising: a) obtaining or having obtained a sample from the subject and identifying the subject as having COC1 ACC by measuring G0S2 methylation levels and BUB1b and PINK1 expression levels in the sample; b) if the subject has a G0S2 methylation level and a BUB1B-PINK1 expression score indicative of the presence of COC1 ACC, administering to the subject an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor such as linsitinib); The present invention provides a method comprising:

[0011] Another embodiment is a method for treating ACC in a subject, comprising: a) determining the G0S2 methylation level and the expression levels of BUB1B and PINK1 in a sample from the subject; b) identifying subjects with a G0S2 methylation level below a threshold level and a BUB1B-PINK1 expression score above a threshold level in the sample as having COC1 ACC; c) administering to subjects identified as having COC1 ACC an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor); The present invention provides a method comprising:

[0012] Also provided herein is the use of an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) to treat ACC in a subject identified as having COC1 ACC, or an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) for use in treating ACC in a subject identified as having COC1 ACC.

[0013] Certain embodiments include a method for treating ACC, comprising: The method includes administering an agent that blocks IGF1R signaling to a subject identified as having a G0S2 methylation level below a threshold level (e.g., 4.696%) and a BUB1B-PINK1 expression score above a threshold level in a sample isolated from the subject.

[0014] In some embodiments, there is provided a method for treating ACC in a subject, comprising: a) obtaining or having obtained a sample from the subject and determining the level of G0S2 methylation and the level of BUB1B-PINK1 expression score in the sample, thereby identifying the subject as having a G0S2 methylation level below a threshold level and a BUB1B-PINK1 expression score above a threshold level; b) administering to the subject an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) if the subject has a G0S2 methylation level below a threshold level (e.g., 4.696%) and a BUB1B-PINK1 expression score above a threshold level. The present invention provides a method comprising:

[0015] Yet another embodiment is a method for treating ACC in a subject, comprising: a) determining the G0S2 methylation level and the expression levels of BUB1B and PINK1 in a sample from the subject; b) identifying subjects with a G0S2 methylation level below a threshold level (e.g., 4.696%) in the sample and a BUB1B-PINK1 expression score above the threshold level; c) administering an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) to subjects identified as having a G0S2 methylation level below a threshold level (e.g., 4.696%) and a BUB1B-PINK1 expression score above a threshold level; The present invention provides a method comprising:

[0016] In further embodiments, there is provided the use of an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) to treat ACC in a subject identified as having a G0S2 methylation level below a threshold level and a BUB1B-PINK1 expression score above a threshold level, or an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) for use in treating ACC in a subject identified as having a G0S2 methylation level below a threshold level (e.g., 4.696%) and a BUB1B-PINK1 expression score above a threshold level.

[0017] Also provided is a method for treating adrenocortical carcinoma (ACC), comprising: a) contacting a sample from a subject diagnosed with ACC with a reagent for determining at least one of the expression level of BUB1b, PINK1, and the expression level and methylation status of G0S2; b) characterizing ACC as molecular subgroup 1 (COC1), cluster 2 (COC2), or cluster 3 (COC3) based on the BUB1B-PINK1 expression score and the level of G0S2 expression or methylation; c) administering to the subject at least one treatment customized for the molecule, e.g. administering to a subject confirmed to have COC3 cancer one or more of an inhibitor of a cell cycle effector protein, an inhibitor of a DNA repair protein, a Wnt inhibitor, an NR5A1 inhibitor, or an inhibitor of an epigenetic writer; administering one or more of a Wnt inhibitor or an NR5A1 inhibitor to a subject confirmed to have COC2 cancer; or administering one or more agents that block IGF1R signaling (e.g., IGF1R inhibitors) to a subject identified as having COC1 cancer; In some embodiments, the method further comprises: The method includes administering to a subject identified as having COC2 or COC3 cancer an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and / or an immune checkpoint inhibitor (in combination with an NR5A1 inhibitor, a glucocorticoid synthesis / metabolism inhibitor, or a glucocorticoid receptor inhibitor). In some embodiments, the method further includes administering to the subject identified as having COC1 an immune checkpoint inhibitor.

[0018] In some embodiments, characterization includes determining a BUB1B-PINK1 expression score. The present disclosure is not limited to a particular cutoff value or threshold for characterizing ACC. For example, in some embodiments, a BUB1B-PINK1 expression score above a threshold level and a G0S2 methylation level below a threshold level (e.g., 4.696%) indicates COC1. In some embodiments, a BUB1B-PINK1 expression score below a threshold level and a G0S2 methylation level below a threshold level (e.g., 4.696%) indicates COC2. In some embodiments, the presence of G0S2 methylation above 4.696% (e.g., above 4.696%, 4.7%, 4.8%, 4.9%, or 5.0%) indicates COC3. In some embodiments, the BUB1B-PINK1 expression score threshold level is 1.4 to 1.8 (e.g., 1.4, 1.5, 1.6, 1.7, or 1.8). In some embodiments, the BUB1B-PINK1 expression score and G0S2 methylation value shown in Table 1 are utilized to characterize ACC. In some embodiments, the biological sample is a tissue sample, a biopsy sample, a blood sample, or a urine sample. In some embodiments, the reagent is one or more nucleic acid probes that hybridize to at least one of BUB1B, PINK1, and G0S2, one or more nucleic acid primers for amplification or extension of at least one of BUB1B, PINK1, and G0S2, or one or more nucleic acid primers that specifically bind to methylated G0S2 nucleic acids. In some embodiments, an amplification assay (e.g., real-time PCR) is used to measure the expression of BUB1B, PINK, and G0S2.

[0019] The present disclosure is not limited to a specific target gene or treatment. In some embodiments, the therapeutic agent is an antibody, a nucleic acid, or a small molecule. For example, in some embodiments, the cell cycle effector protein is CDK4 / 6, PLK1, MELK, or AURK, and the inhibitor is palbociclib. In some embodiments, the DNA repair protein is WEE1 or PARP, and the inhibitor is olaparib or adavosertib. In some embodiments, the epigenetic driver is EZH2 and / or DNMT1, and the inhibitor is 3-deazaneplanocin A, EPZ005687, EPZ-6438 (tazemetostat), decitabine, or 5-azacytidine. In some embodiments, the IGF1R inhibitor is lincitinib, cixutumumab, ganitumab, figitumumab, dalotuzumab, istiratumab, dusigitusumab, or teprotumumab. In some embodiments, the Wnt inhibitor is WNT974 or PRI-724. In some embodiments, the immune checkpoint inhibitor is ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, or cemiplimab. In some embodiments, the NR5A1 inhibitor is SID 7969543, 45594 [4-(heptyloxy)phenol], or octyloxyphenyl (OOP).

[0020] A further embodiment is a method for characterizing adrenocortical carcinoma (ACC), comprising: a) contacting a sample from a subject diagnosed with ACC with a reagent for determining the expression level of at least one of BUB1b, PINK1, and G0S2, and the methylation status of G0S2; b) characterizing ACC as molecular subgroup COC1 if a BUB1B-PINK1 expression score above a threshold level and G0S2 methylation below a threshold level (e.g., 4.696%) are identified; If a BUB1B-PINK1 expression score below a threshold level and G0S2 methylation below a threshold level (e.g., 4.696%) were confirmed, the ACC was characterized as COC2; characterizing the ACC as COC3 if the presence of G0S2 methylation above a threshold level (e.g., 4.696%) is confirmed; The present invention provides a method comprising:

[0021] A further embodiment is Use of at least one treatment selected from an inhibitor of a cell cycle effector protein, an inhibitor of a DNA repair protein, a Wnt inhibitor, an NR5A1 inhibitor, or an epigenetic writer inhibitor to treat ACC in a subject confirmed to have COC3 cancer; The use of at least one treatment selected from a Wnt inhibitor and an NR5A1 inhibitor to treat ACC in a subject confirmed to have COC2 cancer; or The present invention provides the use of at least one treatment selected from an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and a checkpoint inhibitor to treat ACC in a subject confirmed to have COC1 cancer.

[0022] Still other embodiments include at least one treatment selected from an inhibitor of a cell cycle effector protein, an inhibitor of a DNA repair protein, a Wnt inhibitor, a Wnt inhibitor, an NR5A1 inhibitor, or an epigenetic writer inhibitor for use in treating ACC in a subject confirmed to have COC3 cancer; At least one treatment selected from a Wnt inhibitor or an NR5A1 inhibitor for use in treating ACC in a subject confirmed to have COC2 cancer; or Agents that block IGF1R signaling (eg, IGF1R inhibitors) are provided for use in treating ACC in a subject identified as having COC1 cancer.

[0023] Certain embodiments provide a method for screening for a treatment of adrenocortical carcinoma (ACC), comprising: a) contacting a sample from a subject diagnosed with ACC with a reagent for determining the expression level of at least one of BUB1b, PINK1, and G0S2 and the methylation status of G0S2; b) characterizing ACC as molecular subgroups COC1, COC2, or COC3 based on the expression levels of BUB1b, PINK1, and G0S2 and the methylation status of G0S2; c) administering at least one treatment selected from an inhibitor of a cell cycle effector protein, an inhibitor of a DNA repair protein, a Wnt inhibitor, an NR5A1 inhibitor, or an epigenetic writer inhibitor to a subject confirmed to have COC3 cancer; administering at least one treatment selected from a Wnt inhibitor or an NR5A1 inhibitor to a subject confirmed to have COC2 cancer; or administering to a subject identified as having COC1 cancer at least one treatment selected from an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and / or a checkpoint inhibitor; d) assessing the effect of treatment on one or more signs or symptoms of ACC; and The present invention provides a method comprising:

[0024] Certain embodiments provide a method for selecting a treatment for adrenocortical carcinoma (ACC), comprising: a) contacting a sample from a subject diagnosed with ACC with a reagent for determining the expression level of at least one of BUB1b, PINK1, and G0S2 and the methylation status of G0S2; b) characterizing ACC as molecular subgroups COC1, COC2, or COC3 based on the expression levels of BUB1b, PINK1, and G0S2 and the methylation status of G0S2; c) for a subject confirmed to have COC3 cancer, selecting at least one treatment selected from an inhibitor of a cell cycle effector protein, an inhibitor of a DNA repair protein, a Wnt inhibitor, an NR5A1 inhibitor, or an epigenetic light receptor inhibitor; selecting at least one treatment selected from a Wnt inhibitor and an NR5A1 inhibitor for a subject confirmed to have COC2 cancer; selecting at least one treatment selected from an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and an immune checkpoint inhibitor for a subject confirmed to have COC1 cancer; The present invention provides a method comprising:

[0025] Additional embodiments will be apparent to those skilled in the relevant art(s) based on the teachings contained herein.

[0026] DESCRIPTION OF THE DRAWINGS Figure 1 shows that ACC-TCGA identifies three distinct multi-platform molecular subtypes of ACC (COC1-COC3) that can be refined down to single platform features. A. Specific types of somatic copy number alteration profiles (SCNAs), CpG island methylator phenotypes (CIMPs), and mRNA subtypes converge in each COC. B. Heatmap showing SCNA, CIMP and mRNA classification for each sample with COC assignment in ACC-TCGA. C. Dominant SCNA, CIMP, and mRNA groups for each COC.

[0027] Figure 2 shows that COC groups from ACC-TCGA are associated with distinct somatic alteration profiles and activation of transcriptional programs. A. COC3 tumors have a high frequency of driver somatic alterations that result in constitutive cell cycle activation (“MUT”) and high expression of cell cycle genes as measured by cell cycle score. B. COC2-3 tumors have a high frequency of driver somatic alterations that result in constitutive Wnt pathway activation (“MUT”) and high expression of Wnt pathway targets as measured by Wnt Score. C. Left - COC3 tumors frequently harbor an abnormal epigenetic landscape characterized by non-physiological DNA methylation directed at CpG islands and high expression of epigenetic enzymes as measured by "CIMP high," an epigenetics score. Intermediate- COC2+3 tumors have a predominance of "steroid-high" and "steroid-high / proliferative" transcriptional programs and high expression of steroidogenic enzymes as measured by the "steroid score." Right - ACC-TCGA confirmed that bulk ACC is mostly immune-deficient compared to other cancers, but COC1 has a high degree of immune infiltration as measured by the "immune score."

[0028] FIG. 3 shows the heat maps of FIGS. 1 and 2 for each sample. A. Gene expression levels from ACC-TCGA RNA-seq data are color-coded by row z-score (white to black). B. This heatmap depicts the same information as A, except in this case, genes are collapsed into corresponding epigenetic ("EPIG"), cell cycle ("CELLCYCLE"), steroid ("Steroid"), Wnt ("Wnt"), and immune ("Immune") scores, and the scores are color-coded by their actual value.

[0029] Figure 4 shows that the BUB1B-PINK1 score distinguishes patients with COC1 disease from those with COC2 and COC3 tumors in the ACC-TCGA.

[0030] FIG. 5 shows that in ACC-TCGA, COC1 patients with non-CIMP high progressive disease had statistically different BUB1B-PINK1 than COC2 patients with non-CIMP high progressive disease.

[0031] Figure 6. BUB1B‐PINK1 threshold allows reasonable discrimination between COC1 and COC2‐3 tumors in patients with non‐CIMP‐high advanced disease.

[0032] Figure 7. Cumulative distribution function of BUB1B-PINK1 scores in ACC-TCGA allows validation of comparable BUB1B-PINK1 cutoffs by qPCR in the FMUSP+UM population.

[0033] Figure 8. Combined BUB1B-PINK1 and CIMP status faithfully recapitulates COC in patients with advanced disease.

[0034] Figure 9. Progression-free survival curves for patients enrolled in the Fassnacht et al. Lancet Oncology 2015 study.

[0035] Figure 10. Breakpoint analysis of exome sequencing data from responders and non-responders treated with linsitinib. Each point in this figure represents the total number of breakpoints identified in sequenced tumors from patients categorized by "responder" or "non-responder" status.

[0036] Figure 11. Noisy ACC patients progress on linsitinib. Top left: B-allele frequency profile for each autosome (column) of each patient (row) classified as "responder" or "non-responder." Bottom left: zoom of chromosome 1 for each patient. Right: chromosome 1 from patient samples representing each SCNA class from ACC-TCGA.

[0037] Figure 12. Lethal CIMP-high tumors from ACC-TCGA often have noisy copy number / LOH profiles. A, adapted from Mohan & Lerario et al. Clinical Cancer Research 2019, shows that CIMP-high tumors in ACC-TCGA are routinely lethal. B, shows that CIMP-high tumors from ACC-TCGA are significantly enriched for noisy copy number / LOH profiles, while non-CIMP-high tumors from ACC-TCGA are significantly enriched for chromosome copy number / LOH profiles, consistent with COC3.

[0038] [Definition] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0039] The term "sensitivity" as used herein is defined as a statistical measure of the performance of an assay (e.g., method, test) calculated by dividing the number of true positives by the sum of the true positives and false negatives.

[0040] The term "specificity" as used herein is defined as a statistical measure of the performance of an assay (e.g., method, test) calculated by dividing the number of true negatives by the sum of the true negatives and false positives.

[0041] As used herein, the term "informative" or "informativeness" refers to the quality of a marker or panel of markers, and in particular the likelihood of finding a marker (or panel of markers) in a positive sample.

[0042] As used herein, the term "metastasis" refers to the process by which cancer cells originating from one organ or part of the body are relocated to another part of the body and continue to replicate. The metastatic cells may subsequently form tumors and further metastasize. Thus, metastasis refers to the spread of cancer from the site where it first developed to other parts of the body. As used herein, the term "metastatic ACC cancer cells" refers to metastatic ACC cancer cells.

[0043] The term "neoplasm" as used herein refers to any new and abnormal growth of tissue. Therefore, neoplasms can be non-malignant neoplasms, pre-malignant neoplasms, or malignant neoplasms. The term "neoplasm-specific marker" refers to any biological substance that can be used to indicate the presence of neoplasms. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells.

[0044] As used herein, the term "amplicon" refers to a nucleic acid generated using a primer pair. An amplicon is typically single-stranded DNA (e.g., the result of asymmetric amplification), but may also be RNA or dsDNA.

[0045] The term "amplify" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide or portion of a polynucleotide, where an amplification product or amplicon is generally detectable and typically starts from a small amount of the polynucleotide (e.g., a single polynucleotide molecule). Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The production of multiple DNA copies from one or a few copies of a target or template DNA molecule during polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810; incorporated herein by reference in its entirety) is a form of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611, which is incorporated herein by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408, which is incorporated herein by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, which is incorporated herein by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671, which are incorporated herein by reference in their entirety), inter-sequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, which is incorporated herein by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, Ret al., Nucleic Acids Res., 16:8186, which is incorporated herein by reference in its entirety), and ligation-mediated PCR (see, e.g., Guilfoyle, Ret al., Nucleic Acids Res., 16:8186, which is incorporated herein by reference in its entirety). 25:1854-1858 (1997); see U.S. Pat. No. 5,508,169), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826, which is incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Res., 30(12); e57), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Res., 16(23) 1141-1156; Ballabio, et al., (1990) Hayden, BMC Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80, which are incorporated herein by reference in their entireties), nested PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Res., 16(15) 7351-7367, which are incorporated herein by reference in their entireties), real-time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030;), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193, which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485, which are incorporated herein by reference in their entireties). Polynucleotide amplification can also be achieved using digital PCR (see, e.g., Kalinina et al., Nucleic Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA; 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525, which are incorporated by reference in their entireties).

[0046] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing rules. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'." Complementarity can be "partial," in which only some of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0047] As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring, as in a purified restriction digest, or synthetically produced, that can act as a point of initiation of synthesis under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (e.g., when placed in the presence of nucleotides and an inducing agent, such as a biocatalyst (e.g., DNA polymerase), and at a suitable temperature and pH). Primers are typically single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer is sufficiently long to initiate synthesis of an extension product in the presence of an inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer, and use of the method. In certain embodiments, the primer is a capture primer.

[0048] As used herein, the term "nucleic acid molecule" refers to any nucleic acid-containing molecule, including, but not limited to, DNA or RNA. This term includes, but is not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl and 2,6-diaminopurine.

[0049] As used herein, the term "nucleobase" is synonymous with other terms used in the art, including "nucleotide," "deoxynucleotide," "nucleotide residue," "deoxynucleotide residue," "nucleotide triphosphate (NTP)," or deoxynucleotide triphosphate (dNTP).

[0050] An "oligonucleotide" refers to a nucleic acid containing at least two nucleic acid monomer units (e.g., nucleotides), typically more than three monomer units, and more typically more than ten monomer units. The exact size of an oligonucleotide generally depends on various factors, including the ultimate function or use of the oligonucleotide. To further illustrate, oligonucleotides are typically less than 200 residues in length (e.g., 15-100), although as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is referred to as a "24-mer." Typically, the nucleoside monomers are linked by phosphodiester bonds or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, e.g., H + , NH4 + , Na +and the like, if such a counterion is present. Furthermore, oligonucleotides are typically single-stranded. Oligonucleotides may optionally be prepared by isolation of existing or naturally occurring sequences, DNA replication or amplification, reverse transcription, cloning of appropriate sequences and restriction digestion, or automated synthesis using methods such as the phosphotriester method of Narang et al. (1979) Meth Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862; the triester method of Matteucci et al. (1981) J Am Chem Soc. 103:3185-3191; automated synthesis; or the method described in "PROCESS FOR PREPARING SYNTHESIS" by Caruthers et al., published July 3, 1984. No. 4,458,066, entitled "Polynucleotides and Their Synthesis," or other methods known to those skilled in the art. All of these references are incorporated by reference.

[0051] The "sequence" of a biopolymer refers to the order and identity of the monomeric units (e.g., nucleotides) in the biopolymer. The sequence of a nucleic acid (e.g., base sequence) is typically read in the 5' to 3' direction.

[0052] As used herein, "methylation" refers to cytosine methylation at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is unmethylated because in vitro DNA amplification methods do not retain the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template is unmethylated or methylated, respectively.

[0053] "Methylation state" refers to the presence, absence, and / or amount of methylation at a particular nucleotide or nucleotides within a portion of DNA. The methylation state of a particular DNA sequence (e.g., a genetic marker or DNA region described herein) can indicate the methylation state of every base in the sequence, or it can indicate the methylation state of a subset of base pairs (e.g., one or more cytosines) in the sequence, or it can indicate information about the local methylation concentration within the sequence without providing precise information about where methylation occurs within the sequence. The methylation state can optionally be expressed or indicated by a "methylation value." Methylation values ​​can be generated, for example, by quantifying the amount of intact DNA present after restriction digestion with a methylation-dependent restriction enzyme, by comparing amplification profiles after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated DNA. Thus, for example, the methylation value represents the methylation state and can therefore be used as a quantitative indicator of the methylation state across multiple copies of a gene locus. This is particularly useful when it is desirable to compare the methylation state of sequences in a sample to a threshold or reference value.

[0054] As used herein, the term "subject" refers to any animal (e.g., mammal) that is the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0055] As used herein, the term "non-human animal" refers to all non-human animals, including, but not limited to, vertebrates such as rodents, non-human primates, ovine, bovine, ruminant, lagomorph, porcine, caprine, equine, canine, feline, and goat.

[0056] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including, but not limited to, mRNA, tRNA, and rRNA), or precursor. The polypeptide, RNA, or precursor can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment is retained. The term also encompasses the coding region of a structural gene and the encompassing sequences located adjacent to the coding region, at both the 5' and 3' ends, approximately 1 kb from either end, so that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" includes both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript; therefore, introns are absent in the messenger RNA (mRNA) processed transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0057] As used herein, the term "locus" refers to a nucleic acid sequence on a chromosome or on a linkage map, and includes coding sequences and 5' and 3' sequences involved in the regulation of a gene.

[0058] Detailed Description of the Invention The present disclosure relates to compositions, systems, and methods for treating cancer. In particular, the present disclosure relates to compositions, systems, and methods for stratifying and treating adrenocortical carcinoma using gene expression and methylation profiles, as well as drugs that have utility in patients stratified by these means.

[0059] Recent comprehensive genomics studies, such as the Cancer Genome Atlas Study on ACC (ACC-TCGA), have demonstrated that ACC may be better stratified using molecular stratification rather than proliferation measurements (KI67 or mitotic counts). ACC-TCGA demonstrated that ACC is a molecularly heterogeneous disease, primarily composed of three distinct molecular subtypes: COC1, COC2, and COC3 (Zheng et al., Cancer Cell 2016). Notably, these molecular subtypes are characterized by distinct patterns of somatic alterations, activation of unique transcriptional programs, and marked alterations in epigenetic patterning. Importantly, COC1-3 status predicts disease course under standard treatment, with patients with COC1 disease having a predominantly favorable prognosis, patients with COC2 disease having an intermediate prognosis, and patients with COC3 disease having a uniformly poor prognosis.

[0060] Thus, while stratification of ACC based on molecular subtyping seems promising, strategies for incorporating molecular classes into clinically feasible tests remain elusive. An ideal molecular biomarker would be rapidly measurable by a simple assay and reliably capture distinct molecular classes. The ACC-TCGA revealed that COC3 ACC is characterized by a genome-wide pattern of DNA methylation targeting a CpG island known as "CIMP-high"; this data indicates that DNA methylation-based molecular biomarkers enable reliable confirmation of CIMP-high / COC3 tumors. Indeed, it was recently demonstrated that accurate differentiation between CIMP-high and non-CIMP-high ACC can be achieved using a simple overnight restriction digestion / qPCR-based molecular assay to quantify CpG island methylation at the G0S2 locus (Mohan, Lerario, et al., Clinical Cancer Research 2019). Furthermore, the inclusion of a second qPCR-based assay to measure BUB1B and PINK1 mRNA expression and calculate a score resulting from subtraction of BUB1B-PINK1 Ct values ​​demonstrated a further stratification of ACC into "high-risk," "intermediate-risk," and "low-risk" prognostic groups based on the likelihood of disease progression after surgery and the development of metastatic disease at any time. Thus, "high-risk" patients almost exclusively relapse after complete surgical resection and exhibit rapid progression of metastatic disease, while "low-risk" patients never relapse or develop metastatic disease. Thus, these categories provide a molecular risk stratification approach based on these molecular biomarkers for the clinical management of ACC patients.

[0061] The above-mentioned molecular stratification strategy for prognosing ACC is described in co-pending patent application WO 2019 / 108568 (the entirety of which is incorporated herein by reference). However, such classification methods do not provide customized treatment methods based on molecular classification. Therefore, provided herein is a method for using COC1-3 classification to determine treatment course actions, screen candidate therapeutic agents for efficacy, and treat ACC based on its associated molecular marker ACC classification.

[0062] One of such classes of drugs is the agent that blocks IGF1R signal transduction.As used herein, the term "agent that blocks IGF1R signal transduction" refers to any agent that blocks the signal transduction by or through IGF1R.For example, it includes, but is not limited to, the inhibitor or enhancer of the upstream or downstream signal transduction partner, modulator, or ligand of IGF1R.For example, in some embodiments, this agent reduces the level, availability, or access of ligand (e.g., IGF2, IGF1) to IGF1R receptor, binds (e.g., extracellularly or intracellularly), and antagonizes IGF1R (e.g., antibody or small molecule kinase inhibitor) and / or downstream IGF1R signal transduction pathway.

[0063] In some embodiments, the drug is an IGF1R inhibitor.Linsitinib is an IGF1R inhibitor and has been studied in patients with ACC, but has not reached the primary endpoint of the GALACCTIC Phase 3 clinical trial and has been discontinued.However, in a small group of patients, meaningful and sustained responses have been observed, indicating drug activity, while drug toxicity has been limited and manageable.Unfortunately, before the present disclosure, there was no method to identify this subgroup of patients who respond to IGF1R inhibitors such as linsitinib, and therefore, patients who will unexpectedly benefit from treatment with IGF1R inhibitors such as linsitinib.

[0064] In particular, the first phase 3 trial evaluating the efficacy of linsitinib (also known as OSI-906) in patients with advanced adrenocortical carcinoma (ACC) was published by Fassnacht et al. in Lancet Oncology 2015. They reported no statistically significant differences in Kaplan-Meier progression-free survival curves between linsitinib-treated and placebo-treated patients. However, upon review of this data, they observed that long-term durable responses (progression-free survival >150 days) were observed in a small subset of patients treated with linsitinib but not in those treated with placebo.

[0065] During development of embodiments of the present invention, detailed analysis of data from this clinical trial, including molecular analysis (described in detail in Example 2), unexpectedly identified a unique patient population predicted to respond to treatment with an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor such as linsitinib), while excluding patients unlikely to respond to such agents. As described in Example 2, molecular analysis identified responders in the trial as having molecular markers indicative of COC1 ACC. This provides a specific patient population for treatment with an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor such as linsitinib). Furthermore, if this treatment had been employed in a clinical trial in which patients were first tested to assess whether they had COC1 ACC and were enrolled only if they did, this failed clinical trial would conversely have demonstrated clinically meaningful efficacy of linsitinib compared to placebo.

[0066] For example, in some embodiments, a subject with COC1 ACC is administered an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor). In some embodiments, as described in more detail below, a subject with G0S2 methylation below a threshold level is administered an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor). In some embodiments, a subject with G0S2 methylation and a BUB1B-PINK1 score above a threshold limit is administered an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor). Furthermore, in some embodiments, a subject lacking molecular markers indicative of a response to an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) is provided with an alternative treatment.

[0067] Thus, the present invention provides compositions and methods for treating specific subsets of ACC patients with significantly improved efficacy and demonstrated safety (see, e.g., Example 3) compared to the current state of the art, which will result in improved patient care and provide new treatment options for this difficult-to-treat disease.

[0068] The following description identifies patients for treatment with particular therapies and describes compositions and methods for providing such therapies.

[0069] I. Identifying ACC Classification As described herein, in some embodiments, compositions and methods are provided for classifying ACC based on the expression or level of one or more ACC markers (e.g., for BUB1B, PINK1, or G0S2) and the methylation status of G0S2. In some embodiments, expression and / or methylation scores are used to characterize ACC. In some embodiments, characterization includes determining a BUB1B-PINK1 expression score.

[0070] Exemplary detection and scoring methods are described below.

[0071] A. Detection Assays In some embodiments, RNA is detected by Northern blot analysis, which involves the separation of RNA and hybridization of a complementary labeled probe.

[0072] In some embodiments, RNA (or corresponding cDNA) is detected by hybridization to an oligonucleotide probe. A variety of hybridization assays using different techniques for hybridization and detection are available. For example, in some embodiments, the TaqMan assay (PE Biosystems, Foster City, CA; see, e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each incorporated herein by reference) is utilized. This assay is performed during a PCR reaction. The TaqMan assay utilizes the 5'-3' exonuclease activity of AMPLITAQ GOLD DNA polymerase. The PCR reaction includes a probe consisting of an oligonucleotide bearing a 5'-reporter dye (e.g., a fluorescent dye) and a 3'-quencher dye. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of AMPLITAQ GOLD polymerase cleaves the probe between the reporter and quencher dyes. Separation of the reporter dye from the quencher dye results in an increase in fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorometer.

[0073] In some embodiments, microarrays, including but not limited to DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays); protein microarrays; tissue microarrays; transfection or cell microarrays; chemical compound microarrays; and antibody microarrays, are utilized to measure cancer marker mRNA levels. DNA microarrays, commonly known as gene chips, DNA chips, or biochips, are collections of microscopic DNA spots attached to a solid support (e.g., glass, plastic, or silicon chips) that form an array for simultaneously profiling or monitoring the expression levels of thousands of genes. The immobilized DNA segments are known as probes, thousands of which can be used on a single DNA microarray. Microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells. Microarrays can be fabricated using a variety of techniques, including, but not limited to, printing with finely pointed pins on glass slides; photolithography using prefabricated masks; photolithography using dynamic micromirror devices; inkjet printing; or electrochemistry on microelectrode arrays.

[0074] In yet other embodiments, reverse transcriptase PCR (RT-PCR) is used to detect RNA expression. In RT-PCR, RNA is enzymatically converted into complementary DNA, or "cDNA," using reverse transcriptase. The cDNA is then used as a template for the PCR reaction. PCR products can be detected by any suitable method, including, but not limited to, gel electrophoresis and staining with a DNA-specific stain or hybridization to a labeled probe. In some embodiments, quantitative reverse transcriptase PCR using a standardized mixture of competitive templates, as described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978 (each of which is incorporated herein by reference), is utilized.

[0075] In some embodiments, cancer markers are detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Illustrative, non-limiting examples of detection methods are described below.

[0076] One exemplary detection method, hybridization protection assay (HPA), involves hybridizing a chemiluminescent oligonucleotide probe (e.g., an acridinium ester-labeled (AE) probe) to a target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probes, and measuring the chemiluminescence generated from the remaining probes with a luminometer. See, e.g., U.S. Pat. No. 5,283,174; Nelson et al., Nonisotopic Probing, Blotting, and Sequencing, ch. 17 (Larry J. Kricka ed., 2nd ed. 1995, each of which is incorporated herein by reference in its entirety).

[0077] The interaction between two molecules can also be detected, for example, using fluorescence energy transfer (FRET) (see, e.g., Lakowiec et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,968,103; each of which is incorporated herein by reference). The fluorophore label is selected so that the emitted fluorescent energy of the first donor molecule is absorbed by a fluorescent label on a second "acceptor" molecule, which can then fluoresce due to the absorbed energy.

[0078] Alternatively, the "donor" protein molecule can simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light so that the "acceptor" molecule label can be distinguished from that of the "donor." The efficiency of energy transfer between the labels is related to the distance separating the molecules, allowing for assessment of the spatial relationship between the molecules. In situations where binding occurs between molecules, the fluorescence emission of the "acceptor" molecule label should be maximal. FRET binding events can be conveniently measured by fluorescence detection means.

[0079] Another example of a detection probe with self-complementarity is a "molecular beacon." A molecular beacon comprises a nucleic acid molecule having a target-complementary sequence, an affinity pair (or nucleic acid arm) that holds the probe in a closed conformation in the absence of the target sequence present in the amplification reaction, and a label pair that interacts when the probe is in the closed conformation. Hybridization between the target sequence and the target-complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to the reduced interaction of the label pair, which can be, for example, a fluorophore and a quencher (e.g., for DABCYL and EDANS). Molecular beacons are disclosed, for example, in U.S. Patent Nos. 5,925,517 and 6,150,097, the entire contents of which are incorporated herein by reference.

[0080] As a non-limiting example, probe binding pairs having interactive labels (e.g., those disclosed in U.S. Patent No. 5,928,862), which is incorporated herein by reference in its entirety, can be adapted for use in the methods of the presently disclosed embodiments. Probe devices used to detect single nucleotide polymorphisms (SNPs) can also be utilized in the present invention. Additional detection systems include "molecular switches," such as those disclosed in U.S. Publication No. 20050042638, which is incorporated herein by reference in its entirety. Other probes, such as those containing intercalating dyes and / or fluorescent dyes, are also useful for detecting amplification products in the methods of the presently disclosed embodiments. See, for example, U.S. Patent No. 5,814,447, which is incorporated herein by reference in its entirety.

[0081] In some embodiments, nucleic acid sequencing is used for detection. In some embodiments, sequencing is second-generation (also known as Next Generation or Next-Gen), third-generation (also known as Next-Next-Gen), or fourth-generation (also known as N3-Gen) sequencing technology, including but not limited to pyrosequencing, sequencing by ligation, single molecule sequencing, sequencing by synthesis (SBS), semiconductor sequencing, massively parallel clone, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide an overview of some such technologies in Genomics, 92: 255 (2008) (incorporated herein in its entirety by reference). Those skilled in the art will recognize that RNA is usually reverse transcribed into DNA before sequencing, because RNA is less stable in cells and is experimentally prone to nuclease attack.

[0082] DNA sequencing techniques include fluorescence-based sequencing methodologies (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, NY; incorporated herein by reference in its entirety). In some embodiments, the sequencing is automated sequencing. In some embodiments, the sequencing is parallel sequencing of divided amplicons (Kevin McKernan et al., PCT Publication No. WO2006084132, incorporated herein by reference in its entirety). In some embodiments, the sequencing is DNA sequencing by parallel oligonucleotide extension (see, e.g., U.S. Patent No. 5,750,341 to Macevicz et al. and U.S. Patent No. 6,306,597 to Macevicz et al., both of which are incorporated herein by reference in their entirety).Further examples of sequencing techniques include the Church polony technique (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Patent Nos. 6,432,360, 6,485,944, and 6,511,803; which are incorporated herein by reference in their entireties), the 454 picotitor pyrosequencing technique (Margulies et al., 2005 Nature 437, 376-380; US 20050130173; incorporated herein by reference in its entirety), Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6,373-382; U.S. Patent No. 6,787,308; U.S. Patent No. 6,833,246; incorporated herein by reference in its entirety), Lynx massively parallel sequencing technology (Brenner et al. (2000) Nat. Biotechnol. 18:630-634; U.S. Patent No. 5,695,934, U.S. Patent No. 5,714,330; incorporated herein by reference in its entirety), and Adessi PCR colony technology (Adessi et al. (2000) Nucleic Acid Res. 28, E87; WO 00018957; incorporated herein by reference in its entirety).

[0083] A variety of nucleic acid sequencing methods are contemplated for use in the methods of the present invention, including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. See, for example, Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al., Proc. Natl. Acad. Sci. USA 74:560-564 (1977); Drmanac et al., Nat. Biotechnol. 16:54-58 (1998); (1998); Kato, Int. J. Clin. Exp. Med. 2:193-202 (2009); Ronaghi et al., Anal. Biochem. 242:84-89 (1996); Margulies et al., Nature 437:376-380 (2005); Ruparel et al., Proc. Natl. Acad. Sci. USA 102:5932-5937 (2005), and Harris et al., Science 320:106-109 (2008); Levene et al., Science 299:682-686 (2003); Korlach et al., Proc. Natl. Acad. Sci. USA 105:1176-1181 (2008); Branton et al., Nat. Biotechnol. 26(10):1146-53 (2008); Eid et al., Science 323:133-138 (2009); each of which is incorporated herein by reference in its entirety.

[0084] Next-generation sequencing (NGS) methods share common features of massively parallel, high-throughput strategies, with the goal of lower cost compared to older sequencing methods (see, e.g., Levy and Meyers, Annual Review of Genomics and Human Genetics Volume 17, 2016 pp 95-115; incorporated herein by reference in its entirety). Numerous commercial platforms for NGS are available (see, e.g., Levy and Meyers, supra).

[0085] In mammals, methylation occurs only at cytosine residues, more specifically, at cytosine residues adjacent to guanine residues (i.e., the sequence CG, often denoted "CpG"). Detecting and mapping sites of DNA methylation is an essential step for understanding epigenetic gene regulation and providing diagnostic tools for identifying cancer and other pathologies associated with misregulation of genes.

[0086] Mapping the DNA methylation status at specific sites is currently achieved by the bisulfite method described by Frommer et al. for the detection of 5-methylcytosine in DNA (Proc. Natl. Acad. Sci. USA 89: 1827-31 (1992), expressly incorporated herein by reference in its entirety for all purposes), or variations thereof. The bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine reacts with hydrogen sulfite ions (also known as bisulfite), but 5-methylcytosine does not. This reaction typically occurs according to the following steps: first, cytosine reacts with bisulfite to form sulfonated cytosine; second, spontaneous deamination of the sulfonated reaction intermediate yields sulfonated uracil; and finally, the sulfonated uracil is desulfonated to form uracil under alkaline conditions. Uracil base pairs with adenine (and thus behaves like thymine), whereas 5-methylcytosine base pairs with guanine (and thus behaves like cytosine), allowing for the differentiation of methylated from unmethylated cytosine, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98) or methylation-specific PCR (MSP), as disclosed, for example, in U.S. Pat. No. 5,786,146.

[0087] The methylation status of a gene is often expressed as the proportion or percentage of individual strands of DNA methylated at a particular site (e.g., at a single nucleotide, or over a longer sequence of interest, e.g., up to a ∼100 bp subsequence of DNA) relative to the total population of DNA in a sample containing that particular site. Traditionally, the amount of unmethylated (e.g., native) gene is determined by PCR using a calibrator. A known amount of DNA is then bisulfite-treated, and the resulting methylation-specific sequence is determined using either real-time PCR or equivalent exponential amplification.

[0088] For example, conventional methods generally involve generating a calibration curve for an unmethylated target by using an external standard. The calibration curve is constructed from at least two points and is used to measure the real-time C of unmethylated DNA. t The values ​​are related to known quantitative standards. A second standard curve for the methylated target is then constructed from at least two points and an external standard. This second standard curve represents the C of methylated DNA. t to a known quantitative standard. Test samples C are then calculated for the methylated and unmethylated populations. t The genome equivalent of DNA is calculated from the standard curve generated by the first two steps. The percentage of methylation at a target site is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, e.g., (number of methylated DNAs) / (number of methylated DNAs + number of unmethylated DNAs) × 100.

[0089] The present disclosure is not limited to methods for measuring the methylation status of genes. For example, in some embodiments, the methylation status is measured by genome scanning. For example, one method includes restriction landmark genome scanning (Kawai et al., Mol. Cell. Biol. 14:7421-7427, 1994), and another example includes methylation-sensitive arbitrarily primed PCR (Gonzalgo et al., Cancer Res. 57:594-599, 1997). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with a methylation-sensitive restriction enzyme, followed by Southern analysis of the region of interest (digestion-Southern). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that includes digesting genomic DNA with a methylation-sensitive restriction enzyme prior to PCR (e.g., qPCR) amplification (Singer-Sam et al., Nucl. Acids Res. 18:687, 1990, or commercially available from Qiagen, Hilden, DE). Additionally, other techniques utilizing bisulfite treatment of DNA as a starting point for methylation analysis have been reported, including methylation-specific PCR (MSP) (Herman et al. Proc Natl Acad Sci USA 93:9821-9826, 1992) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby, Nucl. Acids Res. 24:5058-5059, 1996; and Xiong and Laird, Nucl. Acids Res. 25:2532-2534, 1997). PCR techniques have been developed for the detection of genetic mutations (Kuppuswamy et al., Proc. Natl. Acad. Sci. 55 USA 88:1143-1147, 1991) and quantification of allele-specific expression (Szabo and Mann, Genes Dev. 9:3097-3108, 1995; and Singer-Sam et al., PCR Methods Appl. 1:160-163, 1992). Such techniques use an internal primer that anneals to the PCR-generated template and terminates immediately 5' of the single nucleotide being assayed.In some embodiments, a method using a "quantitative Ms-SNuPE assay" such as that described in US Pat. No. 7,037,650 is used.

[0090] Compositions for use in the methods described herein include, but are not limited to, kits containing one or more reagents for determining the expression levels of BUB1B, PINK1, and G0S2 and the methylation status of G0S2, as described above. In some embodiments, the reagents are, for example, nucleic acid probes that hybridize to BUB1B, PINK1, and G0S2, one or more nucleic acid primers for amplifying or extending BUB1B, PINK1, and G0S2, one or more methylation-specific restriction enzymes, or one or more nucleic acid primers that specifically bind to methylated G0S2 nucleic acids.

[0091] The probes may also be provided in the form of an array. In a preferred embodiment, the kit contains all the components necessary to perform the detection assay, including all controls, instructions for performing the assay, and any necessary software for analysis and presentation of results.

[0092] [B. COC classification] As described herein, the expression levels of BUB1B and PINK1 and the methylation level of G0S2 are used to classify COC into subtypes COC1 to 3.

[0093] The present disclosure is not limited to a specific cutoff value or threshold for characterizing ACC. For example, in some embodiments, a BUB1B-PINK1 expression score above a threshold level and a G0S2 methylation below a threshold level (e.g., less than 4.696%, 4.6%, or 4.5%) indicates COC1. In some embodiments, the cutoff value for the G0S2 methylation level is less than 4.696% ± 1%, 5%, or 10%.

[0094] In some embodiments, a BUB1B-PINK1 expression score below a threshold level and a G0S2 methylation below a threshold level (e.g., less than 4.696%, 4.6%, or 4.5%) indicates COC2. In some embodiments, the cutoff value for the G0S2 methylation level is less than 4.696% ± 1%, 5%, or 10%.

[0095] In some embodiments, the presence of G0S2 methylation above a threshold level (e.g., greater than 4.696%, 4.6%, 4.7%, 4.8%, 4.9%, or 5.0%) is indicative of COC3. In some embodiments, the cutoff value for G0S2 methylation level is less than 4.696% ± 1%, 5%, or 10%.

[0096] The present invention is not limited to a particular threshold level for the BUB1B-PINK1 expression score (e.g., for distinguishing between COC1 and COC2). In some embodiments, the BUB1B-PINK1 expression score threshold level is 1.4 to 1.8 (e.g., 1.4, 1.5, 1.6, 1.7, or 1.8). In some embodiments, the BUB1B-PINK1 expression score threshold is 1.646 ± 1%, 5%, or 10%.

[0097] In some embodiments, the BUB1B-PINK1 expression score is based on subtraction of the delta Ct values ​​of BUB1B and PINK1.

[0098] In some embodiments, when BUB1B and PINK1 are measured using alternative techniques, including RNA-seq, the BUB1B-PINK1 expression score is determined by subtraction of gene expression z-scores.

[0099] In some embodiments, the BUB1B-PINK1 expression score and G0S2 methylation value (e.g., ±1%, ±5%, or 10%) shown in Table 1 are used to characterize ACC.

[0100] If a higher-resolution approach is used to assess G0S2 methylation, the cutoff value for classifying a sample as having G0S2 methylation may change. This is because higher-resolution approaches to assess G0S2 methylation do not reduce methylation to a single value but rather measure multiple CpG residues present within and around the G0S2 locus. Notably, the distribution of average methylation across the G0S2 locus in ACC patient populations is bimodal and does not vary depending on the measurement strategy employed, as demonstrated by Mohan & Lerario et al., Clinical Cancer Research 2019 (incorporated herein by reference in its entirety). If a user is using a higher-resolution approach to measure G0S2 methylation, including but not limited to genome-wide DNA methylation arrays, or next-generation sequencing-based approaches, including targeted bisulfite sequencing, a different method may be used to classify samples as having G0S2 methylation levels above or below the threshold level. For example, in some embodiments, unsupervised full hierarchical clustering using Euclidean distance to the logit-transformed methylation beta values ​​(or equivalent values) of CpG residues present in and around the G0S2 locus is used to classify samples as being above or below a methylation threshold (e.g., samples at the lower end of the methylation distribution are classified as below the threshold, and samples at the higher end of the methylation distribution are classified as above the threshold), as described in Mohan & Lerario et al. Clinical Cancer Research 2019.

[0101] Also, as described herein, in some embodiments, when a higher-resolution approach is applied to assess the BUB1B-PINK1 score (including, but not limited to, targeted RNA-seq panels), the BUB1B-PINK1 expression score is calculated using expression z-score subtraction and calibrated according to expression percentiles. Note that when the BUB1B-PINK1 expression score is calculated using z-score differences, the direction of the score is reversed: samples with G0S2 methylation below the threshold level and a BUB1B-PINK1 expression score above the threshold level are classified as COC2, while samples with G0S2 methylation below the threshold level and a BUB1B-PINK1 expression score below the threshold level are classified as COC1. For example, in some embodiments, the threshold level of the BUB1B-PINK1 expression score for stratifying patients is the 44th percentile for non-CIMP-high patients with a history of metastatic disease.

[0102] In some embodiments, the ACC classification (eg, COC1-3) is used to recommend and / or administer a treatment or candidate treatment to a subject with ACC.

[0103] In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the expression level or methylation level of one or more given markers) into data of predictive value to the clinician. The clinician can access the predictive data using any appropriate means. Thus, in some preferred embodiments, the present disclosure provides the additional benefit that clinicians, who are unlikely to be trained in genetics or molecular biology, do not need to understand the raw data. The data is presented directly to the clinician in the most useful form. The clinician can then immediately use the information to optimize the subject's care.

[0104] The present disclosure contemplates any method for receiving, processing, and transmitting information between the laboratory performing the assay, the information provider, the medical individual, and the subject. For example, in an embodiment of the present disclosure, a sample (e.g., a biopsy or blood or urine sample) is obtained from a subject and submitted to a profiling service (e.g., a clinical laboratory in a medical facility, a genomic profiling business, etc.) located anywhere in the world (e.g., a country other than the country where the subject is located or the country where the information will ultimately be used) to generate raw data. If the sample contains tissue or other biological samples, the subject can visit a medical center to obtain the sample and send it to the profiling center, or the subject can collect the sample (e.g., a urine sample) themselves and send it directly to the profiling center. If the sample contains previously determined biological information, the information can be sent directly to the profiling service by the subject (e.g., an information card containing the information can be scanned by a computer, and the data can be transmitted to the profiling center's computer using an electronic communication system). Once received by the profiling service, the sample is processed to generate a profile (ie, expression or methylation data) specific to the diagnostic or prognostic information desired for the subject.

[0105] The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format can represent a diagnosis or risk assessment for the subject, along with a recommendation for a particular treatment option. The data may be displayed to the clinician in any suitable manner. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of treatment) or displayed to the clinician on a computer monitor.

[0106] In some embodiments, information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to convert the raw data into information useful to clinicians or patients. A central processing facility offers the advantages of privacy (all data is stored at the central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data after the subject's treatment. For example, the central facility can provide the data to clinicians, subjects, or researchers using electronic communication devices.

[0107] In some embodiments, the subject can directly access the data using an electronic communication system. The subject can select further intervention or counseling based on the results. In some embodiments, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or exclusion of markers as informative indicators of a particular condition or disease stage, or as companion diagnostics to determine therapeutic course of action. In some embodiments, the results are used to select candidate therapies for drug screening or clinical trials.

[0108] II. Treatment of ACC In some embodiments, molecular classification of ACC is used to recommend and administer treatment for ACC. COC1-3 molecular subtypes are characterized by the enrichment of distinct transcriptional programs that are targeted by these pharmacological agents individually or in combination therapy. Therefore, classifying ACC into COC1-3 allows for the pre-selection of patient populations that are most likely to respond to a given targeted drug (or combination of targeted drugs), providing treatment methods with such drugs, and increasing the effectiveness of such drugs by focusing their use on patients who are most likely to benefit. Similarly, such focused use reduces or eliminates the use of drugs in patients who are unlikely to benefit, thereby reducing or avoiding unnecessary drug toxicity, saving money, and avoiding patient dissatisfaction when drugs are ineffective.

[0109] For example, Figure 1 shows that ACC-TCGA (Zheng et al., Cancer Cell 2016) identifies three distinct multi-platform molecular subtypes of ACC (COC1-COC3) that can be refined into single-platform signatures. Specific types of somatic copy number alteration profiles (SCNAs) derived from SNP array profiling, CpG island methylator phenotypes (CIMPs) derived from array-based DNA methylation profiling, and mRNA subtypes derived from RNA-seq transcriptome profiling converge for each COC, as measured by chi-squared test (A) or heatmap (B). Figure 1C shows the dominant SCNAs, CIMPs, and mRNA groups for each COC. Notably, as described herein, and particularly in relation to Examples 1-2, COC3 tumors have a CIMP-high DNA methylation profile and a noisy SCNA profile. Each COC is associated with a distinct prognosis - COC1 (favorable, indolent disease dynamics), COC2 (intermediate, moderate disease dynamics), and COC3 (poor, rapidly progressive disease dynamics).

[0110] Analysis of ACC-TCGA data revealed that 90% of ACC tumors across all three molecular classes of ACC (COC1, COC2, and COC3) exhibit high expression of IGF2 (and thus IGF1R-induced signaling) (Zheng et al. Cancer Cell 2016). Meanwhile, COC3 / CIMP-high tumors are characterized by a stronger induction of E2F-dependent transcription (including cell cycle genes, DNA repair genes, and epigenetic writers such as EZH2 and DNMT1), high levels of NR5A1-dependent steroidogenesis, and high levels of canonical Wnt signaling activation. Furthermore, COC3 / CIMP-high ACC exhibited lower expression of immune cell transcriptional markers, consistent with immune cell exclusion in these tumors.

[0111] Furthermore, as shown in Figures 2 and 3, COC populations derived from ACC-TCGA are associated with distinct somatic alteration profiles and activation of transcriptional programs. COC3 tumors have a higher frequency of driver somatic alterations leading to constitutive cell cycle activation ("MUT"). Consistent with this enrichment, COC3 tumors also have a higher cell cycle score, supporting cell cycle-targeted therapy in COC3 tumors. This cell cycle score is derived from the expression of E2F target genes known to have bona fide cell cycle and higher expression in cycling cells (TOP2A, MKI67, BUB1B, AURKB, E2F2, PLK1, FOXM1) using ACC-TCGA (Zheng et al. Cancer Cell 2016) RNA-seq data and GSVA (Hanzelmann et al. BMC Bioinformatics 2013). COC2-3 tumors have a higher frequency of driver somatic alterations leading to constitutive Wnt pathway activation ("MUT"). Consistent with this enrichment, both COC2 and COC3 tumors had a high Wnt score, supporting Wnt pathway-targeted therapy in COC2+3 tumors. The Wnt score was derived from the expression of bona fide Wnt pathway target genes (AXIN2, LEF1, NKD1, LGR5, APCDD1, BMP4) using ACC-TCGA RNA-seq data and GSVA.

[0112] COC3 tumors often harbor an abnormal epigenetic landscape characterized by nonphysiological DNA methylation directed toward CpG islands, "high CIMP." COC3 tumors also possess a high epigenetic score (derived using ACC-TCGA RNA-seq and GSVA) ​​consisting of three genes involved in this program (DNMT1, DNMT3A, and EZH2), supporting a role for targeted epigenetic therapies (including DNA methyltransferase inhibitors and EZH2 inhibitors) in COC3 tumors.

[0113] COC2 and COC3 tumors are dominated by "steroid-high" and "steroid-high / proliferative" transcriptional programs, as identified by ACC-TCGA. This program is characterized by high expression of the adrenocortical transcription factor SF1 (encoded by NR5A1) and elevated expression of steroidogenic enzymes. Consistent with this, the ACC-TCGA RNA-seq / GSVA-derived steroid score, comprised of genes involved in this program (NR5A1, MC2R, MRAP, CYP17A1, HSD3B2, CYP21A2, and CYP11B1), is higher in COC2 and COC3 tumors. This data supports therapeutic targeting of SF1 and steroidogenesis in COC2 and COC3. ACC-TCGA confirmed that the majority of ACCs are immunocompromised compared with other cancers. As shown in Figure 2, COC1 tumors have a higher degree of immune infiltration (measured by the ACC-TCGA RNA-seq / GSVA-derived immune score, consisting of expression of the immune genes CD3D, CD3G, CD4, CD8A, CD8B, CD274, and PDCD1). This supports T cell-targeted immunotherapy, referred to herein as "immune checkpoint therapy," in COC1 tumors. Finally, cortisol / glucocorticoids (the production of which is controlled by the genes used to derive the steroid score) are immunosuppressive. Thus, in some embodiments, a combination of steroidogenesis inhibition and immunotherapy is utilized to treat patients with COC2 and COC3 tumors.

[0114] Figure 3 provides a heatmap displaying information from Figures 1 and 2 by sample, including additional information on clinical cortisol production ("Cortisol") and death at the time of study ("Death"). Each sample is represented by a column, and the dendrogram shows unsupervised hierarchical clustering performed on the samples based on gene expression. Alterations in the Wnt pathway are indicated by the "Wnt_mut" track, and cell cycle alterations are indicated by the "CC" track. Gene expression levels from the ACC-TCGA RNA-seq data are color-coded by row z-score (white to black). Patients with COC3 tumors have more aggressive disease ("Death") and higher tumor expression of epigenetic (DNMT1, DNMT3A, EZH2) and cell cycle genes (TOP2A, MKI67, BUB1B, AURKB, E2F2, PLK1, FOXM1). Patients with COC2 and COC3 disease have clinical cortisol production, more aggressive disease, and higher tumor expression of steroid (NR5A1, MC2R, MRAP, CYP17A1, HSD3B2, CYP21A2, CYP11B1) and Wnt pathway (AXIN2, LEF1, NKD1, LGR5, APCDD1, BMP4) genes. Patients with COC1 tumors are less aggressive, produce cortisol less frequently, and have higher expression of genes associated with immune infiltration and activation of targetable immune checkpoints (CD3D, CD3G, CD4, CD8A, CD8B, CD274, PDCD1).

[0115] Figure 3B shows a heatmap in which genes are collapsed into corresponding epigenetics ("EPIG"), cell cycle ("CELLCYCLE"), steroid ("STEROID"), Wnt ("Wnt"), and immune ("IMMUNE") scores. The scale represents the actual score value (white to black). Unsupervised hierarchical clustering was performed on samples and scores based on the score values, demonstrating coordinated regulation of epigenetics, cell cycle, steroid, and Wnt scores, and their correlation with the immune score.

[0116] COC1 tumors are characterized by low levels of E2F, low levels of Wnt-dependent transcriptional programs, and rare somatic alterations that, when present, lead to constitutive cell cycle activation and constitutive Wnt pathway activation, as observed in COC3 (constitutive cell cycle activation and constitutive Wnt pathway activation) and COC2 (constitutive Wnt pathway activation) (Figure 2, Figure 3), indicating that IGF1R-dependent signaling is the predominant oncogenic pathway for this molecular class. Furthermore, COC1 tumors are uniquely characterized by increased expression of immune-related genes. Therefore, pharmacological agents targeting COC1 tumors include agents that block IGF1R signaling (e.g., IGF1R inhibitors) and / or immune checkpoint inhibitors.

[0117] Thus, in some particular embodiments, the present invention provides compositions and methods for treating COC1 (e.g., tumors having a G0S2 methylation level below a threshold level and a BUB1B-PINK1 expression score above a threshold level) with an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor, such as, but not limited to, linsitinib, cixutumumab, ganitumab, figitumumab, dalotuzumab, istiratumab, dusigizumab, or teprotumab). In some specific embodiments, subjects with G0S2 methylation below the threshold level and a BUB1B-PINK1 expression score above the threshold level are treated with linsitinib, and subjects with G0S2 methylation above the threshold level and a BUB1B-PINK1 expression score below the threshold level are not treated with linsitinib, another IGF1R inhibitor, or an agent that blocks IGF1R signaling.

[0118] In some embodiments, in addition to or as an alternative to an agent that blocks IGF1R signaling, COC1 tumors are treated with an immune checkpoint inhibitor (e.g., an agent that targets CTLA-4, PD-1, or PD-L1).

[0119] In some embodiments, molecular targets in elevated COC3 / CIMP include, but are not limited to, cell cycle effectors (e.g., one or more of CDK4 / 6, PLK1, MELK, or AURKB), DNA repair proteins (e.g., WEE1 and / or PARP), agents that alter Wnt signaling (e.g., one or more of β-catenin, CBP, TCF, LEF, Wnt ligands, APC / GSK3β destruction complex, and / or porcupine), epigenetic writers (e.g., EZH2 and / or DNMT1), and NR5A1.

[0120] On the other hand, COC3 / CIMP-high tumors are less likely to respond to immune checkpoint inhibitors or agents that block IGF1R signaling (e.g., IGF1R inhibitors) as monotherapy. However, in some embodiments, in addition to one or more of the targets listed above, COC3 tumors are further treated with an agent that blocks IGF1R signaling and / or immune checkpoint therapy in combination with an NR5A1 inhibitor, a glucocorticoid synthesis / metabolism inhibitor, or a glucocorticoid receptor inhibitor.

[0121] Similarly, COC2 tumors are characterized by high canonical Wnt signaling activation and high levels of NR5A1-dependent transcription. Therapeutic agents targeting COC2 tumors include, but are not limited to, Wnt inhibitors and / or NR5A1 antagonists. In some embodiments, in addition to one or more of the targets listed above, COC2 tumors are further treated with an agent that blocks IGF1R signaling and / or immune checkpoint therapy in combination with an NR5A1 inhibitor, a glucocorticoid synthesis / metabolism inhibitor, or a glucocorticoid receptor inhibitor.

[0122] The present disclosure is not limited to a specific target gene or treatment. In some embodiments, the inhibitor is an antibody, a nucleic acid, or a small molecule. For example, in some embodiments, the cell cycle effector protein is CDK4 / 6, PLK1, MELK, or AURK, and the inhibitor is palbociclib (Pfizer, New York, NY). In some embodiments, the DNA repair protein is WEE1 or PARP, and the inhibitor is adavosertib (Merck) or olaparib (Merck, Kenilworth, New Jersey). In some embodiments, the IGF1R inhibitor is linsitinib (Achemtek, Worcester, MA), cixutumumab (McKian et al., Expert Opin Investig Drugs. 2009 Jul;18(7):1025-1033), figitumumab (Pfizer), ganitumab (Amgen, Thousand Oaks, CA), teprotumumab (Horizon Pharma), dalotuzumab (Merck / Pierre Fabre), istiratumab (Merrimack Pharma), or dusigizumab (Medimmune / AstraZeneca). In some embodiments, the Wnt inhibitor is WNT974 (Novartis, Basel, Switzerland) or PRI-724 (Prism Pharma). In some embodiments, the immune checkpoint inhibitor is ipilimumab (Bristol-Myers Squibb, New York, NY), nivolumab (Bristol-Myers Squibb, New York, NY), or nivolumab (Bristol-Myers Squibb). Squibb), pembrolizumab (Merck), or atezolizumab (Hoffmann-La Roche, Basel, Switzerland). In some embodiments, the NR5A1 inhibitor is SID 7969543 (Bio-Techne Corporation, Minneapolis, MN), 45594 [4-(heptyloxy)phenol], or octyloxyphenyl (OOP) (both available from ACADIA Pharmaceuticals, San Diego, CA).In some embodiments, the epigenetic writer is EZH2 and / or DNMT1 and the inhibitor is 3-deazaneplanocin A (Cayman, Ann Arbor, MI), EPZ005687 (Epizyme, Cambridge, MA), EPZ6438 / tazemetostat (Epizyme), or 5-azacytidine (Tocris, Minneapolis, MN). In some embodiments, the immune checkpoint inhibitor is ipilimumab (Bristol-Myers Squibb, New York, NY), nivolumab (Bristol-Myers Squibb, NY), pembrolizumab (Merck, Kenilworth, NJ), atezolizumab (Roche, Basel, Switzerland), avelumab (Merck, Kenilworth, NJ), durvalumab (AstraZeneca, Cambridge, United Kingdom), and cemiplimab (Regeneron, Tarrytown, NY). In some embodiments, the glucocorticoid synthesis / metabolism inhibitor or glucocorticoid receptor inhibitor is aminoglutethimide, oscilostat (Novartis, Basel, Switzerland), metyrapone (Catalent Deutschland Eberbach GmbH, Eberbach, Germany), mifepristone (Danco Laboratories, New York, NY), or ketoconazole.

[0123] In some embodiments, one or more (e.g., 1, 2, 3, 4, or more) inhibitors targeting the same gene or COC subtype are administered to a subject. In some embodiments, a combination of therapies directed to a specific COC subtype is used in combination. For example, in one non-limiting example, a subject with COC3 cancer is administered one or more of the above-mentioned therapies targeting COC3 in combination with a Wnt inhibitor or NR5A1 inhibitor; and an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and / or an immune checkpoint inhibitor. In another example, a subject with COC2 cancer is administered the above-mentioned COC2-targeting therapy and an agent that blocks IGF1R signaling (e.g., an IGF1R inhibitor) and / or an immune checkpoint inhibitor in combination with an NR5A1 inhibitor or a glucocorticoid synthesis / metabolism inhibitor or a glucocorticoid receptor inhibitor. Further combination therapies are described herein.

[0124] In some embodiments, the inhibitor is nucleic acid.The exemplary nucleic acid suitable for inhibiting the expression of the described marker (for example, by preventing the expression of the marker) includes, but is not limited to, antisense nucleic acid and RNAi.In some embodiments, the nucleic acid treatment is complementary to and hybridizes with at least part (for example, at least 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides) of the marker described herein.

[0125] In some embodiments, compositions containing oligomeric antisense compounds, particularly oligonucleotides, are used to regulate the function of nucleic acid molecules encoding the markers described herein, ultimately regulating the amount of expressed marker genes. This is achieved by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding the marker genes. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This regulation of the function of a target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is generally referred to as "antisense." The functions of DNA that are interfered with include replication and transcription. The functions of RNA that are interfered with include all important functions, such as translocation of RNA to the protein translation site, translation of protein from RNA, splicing of RNA to produce one or more mRNA species, and catalytic activities that are involved in or can be promoted by RNA. The overall effect of such interference with target nucleic acid function is to reduce the amount of expressed marker.

[0126] In some embodiments, the nucleic acid is an RNAi nucleic acid. " RNA interference (RNAi) " is the process of sequence-specific, post-transcriptional gene silencing initiated by small interfering RNA (siRNA), shRNA or microRNA (miRNA). During RNAi, RNA induces the degradation of target mRNA, resulting in sequence-specific inhibition of gene expression.

[0127] In "RNA interference" or "RNAi," "small interfering RNA" or "short interfering RNA" or "siRNA" or "short hairpin RNA" or "shRNA" molecules, or "miRNA," nucleotide RNAi (e.g., single-stranded, double-stranded, or hairpin) targets a nucleic acid sequence of interest, such as a marker disclosed herein.

[0128] An "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. RNA used in RNAi "targets" a gene in that the nucleotide sequence of the duplex portion of the RNAi is complementary to the nucleotide sequence of the target gene. In certain embodiments, the RNAi targets a sequence encoding a marker described herein. In some embodiments, the length of the RNAi is less than 30 base pairs. In some embodiments, the RNA can be 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs in length. In some embodiments, the length of the RNAi is 19-32 base pairs in length. In certain embodiments, the length of the RNAi is 19 or 21 base pairs in length.

[0129] In some embodiments, the RNAi comprises a hairpin structure (e.g., shRNA). The hairpin structure may comprise a loop portion located between the two sequences forming the duplex in addition to the duplex portion. The loop length can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure may also comprise a 3' and / or 5' overhang portion. In some embodiments, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0130] "miRNA" or "miR" refers to a non-coding RNA between 18 and 25 nucleobases in length that hybridizes to and regulates the expression of a coding RNA. In certain embodiments, a miRNA is the product of cleavage of a pre-miRNA by the enzyme Dicer. Examples of miRNAs can be found in the miRNA database known as miRBase.

[0131] As used herein, Dicer substrate RNA (DsiRNA) is a chemically synthesized asymmetric 25-mer / 27-mer double-stranded RNA that has increased efficacy in RNA interference compared to conventional RNAi. Traditional 21-mer RNAi molecules are designed to mimic Dicer products, thus bypassing interaction with the enzyme Dicer. Dicer has recently been shown to be a component of RISC and to be involved in RNAi participation in RISC. Dicer substrate RNAi molecules are designed to be optimally processed by Dicer, and by participating in this natural processing pathway, they show increased efficacy. Using this approach, sustained knockdown has been routinely achieved using subnanomolar concentrations (U.S. Patent No. 8,084,599; Kim et al., Nature Biotechnology 23:222 2005; Rose et al., Nucleic Acids Res., 33:4140 2005).

[0132] The transcription unit of an "shRNA" consists of a sense sequence and an antisense sequence linked by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by Exportin-5 and processed by Dicer in the cytoplasm to generate functional RNAi molecules. The "miRNA" stem-loop, consisting of a sense sequence and an antisense sequence linked by a loop of unpaired nucleotides, is typically expressed as part of a larger primary transcript (pri-miRNA). The "miRNA" stem-loop is excised by a Drosha-DGCR8 complex-generated intermediate known as a pre-miRNA. The "miRNA" stem-loop is then exported from the nucleus by Exportin-5 and processed by Dicer in the cytoplasm to generate functional miRNAs or siRNAs.

[0133] "Artificial miRNA" or "artificial miRNA shuttle vector," as used interchangeably herein, refers to a primary miRNA transcript with a double-stranded stem-loop region (at least approximately 9-20 nucleotides) that is excised via Drosha and Dicer processing, replaced with the siRNA sequence of the target gene while retaining the structural elements within the stem-loop necessary for effective Drosha processing. The term "artificial" arises from the fact that flanking sequences (e.g., approximately 35 nucleotides upstream and approximately 40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the RNAi. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially generated miRNA shuttle vectors.

[0134] The RNAi can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six T's.

[0135] The present invention further provides pharmaceutical compositions (e.g., comprising the above-described compounds). The pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including mucous membranes, including ocular and vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal, and transdermal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0136] In some embodiments, one or more targeted therapies are administered in combination with existing therapies for ACC. For example, in some embodiments, subjects with COC3 tumors are administered adjuvant cytotoxic chemotherapy (e.g., one or more of etoposide, doxorubicin, cisplatin, or other cytotoxic agents). In some embodiments, COC classification determination is repeated (e.g., during treatment or after surgery).

[0137] In some embodiments, agents described herein are screened for activity against ACC (eg, in an in vitro drug screening assay or in a clinical study).

[0138] 〔experiment〕 The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.

[0139] Example 1 To prospectively stratify ACC samples into COC1-3 subgroups, we use a modified version of a previously described two-step prognostic strategy (Mohan, Lerario, et al., Clinical Cancer Research 2019). The method described herein distinguishes between COC1, COC2, and COC3. The data reveal that G0S2 hypermethylation is observed almost exclusively in CIMP-high / COC3 (and can therefore be used to confirm this aggressive molecular subtype of ACC), while non-CIMP-high (COC1-2) ACC always exhibits low, indistinguishable levels of G0S2 methylation. However, COC1 and COC2 exhibit different BUB1B-PINK1 scores. Analysis of ACC-TCGA RNA-seq data reliably distinguished COC1 from COC2 and COC3 due to differences in BUB1B and PINK1 z-score expression in recurrent / metastatic tumors (Table 1 and Figures 4-8). This cutoff was used to calculate the corresponding BUB1B-PINK1 score (measured by TaqMan assay) in an independent population of non-CIMP-high metastatic / recurrent tumors (see, for example, Mohan, Lerario, et al., Clinical Cancer Research 2019). This cutoff allows for differentiation between COC1 and COC2+3 tumors using the TaqMan assay. Thus, COC3 ACC is defined as G0S2 methylation >4.696% and any value of BUB1B-PINK1 score, COC2 ACC is characterized by G0S2 methylation <4.696% and a BUB1B-PINK1 score <1.646, and COC1 ACC is characterized by G0S2 methylation <4.696% and a BUB1B-PINK1 score >1.646 (Table 1). The application of this strategy is not limited to the availability of fresh / frozen tissue samples, but is extendable to embedded, frozen, or formalin-fixed, paraffin-embedded (FFPE) samples from ACC samples.

[0140] [Table 1]

[0141] Example 2 The ACC-TCGA (Zheng et al. Cancer Cell 2016) identified three types of ACC: COC1, COC2, and COC3. These types are characterized by distinct molecular features: specific copy number alteration profiles (SCNAs), DNA methylation profiles (CIMPs), and mRNA profiles (mRNAs), as shown in Figure 1.

[0142] COC1, COC2, and COC3 disease progress at different rates. Among all patients enrolled in ACC-TCGA, patients with COC1 disease progressed slowly (median event-free survival not reached), patients with COC2 disease progressed moderately (median event-free survival 38 months), and patients with COC3 disease progressed rapidly (median event-free survival 8 months). However, despite the different rates at which COC1, COC2, and COC3 disease progress, all types of ACC can progress to metastatic disease, and the majority of patients with ACC ultimately develop metastases. Among all patients diagnosed with or with a history of metastatic disease in ACC-TCGA (n = 40), 8 / 40 had COC1 disease (20%), 11 / 40 had COC2 disease (27.5%), and 21 / 40 had COC3 disease (52.5%).

[0143] The unique molecular features that define the COC1, COC2, and COC3 classes of tumors indicate that each ACC type (COC1, COC2, COC3) is uniformly sensitive to treatments or combinations of therapies targeted for use in that particular class.

[0144] A phase 3 trial evaluating the efficacy of OSI-906 / linsitinib in patients with advanced adrenocortical carcinoma (ACC) was published by Fassnacht et al. in Lancet Oncology 2015. In this study, eligible patients were treated with OSI-906 or placebo until they experienced intolerable drug adverse effects or a progression event, as defined by RECIST criteria (Eisenhauer et al. Eur J. Cancer. 2009); patients who experienced intolerable drug adverse effects or a progression event discontinued the study. Fassnacht et al. reported no statistically significant difference in Kaplan-Meier progression-free survival curves between linsitinib-treated and placebo-treated patients. However, disease progression was observed in all placebo-treated patients by 150 days after randomization, resulting in a 25% survival divergence in disease dynamics between the linsitinib and placebo groups, with a subset of patients in the linsitinib group experiencing prolonged progression-free survival (>150 days). This prolonged progression-free survival was not observed in the placebo group, indicating a response to linsitinib (Figure 9).

[0145] Data from this trial demonstrated favorable responses in 6 of 90 patients treated with OSI-906, including 4 patients who achieved partial response (PR) by RECIST criteria (Eisenhauer et al. Eur J.Cancer. 2009) and 2 patients who achieved long-term stable disease (SD; also by RECIST criteria, Eisenhauer et al. Eur J.Cancer. 2009). These individual patients are shown in Table 2.

[0146] [Table 2]

[0147] After the study ended, exome sequencing was performed on tumor DNA from 12 patients treated with OSI-906: six who responded well to OSI-906 (Table 2) and six matched non-responders (Table 3 below). To ensure that responders and non-responders were otherwise comparable, non-responders were matched based on tumor grade, which is currently the gold standard method for prognosticating and classifying ACC. All non-responders progressed ("PD") on linsitinib treatment, as defined by RECIST criteria (Eisenhauer et al. Eur J. Cancer. 2009).

[0148] [Table 3]

[0149] The goal of this exome sequencing study was to identify molecular predictors of response to OSI-906. Exome sequencing data were analyzed to characterize the loss of heterozygosity (LOH) profile of each tumor. A bioinformatics pipeline (samtools mpileup, bcftools coll, and R / CRAN package modifications) was used to generate B-allele frequency profiles for each tumor and identify breakpoints between regions of variable allele frequency.

[0150] Exome sequencing revealed that linsitinib responders had fewer chromosomal breakpoints and a "chromosomal" SCNA profile, while linsitinib non-responders had a "noisy" SCNA profile (Figure 10, Figure 11). This first step demonstrates that linsitinib responders were either COC1 or COC2, while linsitinib non-responders were COC3 (Figure 1).

[0151] To determine whether these linsitinib responders were COC1 or COC2, the next step was to examine the disease dynamics of patients enrolled in the original linsitinib trial. Based on the observed split in the survival curves of patients treated with the drug or placebo, it was hypothesized that it would be possible to determine which class of ACC responded to treatment.

[0152] As expected, based on the molecular studies described above, it was determined that patients with rapidly progressing COC3 disease (which accounts for 52.5% of all metastatic ACC) would not respond to linsitinib treatment. Therefore, there was no difference between the placebo-treated and linsitinib-treated groups prior to the 47.5% survival rate, which is what was observed.

[0153] Furthermore, if patients with only COC2 disease (representing 27.5% of all metastatic ACC) responded to linsitinib treatment, one would expect to observe a difference in survival curves ranging between 47.5% and 20% survival. However, instead, a difference in survival curves was observed only at the beginning of 25% survival, as shown in Figure 9.

[0154] The timing of this branching demonstrated that patients who responded to linsitinib treatment were mostly, if not entirely, composed of patients with COC1, the slowest-growing disease subtype.

[0155] These collective measures used to identify COC1 patients (distinct from COC2 or COC3) further observed that, based on the differences in survival curves from the phase 3 trial, the magnitude and duration of long-term progression-free survival for 4 of 6 linsitinib-treated COC1 patients who achieved a partial response (PR) exceeded previously known expectations for any patient with metastatic ACC, and that the response must have been due to an effect of the therapeutic agent and not simply due to slower natural disease progression.

[0156] Supporting this is the fact that, in contrast, all patients in the placebo group (including those with COC1 tumors), a patient sample with a known natural history of metastatic ACC, progressed by day 150. This result further supports the assertion that patients with inherently slow-growing COC1 disease, if left untreated, will still inevitably experience a progression event by day 150, and serves to support the conclusion that COC1 linsitinib responders were, in fact, responding to the therapeutic effects of the drug.

[0157] Example 3 Considering the findings of Fassnacht et al. (2015) that 6 / 90 patients responded favorably to linsitinib (Table 2, Figure 9), the invention of novel molecular signatures attributed to linsitinib responders disclosed herein (Figures 7-8), and the invention of molecular diagnostic markers that can be used to identify patients with and without these signatures (Figure 12 and Mohan & Lerario et al. 2019), clinical trials evaluating the efficacy of linsitinib using these molecular diagnostic markers will be conducted.

[0158] Patients were stratified for inclusion into the study according to the ACC-TCGA molecular classes detailed in Table 1, and only patients falling into the COC1 class were included. The main advantage of this molecular classification strategy is that in addition to enriching for COC1 (ACC-TCGA) (Table 1; Figures 4-8), disease dynamics were also captured using biomarkers (Table 4 below). The presence of G0S2 methylation above a threshold (a proxy for CIMP-high DNA methylation and therefore COC3 status) was used to exclude COC3 patients. The presence of BUB1B-PINK1 below a threshold in the absence of G0S2 methylation was used to exclude COC2 patients. The presence of BUB1B-PINK1 above a threshold in the absence of G0S2 methylation was used to confirm and enroll COC1 patients. Linsitinib was administered to enrolled subjects as monotherapy for cancer.

[0159] [Table 4]

[0160] Application of molecular biomarkers would exclude from study enrollment 100-25.8% or 74.2% of patients with metastatic disease but not COC1, all of whom are unlikely to respond to IGF2 / IGF1R-targeted monotherapy.

[0161] In such a hypothetical clinical study, for 90 patients deemed eligible for enrollment using the criteria of the previous GALACCTIC trial and with six responders (including, but not limited to, a history of failure of all standard treatments for their disease), the following results are planned: Applying BUB1B-PINK1 and G0S2 methylation would result in 0.742 × 90 patients = 67 patients being excluded, leaving 23 patients enrolled in the trial. Of these 23 patients, the six who responded to treatment would be re-observed. Thus, a response rate of 6 / 23, or 26%, would be observed, representing a clinically meaningful increase in response rate compared to the previously failed trial, for a response rate of 6 / 90 = 6.7%. This 26% response rate, a nearly four-fold increase, would help demonstrate and confirm the clinical benefit of the agent in the study patient population and could be used to support a filing for marketing approval of linsitinib. This study design, size, and numerical degree of improved efficacy are merely exemplary, and as will be understood by one of skill in the art, studies of different design, size, and / or statistical power, as well as results of similarly improved but different numerical response rates, can be used to demonstrate clinically meaningful improvements to linsitinib response rates provided by the methods disclosed herein.

[0162] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art of medicine are intended to be within the scope of the following claims. [Brief explanation of the drawings]

[0163] [Figure 1] We show that ACC-TCGA identifies three distinct multi-platform molecular subtypes of ACC (COC1-COC3) that can be refined down to single platform features. [Figure 2] We show that COC populations from ACC-TCGA are associated with distinct somatic alteration profiles and activation of transcriptional programs. [Figure 3] Heat maps in Figures 1 and 2 are shown for each sample. [Figure 4] We show that the BUB1B‐PINK1 score distinguishes patients with COC1 disease from those with COC2 and COC3 tumors in the ACC‐TCGA. [Figure 5] In ACC-TCGA, we show that COC1 patients with non-CIMP-high progressive disease have a statistically different BUB1B-PINK1 profile than COC2 patients with non-CIMP-high progressive disease. [Figure 6] We show that a BUB1B‐PINK1 threshold allows reasonable discrimination between COC1 and COC2‐3 tumors in patients with non‐CIMP‐high advanced disease. [Figure 7] We show that the cumulative distribution function of BUB1B‐PINK1 scores in ACC‐TCGA allows for the validation of comparable BUB1B‐PINK1 cutoffs by qPCR in the FMUSP+UM population. [Figure 8]We show that combining BUB1B-PINK1 and CIMP status faithfully recapitulates COC in patients with advanced disease. [Figure 9] Progression-free survival curves for patients enrolled in the Fassnacht et al. Lancet Oncology 2015 study. [Figure 10] Breakpoint analysis of exome sequencing data obtained from responders and non-responders treated with linsitinib is shown. [Figure 11] We demonstrate that patients with noisy ACC progress on linsitinib. [Figure 12] We show that lethal CIMP-high tumors from ACC-TCGA often have noisy copy number / LOH profiles.

Claims

1. 1. A pharmaceutical composition for treating adrenocortical carcinoma (ACC), comprising: The pharmaceutical composition is for administration to a subject identified as having COC1 ACC, comprising an agent that blocks IGF1R signaling, the subject is identified as having COC1 ACC by measuring G0S2 methylation levels and determining BUB1B and PINK1 expression levels; the drug is an IGF1R inhibitor, A pharmaceutical composition, wherein the IGF1R inhibitor is linsitinib.

2. The pharmaceutical composition of claim 1, wherein the expression levels of BUB1B and PINK1 are used to calculate a BUB1B-PINK1 expression score.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that a BUB1B-PINK1 expression score above a threshold level cut-off and a G0S2 methylation level below a threshold level are indicative of COC1 ACC.

4. 3. The pharmaceutical composition of claim 2, wherein the threshold level of G0S2 methylation is 4.696 as determined using methylation-sensitive restriction digestion and amplification.

5. 3. The pharmaceutical composition of claim 2, wherein the threshold level of G0S2 methylation is determined using unsupervised full hierarchical clustering using Euclidean distance on logit-transformed methylation β values.

6. 3. The pharmaceutical composition of claim 2, wherein the threshold level of the BUB1B-PINK1 expression score is the 44th percentile of non-CIMP elevated patients with a history of metastatic disease.

7. 7. The pharmaceutical composition of claim 6, wherein the threshold level is 1.

5.

8. 7. The pharmaceutical composition of claim 6, wherein the threshold level is 1.

6.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the biological sample is selected from the group consisting of a tissue sample, a biopsy sample, a blood sample, and a urine sample.

10. Measuring G0S2 methylation and BUB1B and / or PINK1 expression levels a nucleic acid probe that hybridizes to at least one of BUB1B, PINK1, and G0S2; one or more nucleic acid primers for amplification or extension of at least one of BUB1B, PINK1, and G0S2; one or more methylation-specific restriction enzymes, and A pharmaceutical composition according to any one of claims 1 to 9, characterized in that it comprises the use of one or more reagents selected from the group consisting of one or more nucleic acid primers that specifically bind to methylated G0S2 nucleic acids.

11. 1. A pharmaceutical composition for treating ACC in a subject, comprising: The subject is a subject who has been confirmed to have COC1 ACC by obtaining or having obtained a sample from the subject and measuring the G0S2 methylation level and BUB1B-PINK1 expression score in the sample, 1. A pharmaceutical composition comprising an agent that blocks IGF1R signaling, administered to a subject if said subject has a G0S2 methylation level and a BUB1B-PINK1 expression score indicative of the presence of COC1 ACC, the drug is an IGF1R inhibitor, A pharmaceutical composition, wherein the IGF1R inhibitor is linsitinib.

12. 1. A pharmaceutical composition for treating ACC in a subject, comprising: determining a G0S2 methylation level and BUB1B and PINK1 expression levels in a sample from the subject, and identifying a subject having a G0S2 methylation level in the sample below a threshold level and a BUB1B-PINK1 expression score above the threshold level as having COC1 ACC; 1. A pharmaceutical composition for administration to a subject identified as having COC1 ACC, comprising an agent that blocks IGF1R signaling, the drug is an IGF1R inhibitor, A pharmaceutical composition wherein the IGF1R inhibitor is linsitinib.

13. 1. A pharmaceutical composition comprising an agent that blocks IGF1R signaling for use in treating ACC in a subject identified as having COC1 ACC, the subject is identified as having COC1 ACC by measuring G0S2 methylation levels and determining BUB1B and PINK1 expression levels; the drug is an IGF1R inhibitor, A pharmaceutical composition wherein the IGF1R inhibitor is linsitinib.

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