Methods for determining genome copy number variation

Abstract

This specification discloses methods for identifying stable control loci for use in fluorescence in situ hybridization (FISH) assays, and methods for determining whether copy number changes (e.g., amplification) of a target genomic locus (e.g., a gene, e.g., cyclin E1 (CCNE1)) have occurred in a biological sample (e.g., a non-neoplastic sample, a pre-neoplastic lesion, and / or a tumor). In particular, this disclosure provides an expanded repertoire of genomic loci that can serve as stable controls (e.g., stable control loci) for FISH assays aimed at determining copy number changes of a target genomic locus.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying genomic loci that can serve as a control for detecting copy number variations of target genomic loci (e.g., cancer biomarkers, e.g., genes, e.g., CCNE1) using fluorescence in situ hybridization (FISH). [Background technology]

[0002] Fluorescence in situ hybridization (FISH) is a molecular technique commonly used to detect cytogenetic abnormalities in cells, such as amplification of chromosomes, genetic elements, and / or genes. Because cytogenetic abnormalities are common features of cancer cells, FISH is often applied to the diagnosis and prognosis of cancer. For example, FISH is commonly used to detect amplification (e.g., duplication) of cyclin E1 (CCNE1), which is characteristic of many cancers.

[0003] FISH-based methods for detecting gene amplification or deletion utilize two fluorescent probes. The first probe (e.g., target probe) targets a genomic locus of interest to the user (i.e., the target genomic locus), while the second probe (e.g., control probe) targets a distinct control locus (i.e., a stable control locus). The ratio of the fluorescent signals produced by the two FISH probes can indicate amplification or deletion of the target genomic locus. Therefore, it is important to have a control probe that targets a locus with substantially constant (i.e., stable) ploidy across patient samples. However, stable control loci in the context of cancer cells can vary depending on the type of cancer. Therefore, the need to identify stable control loci for each cancer remains in this field. [Overview of the project] [Means for solving the problem]

[0004] In a first aspect, the present invention relates to a method for identifying a stable control locus for use in a fluorescence in situ hybridization FISH assay, the method comprising: (a) determining the ploidy of several chromosome bands within a chromosome obtained from a biological sample of interest; (b) repeating step (a) multiple times, wherein the biological sample is obtained from a different subject each time; (c) determining the weighted average (e.g., mean) ploidy of each chromosome band, wherein a weighted average ploidy of 1.8 to 2.2 copies is considered diploid; (d) determining the percentage of subjects considered diploid for each chromosome band; (e) ranking each chromosome band from high to low according to the percentage of subjects considered diploid; and (f) selecting a chromosome band within the upper quartile of step (e) to identify a stable control locus.

[0005] In some embodiments, the biological sample contains cancer cells. In some embodiments, the subject is a patient diagnosed with cancer. In a second embodiment, the present invention provides a method for determining whether cyclin E1 (CCNE1) is amplified in a cell, the method comprising: (a) contacting a population of cells having chromosomal DNA with a plurality of target probes capable of emitting a first detectable signal and complementary to CCNE1, and a plurality of control probes capable of emitting a second detectable signal and complementary to the CCNE1 control locus, wherein the CCNE1 control locus and CCNE1 are located on the same chromosomal DNA molecule; (b) quantifying the total number of the first and second detectable signals in the cells; (c) determining the signal ratio in the cells; (d) repeating steps (b) and (c) multiple times, wherein the cells are different each time; (e) determining the average (e.g., median) signal ratio of step (d); and (f) determining the amplification of CCNE1 from the average signal ratio, wherein an average signal ratio ≥ 2 indicates that CCNE1 is amplified.

[0006] In some embodiments, multiple target probes bind to the Homo sapiens genome assembly NCBI36 within the q12 band of chromosome (chr19 q12).

[0007] In some embodiments, multiple control probes bind to the Homo sapiens genome assembly NCBI36 at chr19 q13.43, chr19 q13.41, chr19 q13.42, chr19 p13.3, chr19 q13.32, chr19 q13.33, chr19 q13.31, chr19 p13.13, or chr19 q13.13. In some embodiments, multiple control probes bind to the Homo sapiens genome assembly NCBI36 at chr19 q13.43. In some embodiments, multiple control probes bind to the Homo sapiens genome assembly NCBI36 at nucleotide positions 29,644,930 to 29,841,126 of chr19 q13.43.

[0008] In some embodiments, contact is made in vitro. In some embodiments, the cell population is derived from a human tumor sample. In some embodiments, the cell population is derived from a human subject diagnosed with cancer. In some embodiments, the cell population is derived from a human subject at risk of developing cancer. In some embodiments, the cell population is derived from a human subject suspected of having cancer.

[0009] In some embodiments, the average (e.g., intermediate) signal ratio is determined from about 20 to about 70 cells (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70). In some embodiments, the average signal ratio is determined from about 50 cells (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55).

[0010] In some embodiments, multiple target and / or control probes each comprise a polynucleotide having modifications to its base, sugar, and / or backbone. In some embodiments, the modifications are selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate (PS) bonds between nucleosides, and 2'-fluoro (2'-F) modified nucleosides.

[0011] In a third embodiment, the present invention provides a method for determining whether a target genomic locus is amplified in a cell, the method comprising: (a) contacting a population of cells having chromosomal DNA with a plurality of target probes capable of emitting a first detectable signal and complementary to a target genomic locus, and a plurality of control probes capable of emitting a second detectable signal and complementary to a stable control locus, wherein the stable control locus and the target genomic locus are located on the same chromosomal DNA molecule; (b) quantifying the total number of the first and second detectable signals in the cells; (c) determining the signal ratio in the cells; (d) repeating steps (b) and (c) multiple times, with different cells each time; (e) determining the average (e.g., median) signal ratio of step (d); and (f) determining the amplification of the target genomic locus from the average signal ratio, wherein an average signal ratio ≥ 2 indicates that the target genomic locus is amplified.

[0012] In some embodiments, a stable control locus is identified by (a) determining the ploidy of multiple chromosome bands within a chromosome obtained from a biological sample of interest; (b) repeating step (a) multiple times, wherein the biological sample is obtained from a different subject each time; (c) determining the weighted average (e.g., intermediate) ploidy of each chromosome band, wherein a weighted average ploidy of 1.8 to 2.2 copies is considered diploid; (d) determining the percentage of subjects considered diploid for each chromosome band; (e) ranking each chromosome band from high to low according to the percentage of subjects considered diploid; and (f) selecting chromosome bands within the upper quartile of step (e) to identify a stable control locus.

[0013] In some aspects, the biological sample contains cancer cells. In some embodiments, contact is made in vitro. In some embodiments, the cell population is derived from a human tumor sample.

[0014] In some embodiments, the cell population is derived from a human subject diagnosed with cancer. In some embodiments, the cell population is derived from a human subject at risk of developing cancer. In some embodiments, the cell population is derived from a human subject suspected of having cancer.

[0015] In some embodiments, the average (e.g., intermediate) signal ratio is determined from about 30 to about 70 cells (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70). In some embodiments, the average signal ratio is determined from about 50 cells (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55).

[0016] In some embodiments, multiple target and / or control probes each comprise a polynucleotide having modifications to its base, sugar, and / or backbone. In some embodiments, the modifications are selected from 2'-O-methyl (2'-O-Me) modified nucleosides, phosphorothioate (PS) bonds between nucleosides, and 2'-fluoro (2'-F) modified nucleosides.

[0017] In a fourth embodiment, the present invention relates to a method for identifying a patient who is at risk of having cancer or is suspected of having cancer, the method comprising (a) obtaining a cell sample from a subject and (b) performing the method of the second embodiment, wherein the subject is identified as a patient if it is determined that the CCNE1 gene is amplified in the sample.

[0018] In a fifth aspect, the present invention relates to a method for treating a patient identified in the first or fourth aspect (for example, a patient at risk of having cancer, suspected of having cancer, or diagnosed with cancer), the method comprising administering an effective amount of membrane-bound tyrosine and a threonine-specific cdc2 inhibitory kinase (Myt1) inhibitor.

[0019] In some embodiments, the Myt1 inhibitor is a compound of formula (I):

[0020] [ka]

[0021] or its pharmaceutically acceptable salt, (In the formula, Each of X, Y, and Z is independently N or CR 2 And, R 1 and each R 2 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C2~6 Alkynyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, optionally substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, cyano, -N(R 7 )2, -OR 7 , -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -Q-R 7B and, or R 1 is, R 1 One R adjacent to 2 together with, optionally substituted C 3~6 forms an alkylene, R 3 and each of R 4 is, independently, optionally substituted C 1~6 alkyl or halogen, R 5 is, H or -N(R 7 )2, R 6 is, -C(O)NH(R 8 )、-C(O)R 7A , or -SO2R 7A and, each R 7 is, independently, hydrogen, optionally substituted C 1~6 alkyl, optionally substituted C 6~10 aryl C 1~6 alkyl, optionally substituted C 3~8 cycloalkyl, optionally substituted C 6~10 aryl, optionally substituted C 2~9 heterocyclyl, optionally substituted C 1~9 heteroaryl, optionally substituted C 1~9 heteroaryl C 1~6Alkyl, or -SO2R 7A and, or two R 7 The group, together with the atom to which it is bonded, is an optionally substituted C 2~9 Forming heterocyclines, Each R 7A This is an independently and arbitrarily substituted C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl or optionally substituted C 6~10 It is Ariel, Each R 7B These are independently hydroxyl, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, -N(R 7 )2, -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or optionally substituted alkoxy, Each R 8 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkoxyalkyl groups, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 3~8 Cycloalkyl or optionally substituted C 1~9 It is a heteroaryl, or two R 8 These, together with the atoms to which they are bonded, form an arbitrarily substituted C 2~9 Forming heterocyclines, Q is C which is arbitrarily substituted. 1~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 Alkynylene, optionally substituted C 3~8 Cycloalkylene, optionally substituted C 3~8 Cycloalkenylene, optionally substituted C 6~10 Arrine, optionally substituted C2~9 Heterocyclylene, or optionally substituted C 1~9 It is a heteroarylene.

[0022] In some embodiments, the Myt1 inhibitor has been enriched with respect to the atrop isomer of formula (IA).

[0023] [ka]

[0024] In some embodiments, X is CR 2 In some embodiments, the Myt1 inhibitor is of formula (II).

[0025] [ka]

[0026] In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (IIA).

[0027] [ka]

[0028] In some embodiments, the Myt1 inhibitor is expressed by formula (III):

[0029] [ka]

[0030] (In the formula, R 2A is hydrogen, optionally substituted with C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, optionally substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, -N(R 7 )2-OR 7 , -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -Q-R 7B is one of).

[0031] In some embodiments, the Myt1 inhibitor is enriched for the atropisomer of formula (IIIA).

[0032]

Chemical formula

[0033] In some embodiments, R 2A is hydrogen, optionally substituted C 1~6 alkyl, or halogen. In some embodiments, R 3 is optionally substituted C 1~6 alkyl. In some embodiments, R 3 is halogen. In some embodiments, R 4 is optionally substituted C 1~6 alkyl. In some embodiments, R 4 is halogen (e.g., chlorine).

[0034] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is optionally substituted C 1~6 alkyl. In some embodiments, R 2R is optionally substituted with methyl or optionally substituted with isopropyl. 2 It is a halogen.

[0035] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a halogen. In some embodiments, R 1 is chlorine or bromine. In some embodiments, R 1 is an arbitrarily substituted C 1~6 It is alkyl. In some embodiments, R 1 R is optionally substituted methyl, optionally substituted ethyl, optionally substituted isopropyl, or optionally substituted butyl. In some embodiments, R 1 is an arbitrarily substituted C 1~9 It is a heteroaryl compound. In some embodiments, R 1 R is 1,3-thiazolyl, 1,2-thiazolyl, 1,3-oxazolyl, benzo-1,3-thiazolyl, benzo-1,3-oxazolyl, indolyl, benzimidazolyl, pyridyl, imidazolyl, pyrimidyl, pyrazinyl, pyridadinyl, or pyrazolyl, where R 1 is an arbitrarily substituted C 1~9 They are optionally substituted with substituents as defined for heteroaryls. In some embodiments, R 1 is an arbitrarily substituted C 3~8 It is cycloalkyl. In some embodiments, R 1 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, where R 1 is an arbitrarily substituted C 3~8 They are optionally substituted with substituents as defined for cycloalkyls. In some embodiments, R 1 is an arbitrarily substituted C 2~9 It is a heterocycline. In some embodiments, R 1is 1,2,3,6-tetrahydropyridinyl, piperidinyl, morpholinyl, piperazinyl, thiomorpholinyl, oxa-aza-spiro[3,3]heptane, or oxa-aza-bicyclo[3.2.1]octane, where R 1 is an arbitrarily substituted C 2~9 They are optionally substituted with substituents as defined for heterocyclines. In some embodiments, R 1 is an arbitrarily substituted C 3~8 It is cycloalkyl. In some embodiments, R 1 is optionally substituted cyclohexenyl or optionally substituted cyclopentenyl. In some embodiments, R 1 is an arbitrarily substituted C 6~10 It is an arrow. In some embodiments, R 1 This is a phenyl compound with arbitrary substitutions.

[0036] In some embodiments, R 1 -QR 7B In some embodiments, Q is optionally replaced by C. 2~6 It is an alkynylene. In some embodiments, Q is optionally substituted with C 1~6 It is an alkylene. In some embodiments, Q is optionally substituted with C 6~10 It is arylene. In some embodiments, R 7B is an arbitrarily substituted C 2~9 It is a heterocycline. In some embodiments, R 7B is an arbitrarily substituted C 6~10 It is Ariel.

[0037] In some embodiments, R 1 These are methyl, difluoromethyl, trifluoromethyl, fluorine, chlorine, bromine, amino, hydroxyl, cyano, oxo, -C(O)NH2, -C(O)NH(Me), -C(O)N(Me)2, and -(CH2). n -C(O)OH, and -(CH2) nIt is optionally substituted with one, two, or three groups independently selected from the group consisting of -C(O)Ot-Bu, where n is 0 or 1.

[0038] In some embodiments, R 1 is -N(R 7 )2. In some embodiments, R 1 It is diethylamino. In some embodiments, R 5 is hydrogen. In some embodiments, R 5 is -N(R 7 )2. In some embodiments, R 5 is -NH2. In some embodiments, R 6 is -C(O)NH(R 8 ) is. In some embodiments, R 6 is -C(O)NH2. In some embodiments, R 6 is -C(O)NH(Me). In some embodiments, R 6 is -SO2R 7A In some embodiments, R 6 It is -SO2Me.

[0039] In some embodiments, the Myt1 inhibitor is selected from the group consisting of compounds 1 to 328 in Table 1 and their pharmaceutically acceptable salts. In some embodiments, the Myt1 inhibitor is compound 182 in Table 1.

[0040] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of “or” shall mean “and / or” unless otherwise specified. The use of the term “including,” as well as other forms such as “includes” and “included,” is not limited to these.

[0041] Unless otherwise specified, nucleic acid sequences described herein are oriented 5' to 3' when read from left to right. Nucleic acid sequences may be provided as DNA, as RNA, or as a combination of DNA and RNA (e.g., chimeric nucleic acids).

[0042] The term “comprise” is intended to mean “include.” Furthermore, the use of the term “including,” as well as other forms such as “includes” and “included,” is not limited. Where a term is provided in the singular form, aspects of the invention described by the plural form of that term are also intended. Where used herein, the term “and / or” should be interpreted as a specific disclosure of each of several particular features or components, with or without another feature or component. Accordingly, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Other terms and definitions are defined below.

[0043] The term "approximately" applied to one or more target values ​​means a value that falls within 10% of the stated reference value in either direction (greater than or less than) unless otherwise specified or the context makes it clear (unless such a number exceeds 100% of the possible value).

[0044] As used herein, the term "acyl" represents the group -C(=O)-R (wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heterocyclyl). The acyl may be optionally substituted for each R group as described herein.

[0045] As used herein, the term “alkanoyl” refers to a hydrogen or alkyl group bonded to a parent group via a carbonyl group, exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, propionyl, butyryl, and isobutyryl. Unsubstituted alkanoyl groups contain 1 to 7 carbon atoms. Alkanoyl groups may be unsubstituted or substituted (e.g., optionally substituted C1-7 alkanoyls), as described herein for alkyl groups. The suffix “-oyl” may be added to other groups defined herein, such as aryl, cycloalkyl, and heterocyclyl, to define “aryloyl,” “cycloalkanoyl,” and “(heterocyclyl)oyl.” These groups represent carbonyl groups substituted with aryl, cycloalkyl, or heterocyclyl, respectively. Each of “aryloyl,” “cycloalkanoyl,” and “(heterocyclyl)oil” can be optionally substituted as defined for “aryl,” “cycloalkyl,” or “heterocyclyl,” respectively.

[0046] As used herein, the term “alkenyl” refers to an acyclic, monovalent, linear or branched hydrocarbon group having one, two, or three carbon-carbon double bonds. Non-limiting examples of alkenyl groups include ethenyl, propa-1-enyl, propa-2-enyl, 1-methylethenyl, buta-1-enyl, buta-2-enyl, buta-3-enyl, 1-methylpropa-1-enyl, 2-methylpropa-1-enyl, and 1-methylpropa-2-enyl. Alkenyl groups may be optionally substituted, as defined herein for alkyl groups.

[0047] As used herein, the term “alkenylene” refers to a divalent alkenyl group. An optionally substituted alkenylene is an optionally substituted alkenylene as described herein for alkenyls.

[0048] As used herein, the term "alkoxy" refers to the formula -OR (wherein R is C unless otherwise specified). 1~6 This represents a chemical substituent of an alkyl group. In some embodiments, the alkyl group may be further substituted as defined herein. The term “alkoxy” can be used in combination with other terms defined herein, such as aryl, cycloalkyl, or heterocyclyl, to define the “arylalkoxy,” “cycloalkylalkoxy,” and “(heterocyclyl)alkoxy” groups. These groups each represent an alkoxy substituted with an aryl, cycloalkyl, or heterocyclyl group. Each of “arylalkoxy,” “cycloalkylalkoxy,” and “(heterocyclyl)alkoxy” may be optionally substituted as defined herein for each individual part.

[0049] As used herein, the term "alkoxyalkyl" refers to the formula -LOR (wherein L is C). 1~6 Alkylene, and R is C 1~6This represents a chemical substituent of an alkyl group. An optionally substituted alkoxyalkyl group is an optionally substituted alkoxyalkyl group as described herein for alkyl groups.

[0050] As used herein, the term "alkyl" refers to an acyclic, straight-chain or branched-chain saturated hydrocarbon group, which, unless otherwise specified, has 1 to 12 carbon atoms when unsubstituted. In certain preferred embodiments, the unsubstituted alkyl group has 1 to 6 carbon atoms. Alkyl groups are exemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso-, and tert-butyl; neopentyl, etc., and may be substituted with one, two, or three substituents independently selected from the group consisting of amino; alkoxy; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heterocyclyl; (heterocyclyl)oxy; heteroaryl; hydroxy; nitro; thiol; silyl; cyano; alkylsulfonyl; alkylsulfinyl; alkylsulfenyl; =O; =S; -C(O)R or -SO2R (wherein R is amino); and =NR' (wherein R' is H, alkyl, aryl, or heterocyclyl), or, in the case of alkyl groups of two or more carbons, with four or more substituents, to the extent permitted by the valence. Each substituent may be unsubstituted by itself, or may be substituted with unsubstituted substituents as defined herein for each group, to the extent permitted by the valence.

[0051] As used herein, the term "alkylene" refers to a divalent alkyl group. Optionally substituted alkylenes are alkylenes that are optionally substituted as described herein for alkyl groups.

[0052] As used herein, the term "alkylamino" refers to the formula -N(R N1 )2 or -NHR N1 (In the formula, R N1A group having an alkyl group as defined herein. The alkyl portion of the alkylamino may be optionally substituted as defined for alkyl groups. Each optional substituent on the substituted alkylamino may be unsubstituted itself, or may be substituted with an unsubstituted substituent as defined herein for each respective group, to the extent permitted by its valence.

[0053] As used herein, the term "alkylsulfenyl" refers to a group of the formula -S-(alkyl). Alkylsulfenyl may be optionally substituted as defined for alkyl.

[0054] As used herein, the term "alkylsulfinyl" refers to a group of the formula -S(O)-(alkyl). Alkylsulfinyls may be optionally substituted as defined for alkyls.

[0055] As used herein, the term "alkylsulfonyl" refers to a group of the formula -S(O)2-(alkyl). Alkylsulfonyls may be optionally substituted as defined for alkyls.

[0056] As used herein, the term "alkynyl" refers to a monovalent linear or branched hydrocarbon group of 2 to 6 carbon atoms having at least one carbon-carbon triple bond, exemplified by ethynyl, 1-propynyl, and the like. The alkynyl group may be unsubstituted or substituted (e.g., optionally substituted alkynyl), as defined for alkyl.

[0057] As used herein, the term "alkynylene" refers to a divalent alkynyl group. An optionally substituted alkynylene is an optionally substituted alkynylene as described herein for alkynyl.

[0058] As used herein, the term "amino" refers to -N(R N1 )2(In the formula, if amino is unsubstituted, both R N1is H, or if amino is substituted, each R N1 These are independently H, -OH, -NO2, -N(R) N2 )2, -SO2OR N2 , -SO2R N2 -SOR N2 , -C(O)OR N2 , N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least one R N1 It is not H, but each R N2 A(H, alkyl, or aryl) is independent of B(H, alkyl, or aryl). Each substituent may be unsubstituted by itself or substituted with an unsubstituted substituent as defined herein for each group. In some embodiments, the amino is an unsubstituted amino (i.e., -NH2) or a substituted amino (e.g., -NHR2). N1 )(wherein, R N1 These are independently -OH, SO2OR N2 , -SO2R N2 -SOR N2 ,-COOR N2 , optionally substituted alkyl, or optionally substituted aryl, and each R N2 (which may be an optionally substituted alkyl or optionally substituted aryl). In some embodiments, the substituted amino may be an alkylamino, and the alkyl may be optionally substituted as described herein for alkyl. In some embodiments, the amino group is -NHR N1 (In the formula, R N1 (where is an optionally substituted alkyl group).

[0059] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic carbocyclic system having one or two aromatic rings. An aryl group may contain 6 to 10 carbon atoms. All atoms in an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl. The aryl group may be unsubstituted, or it may be alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; -(CH2) n -C(O)OR A ;-C(O)R; and SO2R (wherein R is amino or alkyl, R A The group may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of (where is H or alkyl and n is 0 or 1). Each substituent may be unsubstituted by itself or substituted with an unsubstituted substituent as defined herein for each group.

[0060] As used herein, the term "arylene" refers to a divalent aryl group. Optionally substituted arylenes are arylenes in which the aryl group is optionally substituted as described herein.

[0061] As used herein, the term "aryloxy" refers to a chemical substituent of the formula -OR (wherein R is an aryl group unless otherwise specified). In an optionally substituted aryloxy, the aryl group is optionally substituted as described herein for aryl groups.

[0062] As used herein, the term "azide" refers to the -N3 group. The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell proliferation. Cancer includes solid tumor carcinomas and non-solid tumor carcinomas, as well as locally advanced or metastatic cancers (e.g., locally advanced or metastatic tumors). Examples of cancer include carcinomas, lymphomas, blastomas, sarcomas, gliomas, mesotheliomas, and leukemia or lymphoid malignancies. Specific examples of cancer include urothelial carcinoma (UC), e.g., locally advanced and metastatic UC (mUC); bladder cancer (e.g., muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC), e.g., BCG-resistant NMIBC); MIBC urothelial bladder cancer (UBC); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); urinary tract cancer; lung cancer, e.g., small cell lung cancer (SCLC), including advanced-stage SCLC (ES-SCLC); and non-small cell lung cancer, including squamous or non-squamous NSCLC. Adenocarcinomas of the lung, or squamous cell carcinomas (e.g., squamous cell carcinoma of the lung), such as lung cell carcinoma (NSCLC), locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC), or recurrent or metastatic NSCLC (e.g., stage IV NSCLC); pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC), e.g., metastatic PDAC); head and neck cancers (e.g., SCCHN, e.g., recurrent / metastatic PD-L1 positive SCCHN, and head and neck squamous cell carcinomas). Hormonal carcinoma (HNSCC); ovarian cancer (OC); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer (GC) (e.g., gastroesophageal junction (GEJ) cancer), and gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer; glioblastoma; urinary tract cancer; liver cancer; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC (e.g., early TNBC (eTNBC)), which are estrogen receptor (ER-), progesterone receptor (PgR-), and HER2 (HER2-) negative); prostate cancer, For example, castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric cancer or stomach cancer, e.g., gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer (e.g., stage IVB, metastatic, recurrent, or persistent cervical cancer, e.g., metastatic and / or recurrent PD-L1 positive cervical cancer); ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC), e.g., locally advanced or metastatic HCC and / or unresectable HCC); liver cancer; colon cancer; rectal cancer;Colorectal cancer (CRC; e.g., microsatellite-stable (MSS) and microsatellite-instability (MSI-Low) CRC); endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial diffuse melanoma, lentiginous melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocyte (SL) NHL; moderate-grade / follicular NHL; moderate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade Examples include high-grade lymphoblastic NHL; high-grade small unsevered cell NHL; giant lesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hair cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorders (PTLD); myelodysplastic syndromes (MDS), as well as facomatosis, edema (including those associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and abnormal angiogenesis associated with associated metastases.

[0063] As used herein, the term "carbocyclic" refers to an optionally substituted C3-16 monocyclic, bicyclic, or tricyclic structure in which a ring, which may be aromatic or non-aromatic, is formed by carbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, cycloalkynyl, and certain aryl groups.

[0064] As used herein, the term "carbonyl" refers to a -C(O)- group. The terms "CCNE1" and "cyclin E1," as used interchangeably in this specification, refer to G1 / S-specific cyclin E1 (gene name: CCNE1).

[0065] As used herein, the "CCNE control locus" is one of the following loci: chr19 q13.43, chr19 q13.41, chr19 q13.42, chr19 p13.3, chr19 q13.32, chr19 q13.33, chr19 q13.31, chr19 p13.13, or chr19 q13.13. The CCNE1 control loci described herein can be used in fluorescence in situ hybridization (FISH) assays when the target genomic locus is or contains chr19 q12 or the cyclin E1 (CCNE1) gene.

[0066] As used herein, the term "cyano" refers to the -CN group. As used herein, the term "cycloalkenyl" means, unless otherwise specified, a non-aromatic carbocyclic group having at least one double bond in the ring and 3 to 10 carbon atoms (e.g., C 3~10 This refers to cycloalkenyls. Non-restrictive examples of cycloalkenyls include cyclopropa-1-enyl, cyclopropa-2-enyl, cyclobuta-1-enyl, cyclobuta-1-enyl, cyclobuta-2-enyl, cyclopenta-1-enyl, cyclopenta-2-enyl, cyclopenta-3-enyl, norbornene-1-yl, norbornene-2-yl, norbornene-5-yl, and norbornene-7-yl. The cycloalkenyl group may be unsubstituted or substituted as described for cycloalkyls (e.g., optionally substituted cycloalkenyls).

[0067] As used herein, the term "cycloalkenylalkyl" refers to an alkyl group substituted with a cycloalkenyl group, as defined herein. The cycloalkenyl and alkyl moieties may be substituted as the individual groups defined herein.

[0068] As used herein, the term "cycloalkenylene" refers to a divalent cycloalkenyl group. Optionally substituted cycloalkenylenes are cycloalkenylenes that are optionally substituted, as described herein for cycloalkyls.

[0069] As used herein, the term "cycloalkoxy" refers to a chemical substituent of the formula -OR (wherein R is a cycloalkyl group unless otherwise specified). In some embodiments, the cycloalkyl group may be further substituted as defined herein.

[0070] As used herein, the term "cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms (e.g., C) unless otherwise specified. 3~C10This refers to a cycloalkyl group. The cycloalkyl group may be monocyclic or bicyclic. The bicyclic cycloalkyl group may be of the bicyclo[pq0]alkyl type (wherein p and q are independently 1, 2, 3, 4, 5, 6, or 7, with the sum of p and q being 2, 3, 4, 5, 6, 7, or 8). Alternatively, the bicyclic cycloalkyl group may include a cross-linked cycloalkyl structure, such as a bicyclo[pqr]alkyl group (wherein r is 1, 2, or 3, and p and q are independently 1, 2, 3, 4, 5, or 6, with the sum of p, q, and r being 3, 4, 5, 6, 7, or 8). The cycloalkyl group may be a spirocyclic group, for example, a spiro[pq]alkyl group (wherein p and q are independently 2, 3, 4, 5, 6, or 7, but the sum of p and q is 4, 5, 6, 7, 8, or 9). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and dekalinyl. Cycloalkyl groups may be unsubstituted, or alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; heteroaryl; hydroxy; nitro; thiol; silyl; cyano; =O; =S; -SO2R (wherein R is an optionally substituted amino); -NR' (wherein R' is H, alkyl, aryl, or heterocyclyl); and -CON(R A )2(in the formula, each R A These are independently H or alkyl, or both R AThe substituents may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of (which, together with the atoms to which they are bonded, form a heterocycline) (e.g., optionally substituted cycloalkyls). Each substituent may be unsubstituted by itself, or may be substituted with an unsubstituted substituent as defined herein for each group.

[0071] As used herein, the term "cycloalkyl alkyl" refers to an alkyl group substituted with a cycloalkyl group as defined herein. The cycloalkyl and alkyl moieties may be optionally substituted with any of the individual groups described herein.

[0072] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. Optionally substituted cycloalkylenes are cycloalkylenes that have been optionally substituted as described herein for cycloalkyl groups.

[0073] As used herein, the term "cycloalkynyl" refers, unless otherwise specified, to a monovalent carbocyclic group having one or two carbon-carbon triple bonds and containing 8 to 12 carbon atoms. Cycloalkynyls may contain one transcyclic bond or bridge. Non-limiting examples of cycloalkynyls include cyclooctinyl, cyclononinyl, cyclodecinyl, and cyclodecadyinyl. The cycloalkynyl group may be unsubstituted or substituted as defined for cycloalkyl (e.g., optionally substituted cycloalkynyl).

[0074] The term “cytogenetic abnormality” is used herein to refer to chromosomal abnormalities or deletions, such as those resulting from chromosomal deletions, duplications, nondisjunctions, inversions, amplifications, and translocations. Abnormalities resulting from chromosomal deletions, duplications, amplifications, and nondisjunctions may result in aneuploidy. As used herein, the term “cytogenetic abnormality” may also refer to chromosomal abnormalities or deletions, such as deletions, duplications, or amplifications of genes, loci, genetic elements, or bands within a chromosome (e.g., chromosomal bands).

[0075] The terms “diagnosed” and “diagnosis” are used herein to refer to the identification or classification of a molecular or pathological condition, disease, or state (e.g., cancer). For example, “diagnosed” may refer to the identification of a particular type of cancer.

[0076] "Disease" or "condition" refers to the state of existence or health condition of a patient or subject that can be treated by the compounds or methods provided herein. The term “target genomic locus” is used herein to refer to a polynucleotide sequence (e.g., one or more genes or chromosomal bands) within one or more chromosomal DNA molecules (e.g., nuclear and / or mitochondrial DNA) that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to the target probe used in the methods described herein. For example, a target genomic locus may be the CCNE1 gene, a region within the CCNE1 gene, a region containing the CCNE1 gene, chr19 q12, or a region within chr19 q12.

[0077] As used herein, the term "halo" refers to a halogen selected from bromine, chlorine, iodine, and fluorine. As used herein, the terms “human epidermal growth factor receptor 2” and “HER2” refer to the erb-b2 receptor tyrosine kinase 2 (ERBB2) gene (e.g., NCBI gene ID: 2064) or its encoded transcript (e.g., GenBank: MW358920.1 or LN812234.1; or NCBI reference sequences: NM_001382796.1, NM_001382800.1,796.1, NM_001382800.1, NM_001382796.1, NM_001382796.1, NM_001382796.1, NM_001382796.1, NM_001382800.1, NM_001382796.1, NM_001382796.1, NM_001382796.1, It is used to refer to 0.1, NM_001382803.1, or NM_001382805.1) or its protein product (e.g., UniProtKB / Swiss-Prot:P04626.1; NCBI reference sequences:NP_001369725.1, NP_001369719.1, NP_001369728.1, or NP_001369731.1; or GenBank:QSQ86066.1).

[0078] As used herein, the terms “HER2+” or “HER2-positive” refer to cells that overexpress HER2 mRNA and / or protein at elevated levels compared to a control (e.g., non-cancerous cells). Generally, HER2+ cells can be identified using immunohistochemistry (IHC) or FISH assays.

[0079] As used herein, the term “heteroalkyl” refers to an alkyl, alkenyl, or alkynyl group interposed once by one or two heteroatoms, twice independently each time by one or two heteroatoms, three times independently each time by one or two heteroatoms, or four times independently each time by one or two heteroatoms. Each heteroatom is independently O, N, or S. In some embodiments, the heteroatoms are O or N. None of the heteroalkyl groups contain two consecutive oxygen or sulfur atoms. Heteroalkyl groups may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl groups). If a heteroalkyl group is substituted and the substituent is bonded to the heteroatom, the substituent is selected according to the properties and valence of the heteroatom. Thus, substituents bonded to the heteroatom to the extent permitted by valence are =O, -N(R N2)2, -SO2OR N3 , -SO2R N2 -SOR N3 ,-COOR N3 , selected from the group consisting of an N protecting group, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, or cyano (wherein each R N2 Each R is independently H, alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl, and each R N3 (These are independently alkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, or heterocyclyl). Each of these substituents may be unsubstituted by itself or substituted with an unsubstituted substituent as defined herein for each group. If a heteroalkyl is substituted and the substituent is bonded to a carbon, the substituent is selected from those described for alkyl, except that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. It is understood that the carbon atom is found at the terminus of the heteroalkyl group.

[0080] As used herein, the term “heteroarylene” refers to a divalent heteroaryl. An optionally substituted heteroarylene is a heteroarylene that has been optionally substituted as described herein for heteroaryls.

[0081] As used herein, the term “heterocyclyl” refers, unless otherwise specified, to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having condensed, bridged, and / or spiro-3, 4, 5, 6, 7, or 8-membered rings having 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, “heterocyclyl” is, unless otherwise specified, a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having condensed or bridged 5, 6, 7, or 8-membered rings having 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyls may be aromatic or non-aromatic. Non-aromatic 5-membered heterocyclyl groups have 0 or 1 double bonds, non-aromatic 6-membered and 7-membered heterocyclyl groups have 0 to 2 double bonds, and non-aromatic 8-membered heterocyclyl groups have 0 to 2 double bonds and / or 0 or 1 carbon-carbon triple bond. Unless otherwise specified, heterocyclyl groups contain 1 to 16 carbon atoms. Certain heterocyclyl groups may contain up to 9 carbon atoms. Examples of non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridadinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, and dithiazolyl. If a heterocyclic system has at least one aromatic resonance structure or at least one aromatic tautomer, such a structure is an aromatic heterocyclyl (i.e., heteroaryl).Non-restrictive examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, prinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, and tetrazolyl. The term "heterocyclyl" also refers to heterocyclic compounds having a bridging polycyclic structure in which one or more carbons and / or heteroatoms bridge two non-adjacent members of a monocyclic ring (e.g., quinuclidine, tropane, or diaza-bicyclo[2.2.2]octane). The term "heterocyclyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocycles is fused to one, two, or three carbon rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocycle. Examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindidine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted, or it may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl; alkenyl; alkynyl; alkoxy; alkoxy; alkylsulfinyl; alkylsulfenyl; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heteroalkyl; heterocyclyl; (heterocyclyl)oxy; hydroxy; nitro; thiol; silyl; cyano; -C(O)R or -SO2R (wherein R is amino or alkyl); =O; =S; =NR' (wherein R' is H, alkyl, aryl, or heterocyclyl). Each substituent may be unsubstituted, or it may be substituted with an unsubstituted substituent as defined herein for the respective group.

[0082] As used herein, the term “heterocyclylene” refers to a divalent heterocyclyl. An optionally substituted heterocyclylene is a heterocyclylene that has been optionally substituted as described herein for heterocyclyl.

[0083] As used herein, the term "(heterocyclyl)oxy" represents a chemical substituent of the formula -OR (wherein R is a heterocyclyl group unless otherwise specified). (Heterocyclyl)oxy may be optionally substituted in the manner described for heterocyclyl.

[0084] The terms "hydroxyl" and "hydroxy" as used interchangeably in this specification refer to the -OH group. In this specification, “Homo sapiens genome assembly” is used to refer to the National Center for Biotechnology Information (NCBI) complete human genome assembly version 36, commonly known as “NCBI36,” which can be found at www.ncbi.nlm.nih.gov / assembly / GCF_000001405.12 / . The NCBI36 human genome assembly is synonymous with the University of California, Santa Cruz's (UCSC) human genome (hg) assembly version 18, commonly known as “hg18.”

[0085] As used herein, "hybridization" or "annealing" of nucleic acids is achieved when one or more nucleoside residues within a polynucleotide base pair with one or more complementary nucleosides to form a stable double helix. Base pairing is typically driven by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed from native and / or modified nucleic acid bases. Hybridization can also include non-Watson-Crick base pairs, such as fluctuation base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs. Nucleic acids do not need to be 100% complementary to undergo hybridization. For example, one nucleic acid may be 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but the two nucleic acids can still form enough base pairs to hybridize with each other.

[0086] As used herein, the term "Myt1" refers to membrane-bound tyrosine and threonine-specific cdc2 inhibitory kinase (Myt1) (gene name PKMYT1). As used herein, the term "Myt1 inhibitor" refers to a substance that, upon contact with the enzyme Myt1, whether in vitro, in cell culture, or in animals, exhibits a measured Myt1 IC50. 50 This represents a compound that reduces Myt1 activity to 10 μM or less (e.g., 5 μM or less or 1 μM or less). For a specific Myt1 inhibitor, Myt1 IC2 is defined as follows: 50 The ion may be 100 nM or less (for example, 10 nM or less, or 3 nM or less), and may be as low as 100 pM or 10 pM. Preferably, Myt1 IC 50 The molecular weight is 1 nm to 1 μM (for example, 1 nM to 750 nM, 1 nM to 500 nM, or 1 nM to 250 nM). More preferably, Myt1 IC 50 This is less than 20 nm (for example, 1 nM to 20 nM).

[0087] As used herein, the term "nitro" represents the -NO2 group. As used herein, the term "oxo" represents a divalent oxygen atom (e.g., the structure of oxo can be shown as =O).

[0088] As used herein, the terms "programmed death ligand 1" and "PD-L1" are used to refer to the CD274 gene (e.g., NCBI gene ID: 29126) or the encoded transcript (e.g., NCBI reference sequence: NR_052005.2, NM_014143.4, NM_001267706.2, XM_047423262.1, or NM_001314029.2) or its protein product (e.g., GenBank: AAI13735.1, AAI13737.1, AAH74984.1, KAI4006593.1; or NCBI reference sequence: NP_001300958.1). The encoded PD-L1 protein functions as an immune inhibitory receptor ligand and is generally expressed in hematopoietic and non-hematopoietic cells such as T cells and B cells, as well as various types of tumor cells.

[0089] As used herein, the terms "PD-L1+" or "PD-L1 positive" are used to refer to cells that overexpress PD-L1 mRNA and / or protein at increased levels relative to a control (e.g., non-cancerous cells). Generally, immunohistochemistry (IHC) or FISH assays can be utilized to identify PD-L1+ cells.

[0090] As used herein, the term "Ph" represents phenyl. As used herein, the term “pharmaceutically acceptable salt” refers to a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or allergic response, and that is commensurate with a reasonable benefit-risk ratio. pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use (Eds. PHStahl and CGWermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting free base groups with suitable organic acids. Typical acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptone, glycerophosphate, hemisulfate, heptone, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfone. Examples include acid salts, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, and valersates.Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations (such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine, but not limited to these).

[0091] As used interchangeably herein, “polynucleotide,” “oligonucleotide,” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. A nucleotide may be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogues thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by synthetic reactions. Therefore, for example, polynucleotides as defined herein include, but are not limited to, single-stranded and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA containing single-stranded and double-stranded regions, single-stranded, or more typically double-stranded, or hybrid molecules containing DNA and RNA that may contain single-stranded and double-stranded regions. Furthermore, as used herein, the term “polynucleotide” refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The strands within such a region may originate from the same molecule or from different molecules. A region may contain all of one or more molecules, but more typically, only some regions of a molecule. One of the molecules in the triple helix region is often an oligonucleotide. The terms "polynucleotide" and "nucleic acid" include mRNA and cDNA in particular.

[0092] Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogues. If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after synthesis, such as by conjugation with labels. Other types of modifications include, for example, "caps," substitutions with one or more naturally occurring nucleotide analogs, internucleotide modifications (e.g., those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.)), those containing pendant moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.)), those containing intercalators (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (e.g., α-anomeric nucleic acids), and unmodified forms of polynucleotides. Furthermore, any of the hydroxyl groups normally present in sugars can be substituted with, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, activated to prepare further binding to further nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also have analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azid-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars, e.g., arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and baseless nucleoside analogs, e.g., methylriboside. One or more phosphodiester bonds can be substituted with alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate is substituted with P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2" ("amidate"), P(O)R, P(O)OR', CO or CH2" ("formacetal") (wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl). Not all links in the polynucleotide need to be identical. The foregoing description applies to all polynucleotides, oligonucleotides, and nucleic acids referred to herein, including RNA and DNA.

[0093] As used herein, the term “protecting group” refers to a group intended to protect a hydroxyl, amino, or carbonyl group from involvement in one or more undesirable reactions during chemical synthesis. As used herein, the term “O-protecting group” refers to a group intended to protect a hydroxyl or carbonyl group from involvement in one or more undesirable reactions during chemical synthesis. As used herein, the term “N-protecting group” refers to a group intended to protect a nitrogen-containing (e.g., amino, amide, heterocyclic NH, or hydrazine) group from involvement in one or more undesirable reactions during chemical synthesis. Commonly used O- and N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Examples of O and N protecting groups include alkanoyl, allyroyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-isopropylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl.

[0094] Examples of O-protecting groups for protecting carbonyl-containing groups include, but are not limited to, acetals, acylars, 1,3-dithiane, 1,3-dioxane, 1,3-dioxolane, and 1,3-dithiolane.

[0095] Other O-protecting groups include substituted alkyl, aryl, and aryl-alkyl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., Examples include, but are not limited to, trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); and carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

[0096] Other N-protecting groups include, but are not limited to, protected or unprotected D,L or D,L-amino acids, such as chiral auxiliary groups like alanine, leucine, and phenylalanine; sulfonyl-containing groups such as benzenesulfonyl and p-toluenesulfonyl; benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl Examples include carbamate-forming groups such as ru-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl; aryl-alkyl groups such as benzyl, p-methoxybenzyl, 2,4-dimethoxybenzyl, triphenylmethyl, and benzyloxymethyl; silylalkyl acetal groups such as [2-(trimethylsilyl)ethoxy]methyl; and silyl groups such as trimethylsilyl. Useful N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, dimethoxybenzyl, [2-(trimethylsilyl)ethoxy]methyl (SEM), tetrahydropyranyl (THP), t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0097] As used herein, the terms “sample” or “biological sample” refer to a composition obtained from or derived from a subject. Compositions may include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, whole blood, blood-derived cells, cerebrospinal fluid, saliva, sputum, sweat, mucus, feces, tumors, tumor lysates, and tissues and tissue extracts, such as homogenized tissues, tumor tissues, cell extracts, and combinations thereof.

[0098] As used herein, “stable control locus” refers to a region within a cancer cell's chromosome (e.g., a chromosomal band) that is less likely to cause duplication events compared to another region within the same chromosome (e.g., another chromosomal band) within the cancer cell. Stable control loci are used as controls or normalizers for target genomic loci analyzed by FISH assays.

[0099] As used herein, the term “subject” refers to a human or non-human animal (e.g., mammal) that is suffering from or at risk of suffering from a disease or condition, as determined by a qualified professional (e.g., a physician or nurse) with or without using known laboratory tests in the art on a sample derived from the subject. Preferably, the subject is human. Non-limiting examples of diseases and conditions include diseases with symptoms of cell overgrowth, such as cancer.

[0100] As used herein, “target genomic locus” refers to any region within a chromosome (e.g., a locus, a chromosomal band, and / or one or more genes or genetic elements) where amplification has been determined. Essentially, the target genomic locus is an experimentally variable element of the FISH assay.

[0101] "Tissue sample" or "cell sample" means a collection of similar cells obtained from the tissue of a subject or individual. Sources of tissue or cell samples may include solid tissues such as fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; any blood component such as blood or plasma; body fluids such as cerebrospinal fluid, amniotic fluid, ascites, peritoneal fluid, or interstitial fluid; or cells from any point in the subject's pregnancy or development. Tissue samples may also be primary cells, cultured cells, or cell lines. Optionally, tissue or cell samples may be obtained from diseased tissue / organs. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. A tissue sample may contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancerous cells and non-cancerous cells). A tissue sample may contain compounds that do not naturally mix with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, waxes, nutrients, and antibiotics. In some embodiments, a sample is a tumor tissue sample. In some embodiments, the tumor tissue sample is a UC tumor tissue sample (e.g., a bladder cancer tumor tissue sample (e.g., a MIBC tumor tissue sample)). In some embodiments, the sample is a transurethral resection (TURBT) sample. In some embodiments, the sample is a cystectomy or nephroureterectomy sample. In other embodiments, the tumor tissue sample is a lung cancer tumor tissue sample (e.g., an early lung cancer tissue sample (e.g., an NSCLC tumor tissue sample (e.g., a stage II, IIIA, or IIIB NSCLC tumor tissue sample), e.g., a squamous or non-squamous NSCLC tumor tissue sample, e.g., a resectable NSCLC tumor tissue sample)). In some embodiments, the sample is a locally advanced, unresectable NSCLC tumor tissue sample (e.g., a stage IIIB NSCLC tumor tissue sample), or a recurrent or metastatic NSCLC tumor tissue sample (e.g., a stage IV NSCLC tumor tissue sample), a pancreatic cancer tumor tissue sample (e.g., a PDAC tumor tissue sample), a metastatic PDAC tumor tissue sample, or a breast cancer tumor tissue sample (e.g., a HER2+ breast cancer tumor tissue sample or a TNBC tumor tissue sample).

[0102] As used herein, the terms “treatment” and “to treat” refer to the medical management of a subject with the intention of improving, relieving, stabilizing, preventing, or curing a disease or condition. This includes active treatment (treatment aimed at improving a disease or condition), causal treatment (treatment directed at the cause of the associated disease or condition), symptomatic treatment (treatment designed to alleviate the symptoms of a disease or condition), preventive treatment (treatment aimed at minimizing, partially or completely inhibiting, the onset of the associated disease or condition), and supportive care (treatment used to complement another therapy).

[0103] As used herein, “tumor” refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive when used herein. [Brief explanation of the drawing]

[0104] [Figure 1] This bar graph shows that cyclin E1 (CCNE1) amplification is particularly frequent in uterine and ovarian cancers, illustrating the prevalence of CCNE1 amplification in tumor types from The Cancer Genome Atlas (TCGA). CCNE1 amplification frequencies were calculated based on Gistic2 values ​​available in the TCGA PanCancer Atlas, hosted on the publicly available cBioPortal website (www.cbioportal.org). Uterine carcinosarcoma, ovarian cancer, and uterine cancer (endometrial cancer of the uterine body) are gynecological tumors. [Figure 2]This dot diagram shows how the ploidy of chromosome (chr) bands changes between chromosomes and tumor types. Each data point in this diagram corresponds to a chromosome band in the genome. The y-axis shows the percentage of patients whose chromosome band ploidy is approximately equal to 2 (e.g., 1.8–2), which is considered diploid. This information was calculated for different tumor indications such as TCGA-OV (ovarian cancer), TCGA-UCEC (uterine cancer, e.g., endometrial cancer of the uterine body), and TCGA-UCS (uterine carcinosarcoma). The x-axis separates the chromosome data. This diagram demonstrates that the diploid frequency of bands changes within chromosomes, between chromosomes, and across tumor indications. Furthermore, this diagram demonstrates the difficulty of identifying chromosome bands that are diploid at a high frequency across indications. [Figure 3] This dot diagram illustrates the ploidy pattern observed in proximal bands within chromosomes. Each data point in this diagram corresponds to a chromosomal band in the genome. The y-axis shows the percentage of patients whose chromosomal band ploidy is approximately equal to 2 copies (e.g., 1.8–2), which is considered diploid. This information was calculated for different tumor indications such as OV, UCEC, and UCS. The x-axis shows the central position of the chromosome, chromosomal arm, and band. This diagram demonstrates that the diploid frequency of bands changes non-randomly along the chromosome. Deviations from the diploid state are caused by copy number change events, which can be large enough to affect multiple bands at once. This demonstrates that investigating the diploid percentage across chromosomes can identify groups of adjacent bands with increased diploid percentages. [Figure 4]This is a schematic diagram of chromosome 19 (chr19) showing the relative positions of the CCNE1 probe used to determine CCNE1 amplification and an exemplary control probe. Chr19 undergoes significant rearrangement events in ovarian cancer. Cytogenetic abnormalities are particularly frequent on the p arm of chr19, where the control probes used in other assays are located. The subtelomere q arm band (e.g., chr19 q13.43) was used as a stable control locus in the experiments described herein because this region is more frequently diploid than other regions on chr19 (e.g., Figures 5 and 6). [Figure 5] This dot plot shows that the band chr19 q13.43 was frequently diploid in the indicated indications. Each data point in this plot corresponds to a chromosomal band (chr19) in chr19. The y-axis shows the percentage of patients with approximately 2 copies (e.g., 1.8-2) of the band, which were considered diploid. The x-axis shows tumor indications such as OV, UCEC, and UCS. [Figure 6] This dot plot shows that the band chr19 q13.43 is diploid at a high frequency in the indicated indications. Each data point in this plot corresponds to a chromosomal band in chr19. The y-axis shows the percentage of patients where the band's copy number is equal to 2, which is called diploid. This information is calculated for different tumor indications such as OV, UCEC, and UCS. The x-axis shows the chromosome, chromosomal arm, and central position of the band. The chromosomal band chr19 q13.43 did not show a large deviation in the diploid state and showed that the chromosomal region was more stable (i.e., less prone to duplication events compared to about 75% of other bands present on the chromosome). This can be compared to the leftmost band in the TCGA-UCS portion of the plot, which has a very high diploid state, but is closer to bands with a much lower diploid state. [Figure 7]Shows the flowchart of the pretreatment and hybridization conditions for the final CCNE1 FISH assay. The CCNE1 FISH assay was optimized for staining of FFPE cell line pellets (left panel) and FFPE tumor tissue specimens (right panel). Pretreatment conditions vary between these two types of samples, but protease and hybridization conditions are common. Representative images of cell pellet and tissue specimen staining are provided. [Figure 8] Shows representative images of ovarian tumor tissue samples and endometrial tumor tissue samples stained by the CCNE1 FISH assay. Here, non-amplified endometrial tumors (upper left panel), endometrium with low-level amplification (upper right panel) and ovarian tumors (lower left panel), and ovarian tumors with high-level amplification (lower right panel) are shown. The corresponding amplification status estimated by SNIPDx® and the average (e.g., median) CCNE1 / control signal per cell estimated by FISH are shown below each image. The cyclin E protein H score estimated by immunohistochemistry (IHC) assay and the pathologist's review are shown for each specimen. [Figure 9A] Outline of the CCNE1 FISH assay optimization and validation process described herein (see, for example, Examples 3 and 4). Assay conditions were optimized in a set of 6 FFPE tumor tissue samples and 6 cell lines, including amplified and non-amplified cases. Multiple iterations of protease pretreatment and hybridization temperature and conditions were tested to select a protocol with the best signal-to-noise ratio. The sensitivity and specificity of the assay were determined using the samples described in Figure 9B. The interassay precision was performed using 5 tumor specimens (1 tissue with high copy number, 1 non-amplified tissue, and 3 specimens with amplification levels close to the threshold criteria described in Figure 9B). The samples were assayed over 5 days. The intra-day precision was determined by assaying 5 tumor samples selected on the same criteria as the inter-day precision experiment 5 times in duplicate on the same day. [Figure 9B]This is a dot plot of CCNE1 total copy number (TCN)-ploidy for formalin-fixed paraffin-embedded (FFPE) tumor samples used in the CCNE1 FISH assay validation set. A total of 51 tissue samples were used to validate the sensitivity and specificity of the CCNE1 FISH assay, including 30 uterine cancers, 3 ovarian cancers, 2 gastrointestinal tumors, 5 colorectal cancers, and 1 triple-negative breast cancer. Ten normal tissue samples from the ovaries (n=5) and colon (n=5) were also included. Tumor samples were profiled using the targeted next-generation sequencing assay SNiPDx® as a baseline for calculating the amplified state CCNE1 copy number. A total of 16 samples from the uterine cancer cohort, 3 samples from the ovarian cancer cohort, and 1 sample from the gastrointestinal cohort were considered amplified by SNiPDx® using a threshold of [CCNE1 total copy number-ploidy] ≥ 4. Conversely, 14 uterine tumor samples, 1 gastrointestinal tumor, 1 triple-negative breast cancer, and 5 colorectal cancer tissues were considered not to have been amplified by SNIPDx®. [Figure 10] This is a scatter plot of the CCNE1 FISH assay showing >95% overall agreement with SNIPDx®. From 41 tumor samples included in the sensitivity / specificity test (30 uterine cancers, 3 ovarian cancers, 2 gastrointestinal tumors, 5 colorectal cancers, and 1 triple-negative breast cancer), 19 were determined to be amplified by both assays, and 20 were reported not to be amplified, resulting in an overall agreement of 95.1% (39 / 41 samples) between the two assays. Of the discrepant samples, one specimen determined to be amplified by SNIPDx® was reported not to be amplified by FISH, and conversely, one tumor specimen considered not to be amplified by SNIPDx® was reported to be amplified by FISH (sensitivity: 95.0% (19 / 20) and specificity: 95.2% (20 / 21)). Amplification detection using SNiPDx® was based on a threshold of [CCNE1 total copy number - ploidy] ≥ 4. The threshold for reporting amplification by FISH is a CCNE1 to control probe ratio of 2 or greater. [Figure 11] This is a dot plot of the intermittent reproducibility of the CCNE1 FISH assay. The intermittent precision assay was performed using five tumor specimens (one high copy number tissue, one non-amplified tissue, and three specimens with amplification levels close to the threshold criteria mentioned above). The samples were assayed in five separate days. The mean (e.g., intermediate) coefficient of variation (CV) for the five samples and over five days was 14.78%. [Figure 12] This is a dot plot of the intraday reproducibility of the CCNE1 FISH assay. The intraday precision assay was performed using five tumor specimens (one tissue with a high copy number, one non-amplified tissue, and three specimens with amplification levels close to the threshold criteria shown in Figure 11). The samples were assayed five times on the same day. The mean (e.g., intermediate) coefficient of variation (CV) across the five samples was 4.47%. [Figure 13] This figure shows the high degree of consistency between the calculation of total CCNE1 copies (TCN) using the FISH assay and the SNIPDx® next-generation signaling (NGS) assay. Top panel: The mean (e.g., median) CCNE1 signal per cell estimated by FISH (vertical axis) is significantly higher in tumor specimens considered to contain CCNE1 amplified by the SNIPDx® NGS assay (median 8.7, range 3.25–21.9) compared to non-amplified tumor tissue (median 2.75, range 1.85–5.95). In normal tissue, the mean number of CCNE1 copies per cell is approximately 2, as expected for diploid and genetically stable specimens. Bottom panel: Correlation analysis between the total CCNE1 copies estimated by FISH (X-axis) and the SNIPDx® NGS assay (Y-axis) shows strong consistency between the two methodologies (Pearson ρ = 0.923; mp value = 9.8e-18). [Modes for carrying out the invention]

[0105] Generally, the inventions described herein provide a method for identifying genomic loci (e.g., stable control loci) that can serve as controls for fluorescence in situ hybridization (FISH) assays. FISH is a technique commonly used to determine amplification of a target genomic locus (e.g., a gene such as CCNE1 (cyclin E1)) in cells (e.g., cancer cells). Generally, a FISH assay utilizes two sets of FISH probes. A first set of probes (e.g., target probes) binds to a target genomic locus (e.g., a gene, e.g., CCNE1, or a portion thereof) and emits a first detectable signal, while a second set of probes (e.g., control probes) binds to a stable control locus (e.g., the CCNE1 control locus) and emits a second detectable signal. The ratio of a first detectable signal to a second detectable signal (e.g., target genomic locus / stable control locus, e.g., CCNE1 / control) indicates amplification of the target genomic locus in cells and, consequently, can inform the likelihood of cancer cells. However, a stable control locus for one type of cancer may not always function for another type of cancer. Therefore, there is a need for a method to identify stable control loci for each type of cancer. Doing so would allow for more accurate results from FISH assays.

[0106] Advantageously, the methods described herein enable the identification of appropriate stable control loci for any given cancer type or set of cancer types. As a non-limiting example, a method for identifying a stable control locus (e.g., a CCNE1 control locus) for a FISH assay to detect CCNE1 amplification is described herein. The identified CCNE1 control locus was found to maintain more consistent ploidy in cancer cells compared to other chromosomal loci used in prior art (see, for example, Figure 4). In other words, the CCNE1 control locus described herein was identified to have relatively low copy number variation (CNV) in tumor cells (e.g., derived from ovarian (OV) and uterine cancer cells).

[0107] Therefore, the methods described herein enable the identification of an extended repertoire of stable control loci (e.g., CCNE1 control locus) that can be used in the FISH method described herein to accurately determine the amplification state of a target genomic locus (e.g., the gene, e.g., CCNE1) in cells, and consequently to inform about the likelihood of cancer cells.

[0108] A. Method for identifying stable control loci This disclosure provides a method for identifying stable control loci for use in FISH assays. Stable control loci are regions within a cancer cell chromosome (e.g., a chromosomal band) that are less likely to cause duplication events compared to other regions within the same chromosome (e.g., other chromosomal bands). Stable control loci can be used as controls in FISH assays because they are considered stable (i.e., the locus has a higher probability of maintaining its wild-type ploidy in mitotic cancer cells compared to other loci on the same chromosome). For example, stable control loci in human cancer cells are usually loci that maintain a diploid state at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more than the diploid state of the target genomic locus. In another example, a stable control locus in human cancer cells is a locus (e.g., a chromosomal band) that maintains a diploid state at a higher frequency than at least 75% (e.g., at least 75%, 80%, 85%, 90%, or 95%) of other loci (e.g., chromosomal bands) on the same chromosome.

[0109] This method first involves determining the ploidy of multiple chromosomal bands within a chromosome obtained from a biological sample of interest. This first step should be repeated multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times), with the biological sample (e.g., tumor) obtained from a different subject (e.g., cancer subject) each time, thereby generating ploidy data from multiple samples for multiple subjects. Ploidy determination can be performed bioinformatically (e.g., as described in Example 1) by downloading and processing ploidy data from The Cancer Genome Atlas (TCGA), which can be found on the NIH Genomic Data Commons Data Portal (portal.gdc.cancer.gov / ). However, if ploidy data is not available for the desired sample type (e.g., a sample with a desired disease or condition), next-generation sequencing (NGS) can be used on those target samples to generate it.

[0110] Next, this method includes the steps of determining the weighted average (e.g., median) ploidy for each chromosomal band within a chromosome, and then determining the percentage of objects that are diploid in each chromosomal band. Diploid objects for a given chromosomal band are those that have a weighted average ploidy of 1.8 to 2.2 copies (e.g., 1.8 copies, 1.9 copies, 2 copies, 2.1 copies, or 2.2 copies) of the chromosomal band.

[0111] Finally, this method involves ranking each chromosome band from high to low based on the proportion of subjects considered diploid. The upper quartile of this rank (e.g., 75) th , 80 th , 85 th , 90 th ,95 th , 96 th , 97 th , 98 th , 99 th , or 100 thAny chromosomal band identified at the percentile can be considered and selected as a stable control locus for use in the FISH assay described in Section B.

[0112] Ideally, identified stable control loci should only be used in FISH assays that analyze target genomic loci from biological samples with similar diseases or conditions. For example, if a stable control locus is identified using an OV tumor sample, that stable control locus should only be used in FISH assays that analyze target genomic loci in the OV tumor sample. In another example, if a stable control locus is identified using OV and UCEC tumor samples, that stable control locus should only be used in FISH assays that analyze target genomic loci in the OV and / or UCEC tumor samples. In yet another example, if a stable control locus is identified using OV, UCEC, and uterine carcinosarcoma (UCS) tumor samples, that stable control locus should only be used in FISH assays that analyze target genomic loci in the OV, UCEC, and / or USC tumor samples.

[0113] B. Method for determining amplification of target genomic loci This disclosure provides a method for determining whether a target genomic locus (e.g., a gene, e.g., CCNE1) within one or more chromosomal DNA molecules in a population of cells is amplified or not. Determining such amplification can provide information about the likelihood of the presence of cancer cells.

[0114] This method first involves contacting a population of cells (e.g., a tumor) containing chromosomal DNA (e.g., nuclear and / or mitochondrial DNA) with a plurality of target probes and a plurality of control probes (e.g., in vitro). The target probes can emit a first detectable signal and are complementary to a target genomic locus (e.g., the gene, e.g., CCNE1), while the control probes can emit a second detectable signal and are complementary to a stable control locus (e.g., the CCNE1 control locus). In the first example, the plurality of target probes may be complementary to the q12 band of chromosome 19 (chr19 q12) for the Homo sapiens genome assembly NCBI36. In the second example, the plurality of target probes may be complementary to the CCNE1 gene or a portion thereof. In either the first or second example, the control probe may be complementary to a stable control locus (e.g., the CCNE1 control locus) selected from the following chr19 q13.43, chr19 q13.41, chr19 q13.42, chr19 p13.3, chr19 q13.32, chr19 q13.33, chr19 q13.31, chr19 p13.13, or chr19 q13.13 (for the Homo sapiens genome assembly NCBI36).

[0115] Next, this method includes the steps of quantifying the total number of first detectable signals in a cell, quantifying the total number of second detectable signals in the same cell, and then dividing the total number of first detectable signals by the total number of second detectable signals to determine the signal ratio for that cell (e.g., CCNE1 / control). This step can be repeated multiple times, with different cells each time, thereby determining multiple signal ratios within a population of cells (e.g., a tumor). For example, this step can be performed on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 The process may be repeated 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 times or more. Preferably, this process is repeated at least 50 times to determine at least 50 (e.g., 50-60, 50-70, 50-80, 50-90, 50-100) signal ratios within the cell population.

[0116] Finally, this method includes the step of determining the mean (e.g., median) signal ratio for a population of cells. If the mean signal ratio is ≥2 (e.g., ≥2, ≥2.5, ≥3, ≥3.5, ≥4, ≥4.5, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10), the target genomic locus (e.g., gene, e.g., CCNE1) is considered amplified. If the mean signal ratio is <2 (e.g., <1.9, <1.8, <1.7, <1.6, <1.5, <1, <0.5), the target genomic locus (e.g., gene, e.g., CCNE1) is not considered amplified. If the target genomic locus (e.g., gene, e.g., CCNE1) is considered amplified, the subject may be treated with an effective dose of a Myt1 inhibitor, as further detailed in Section B below.

[0117] Further details regarding the target genomic locus, stable control locus (e.g., CCNE1 control locus), target probe, control probe, and the type of cells or samples to be analyzed are described below.

[0118] i. Target genome loci A target genomic locus can be any sequence of nucleic acids within one or more chromosomal DNA molecules (e.g., nuclear and / or mitochondrial). Typically, a target genomic locus is located within a single chromosome and contains at least one gene, heritable element, mobile heritable element, exon, and / or intron. For example, a target genomic locus may be the CCNE1 gene, a region containing the CCNE1 gene, or a region containing a portion of the CCNE1 gene. Target genomic loci are generally found within tissue samples (e.g., tumors), cells (e.g., cancer cells), or any population of cells derived from a sample (e.g., tissue).

[0119] The target genome locus (e.g., a gene, e.g., CCNE1) is approximately 5,000 to 500,000 nucleotides long (e.g., approximately 5,000 to 200,000 nucleotides long, approximately 150,000 to 300,000 nucleotides long, or approximately 250,000 to 500,000 nucleotides long), approximately 5,000 to 300,000 nucleotides long (e.g., approximately 5,000 to 150,000 nucleotides long, approximately 100,000 to 200,000 nucleotides long) 00 nucleotides long, or approximately 150,000 to approximately 300,000 nucleotides long), approximately 50,000 to approximately 300,000 nucleotides long (for example, approximately 50,000 to approximately 150,000 nucleotides long, approximately 100,000 to approximately 250,000 nucleotides long, or approximately 200,000 to approximately 300,000 nucleotides long), approximately 100,000 to approximately 300,000 nucleotides long (for example, approximately 150,000 to approximately 275,000 nucleotides long, Approximately 150,000 to 250,000 nucleotides in length, or approximately 150,000 to 225,000 nucleotides in length), approximately 150,000 to 300,000 nucleotides in length (for example, approximately 150,000 to 240,000 nucleotides, approximately 160,000 to 230,000 nucleotides, or approximately 170,000 to 220,000 nucleotides), approximately 150,000 to 250,000 nucleotides in length (for example, approximately 160,000 It may be a sequence of nucleotides with a length of 0 to approximately 240,000 nucleotides, approximately 170,000 to approximately 230,000 nucleotides, or approximately 180,000 to approximately 220,000 nucleotides, or approximately 175,000 to approximately 225,000 nucleotides (for example, approximately 180,000 to approximately 215,000 nucleotides, approximately 190,000 to approximately 210,000 nucleotides, or approximately 195,000 to approximately 205,000 nucleotides).

[0120] For example, the target genome locus (e.g., gene, e.g., CCNE1) has approximately 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, and 1 25,000, 130,000, 135,000, 140,000, 145,000, 150,000, 155,000, 160,000, 165,000, 170,000, 175,000, 180,000, 185,000, 190,000, 195,000, 196,196, 200,000, 205,000, 210,000, 215,000, 220,000, 225,000, 230,000, 235,000, 240,000, 245,000, 25 0,000, 255,000, 260,000, 265,000, 270,000, 275,000, 280,000, 285,000, 290,000, 295,000, 300,000, 305,000, 310,000, 315,000, 320,000, 325,000, 330,000, 335,000, 340,000, 345,000, 350,000, 355,000, 360,000, 365,000, 370,000, 375,000, 380 The nucleotide lengths can be ,000, 385,000, 390,000, 395,000, 400,000, 405,000, 410,000, 415,000, 420,000, 425,000, 430,000, 435,000, 440,000, 445,000, 450,000, 455,000, 460,000, 465,000, 470,000, 475,000, 480,000, 485,000, 490,000, 495,000, and 500,000.

[0121] ii. Stable control loci A stable control locus is a region within a cancer cell's chromosome (e.g., a chromosomal band) that is less likely to cause duplication events compared to other regions within the same chromosome (e.g., other chromosomal bands). Because stable control loci are considered more stable, they can be used as controls for target genomic loci. In other words, stable control loci (e.g., the CCNE1 control locus) have a higher probability of maintaining their wild-type ploidy (e.g., a diploid locus in humans) in dividing cancer cells. For example, stable control loci in human cells are usually loci that maintain a diploid state at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more than the diploid state of the target genomic locus. In another example, a stable control locus in human cells is a locus (e.g., a chromosomal band) that maintains a diploid state at a higher frequency than at least 75% (e.g., at least 75%, 80%, 85%, 90%, or 95%) of other loci (e.g., chromosomal bands) present on the same chromosome. Methods for identifying stable control loci are described in Section A above. Experiments describing the identification of several CCNE1 control loci are described in Example 1 below.

[0122] Ideally, stable control loci should only be used in FISH assays that analyze target genomic loci from biological samples with similar cancers. In other words, if a stable control locus is identified using OV tumor samples, the stable control locus should only be used in FISH assays that analyze target genomic loci in OV tumor samples. In another example, if a stable control locus is identified using OV and UCEC tumor samples, the stable control locus should only be used in FISH assays that analyze target genomic loci in OV and / or UCEC tumor samples. In yet another example, if a stable control locus is identified using OV, UCEC, and UCS tumor samples, the stable control locus should only be used in FISH assays that analyze target genomic loci in OV, UCEC, and / or USC tumor samples.

[0123] A stable control locus (e.g., the CCNE1 control locus) can be a sequence of nucleic acids located on a human chromosome (e.g., the nucleus and / or mitochondria), such as a chromosomal band or a region within a chromosomal band. The stable control locus is located on the same chromosome as the target genomic locus. For example, if the target genomic locus contains the CCNE1 gene (located on chr19), the stable control locus (relative to the Homo sapiens genome assembly NCBI36) may be the CCNE1 control locus selected from chr19 q13.43, chr19 q13.41, chr19 q13.42, chr19 p13.3, chr19 q13.32, chr19 q13.33, chr19 q13.31, chr19 p13.13, or chr19 q13.13. For example, a stable control locus can be found within nucleotides 29,644,930–29,841,126 of chr 19 q13.43 relative to the Homo sapiens genome assembly NCBI36.

[0124] Stable control loci (e.g., CCNE1 control locus) are approximately 5,000 to 500,000 nucleotides long (e.g., approximately 5,000 to 200,000 nucleotides, approximately 150,000 to 300,000 nucleotides, or approximately 250,000 to 500,000 nucleotides), approximately 5,000 to 300,000 nucleotides long (e.g., approximately 5,000 to 150,000 nucleotides, approximately 100,000 to 200,000 nucleotides) Nucleotide length, or approximately 150,000 to approximately 300,000 nucleotides), approximately 50,000 to approximately 300,000 nucleotides (for example, approximately 50,000 to approximately 150,000 nucleotides, approximately 100,000 to approximately 250,000 nucleotides, or approximately 200,000 to approximately 300,000 nucleotides), approximately 100,000 to approximately 300,000 nucleotides (for example, approximately 150,000 to approximately 275,000 nucleotides, approximately 150,000 to approximately 250,000 nucleotides in length, or approximately 150,000 to approximately 225,000 nucleotides in length), approximately 150,000 to approximately 300,000 nucleotides in length (for example, approximately 150,000 to approximately 240,000 nucleotides, approximately 160,000 to approximately 230,000 nucleotides, or approximately 170,000 to approximately 220,000 nucleotides in length), approximately 150,000 to approximately 250,000 nucleotides in length (for example, approximately 160,000 It may be a sequence of nucleotides with a length of approximately 240,000 nucleotides, approximately 170,000 to approximately 230,000 nucleotides, or approximately 180,000 to approximately 220,000 nucleotides, or approximately 175,000 to approximately 225,000 nucleotides (for example, approximately 180,000 to approximately 215,000 nucleotides, approximately 190,000 to approximately 210,000 nucleotides, or approximately 195,000 to approximately 205,000 nucleotides).

[0125] For example, stable control loci (e.g., CCNE1 control locus) have approximately 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, and 125 ,000, 130,000, 135,000, 140,000, 145,000, 150,000, 155,000, 160,000, 165,000, 170,000, 175,000, 180,000, 185,000, 190,000, 195,000, 196,196, 200,000, 205,000, 210,000, 215,000, 220,000, 225,000, 230,000, 235,000, 240,000, 245,000, 250 ,000, 255,000, 260,000, 265,000, 270,000, 275,000, 280,000, 285,000, 290,000, 295,000, 300,000, 305,000, 310,000, 315,000, 320,000, 325,000, 330,000, 335,000, 340,000, 345,000, 350,000, 355,000, 360,000, 365,000, 370,000, 375,000, 380 The nucleotide lengths can be ,000, 385,000, 390,000, 395,000, 400,000, 405,000, 410,000, 415,000, 420,000, 425,000, 430,000, 435,000, 440,000, 445,000, 450,000, 455,000, 460,000, 465,000, 470,000, 475,000, 480,000, 485,000, 490,000, 495,000, and 500,000.

[0126] iii. Sample The target genomic locus (e.g., a gene, e.g., CCNE1) and a stable control locus (e.g., the CCNE1 control locus) may be present in a sample, such as a biological sample derived from the subject (e.g., a human). Non-limiting examples of biological samples include blood, serum, plasma, saliva, cells, cell cultures, tissues (e.g., skin, liver, kidney, heart, lung, muscle), and tumors (e.g., benign or cancerous tumors). The subject may be any mammal, but is not limited to humans, monkeys, cats, dogs, mice, pigs, cattle, horses, sheep, and bats.

[0127] iv. Target probes A target probe is a nucleic acid molecule that hybridizes to a target genomic locus (e.g., a gene, e.g., CCNE1) and emits a first detectable signal (e.g., a fluorescent signal).

[0128] The target probe may be an allele-specific probe that identifies single nucleotide variants (SNVs) within a target genomic locus. The target probe may be a nucleic acid molecule with a length of approximately 5,000 to 40,000 nucleotides (for example, approximately 5,000 to 10,000 nucleotides, approximately 7,000 to 15,000 nucleotides, approximately 5,000 to 20,000 nucleotides, approximately 10,000 to 20,000 nucleotides, approximately 15,000 to 30,000 nucleotides, approximately 20,000 to 35,000 nucleotides, or approximately 25,000 to 40,000 nucleotides). For example, the target probes are approximately 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, and 2 3,000, approximately 24,000, approximately 25,000, approximately 26,000, approximately 27,000, approximately 28,000, approximately 29,000, approximately 30,000, approximately 31,000, approximately 32,000, approximately 33,000, approximately 34,000, approximately 35,000, approximately 36,000, approximately 37,000, approximately 38,000, approximately 39,000, approximately 40,000 nucleotide lengths are not necessarily.

[0129] v. Control probe The control probe may be a nucleic acid molecule that hybridizes to a stable control locus (e.g., the CCNE1 control locus) and emits a first detectable signal (e.g., a fluorescent signal).

[0130] The control probe may be able to hybridize to the Homo sapiens genome assembly NCBI36 within the chr19 q13.43 chromosomal region (e.g., nucleotides 29,644,930–29,841,126 of chr19).

[0131] The control probe may be a nucleic acid molecule with a length of approximately 5,000 to 40,000 nucleotides (for example, approximately 5,000 to 10,000 nucleotides, approximately 7,000 to 15,000 nucleotides, approximately 5,000 to 20,000 nucleotides, approximately 10,000 to 20,000 nucleotides, approximately 15,000 to 30,000 nucleotides, approximately 20,000 to 35,000 nucleotides, or approximately 25,000 to 40,000 nucleotides). For example, the control probes were approximately 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, and 2 3,000, approximately 24,000, approximately 25,000, approximately 26,000, approximately 27,000, approximately 28,000, approximately 29,000, approximately 30,000, approximately 31,000, approximately 32,000, approximately 33,000, approximately 34,000, approximately 35,000, approximately 36,000, approximately 37,000, approximately 38,000, approximately 39,000, approximately 40,000 nucleotide lengths are not necessarily.

[0132] vi. Modified probe The targets and / or control probes described herein are intended to be used in modified forms. Typically, modifications to nucleic acid molecules are introduced to optimize the efficacy or biophysical properties of the molecule (e.g., to improve stability, and / or to target a specific location or cell type). Such modifications may be achieved by generating chimeric nucleic acid molecules containing any combination of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), cross-linked nucleic acid (BNA), and / or peptide nucleic acid (PNA). Furthermore, modifications may include, for example, the incorporation of one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative nucleoside bonds as described herein.

[0133] Nucleoside modification Modifications of the target and / or control probes described herein include the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(-C=C-CH3)uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil Oligonucleotides may also include one or more of the following: ur, cytosine and thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. Oligonucleotides may also include nucleic acid bases in which a purine or pyrimidine base is substituted with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further modifications of oligonucleotides may include nucleic acid bases as disclosed in U.S. Patent No. 3,687,808; Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302.

[0134] sugar modification Modifications of the target and / or control probe described herein may also include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2'-modifications that can modify oligonucleotides include all possible orientations of OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl;O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Other possible sugar substituent groups include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluorine (F). The 2'-sugar substituent may be at the arabino (upper) or ribo (lower) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications can be made at other locations on the interfering RNA molecule, particularly on the 3' terminal nucleoside or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked oligonucleotides. Oligonucleotides may also have sugar mimetic molecules, such as a cyclobutyl moiety instead of pentofuranosyl sugar.

[0135] Nucleoside bond modification Modifications of target and / or control probes described herein may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and 5'-alkylenephosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having a normal 3'-5' bond, their 2'-5' bond analogues, and those having reverse polarity (one or more internucleotide bonds being 3'-3', 5'-5', or 2'-2' bonds).

[0136] C. Treatment method This disclosure provides a method for identifying and / or treating patients who are at risk of having cancer or suspected to have cancer. This method includes identifying a subject as having cancer or suspected to have cancer by performing one of the methods described in Section A above. For example, a sample of cells or tissue (e.g., a tumor or a population of tumor-derived cells) can be obtained from the subject, and the amplification status of a target genomic locus (e.g., a gene, e.g., CCNE1) in the sample can be determined by the method described in Section A. The target genomic locus may be CCNE1. For example, amplification of CCNE1 in a biological sample (e.g., a tumor or a population of tumor-derived cells) indicates an increased likelihood of the subject having cancer or an increased risk of developing cancer compared to a subject without CCNE1 amplification. Such subjects may be treated using the methods described herein. A method for treating a subject at risk of having cancer or suspected to have cancer includes administering an effective amount of a membrane-bound tyrosine and threonine-specific cdc2 inhibitory kinase (Myt1) inhibitor.

[0137] i.Myt1 inhibitors Examples of Myt1 inhibitors that can be used in the method of the present invention include those disclosed in International Publication Nos. 2021 / 195781 and 2023 / 220831, and U.S. Patent Applications Nos. 17 / 937,977, 17 / 961,103, and 18 / 140,302.

[0138] Myt inhibitors include, for example, compounds of formula (I):

[0139] [ka]

[0140] or its pharmaceutically acceptable salt, (In the formula, Each of X, Y, and Z is independently N or CR 2 And, R 1 Each R 2 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, arbitrarily substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, -N(R 7 )2, -OR 7 ,-C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -QR 7B is, or R 1 R 1 One adjacent R 2 Together with the arbitrarily substituted C 3~6Forming alkylenes, R 3 and R 4 Each of these is independently and arbitrarily substituted C 1~6 It is an alkyl or halogen, R 5 is H or -N(R 7 )2, R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO2R 7A And, Each R 7 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, or -SO2R 7A and, or two R 7 The group, together with the atom to which it is bonded, is an optionally substituted C 2~9 Forming heterocyclines, Each R 7A This is an independently and arbitrarily substituted C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl or optionally substituted C 6~10 It is Ariel, Each R 7B These are independently hydroxyl, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, -N(R 7 )2, -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or optionally substituted alkoxy, Each R 8 These are, independently, hydrogen, and optionally substituted C.1~6 Alkyl, optionally substituted C 2~6 Alkoxyalkyl groups, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 3~8 Cycloalkyl or optionally substituted C 1~9 It is a heteroaryl, or two R 8 These, together with the atoms to which they are bonded, form an arbitrarily substituted C 2~9 Forming heterocyclines, Q is C which is arbitrarily substituted. 1~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 Alkynylene, optionally substituted C 3~8 Cycloalkylene, optionally substituted C 3~8 Cycloalkenylene, optionally substituted C 6~10 Arrine, optionally substituted C 2~9 Heterocyclylene, or optionally substituted C 1~9 (It may be a heteroarylene.)

[0141] Preferably, the compound of formula (I) is concentrated with respect to the atropisomer of formula (IA):

[0142] [ka]

[0143] (All variable elements in the formula are as described herein). The compound of the present invention may be, for example, a compound of formula (II):

[0144] [ka]

[0145] (All variable elements in the formula are as described herein). Preferably, the compound of formula (II) is concentrated with respect to the atrop isomer of formula (IIA):

[0146] [ka]

[0147] (All variable elements in the formula are as described herein). The compounds of the present invention include, for example, the compound of formula (III):

[0148] [ka]

[0149] (In the formula, R 2A is a hydrogen atom, and C is optionally substituted. 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, arbitrarily substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, -N(R 7 )2, -OR 7 ,-C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -QR 7B It may be (that).

[0150] Preferably, the compound of formula (III) is concentrated with respect to the atrop isomer of formula (IIIA).

[0151] [ka]

[0152] Myt1 inhibitors include, for example, compounds of formula (IV):

[0153] [ka]

[0154] or its pharmaceutically acceptable salt, (In the formula, each

[0155] [ka]

[0156] These are single or double bonds, One, two, or three X groups are N, and the remaining X groups are C. Each Y is independently N or CR 2 And, Each Z is independently either N or CH. R 1 OH is R 3 is hydrogen, optionally substituted with C 1~6 Alkyl, halogen, or optionally substituted C 3~8 It is cycloalkyl, or R 1 and R 3 Together, -CR 9 =N-NH- forms Each R 2 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, arbitrarily substituted C 2~9 Heterocyclyl C 1~6Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, cyano, -N(R) 7 )2, -OR 7 ,-C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -QR 7B And, R 4 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl or halogen, R 5 is hydrogen, halogen, or -N(R) 7 )2, R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO2R 7A And, Each R 7 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, or -SO2R 7A and, or two R 7 The group, together with the atom to which it is bonded, is an optionally substituted C 2~9 Forming heterocyclines, Each R 7A This is an independently and arbitrarily substituted C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl or optionally substituted C 6~10 It is Ariel, Each R7B These are independently hydroxyl, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, -N(R 7 )2, -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or optionally substituted alkoxy, Each R 8 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkoxyalkyl groups, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 3~8 Cycloalkyl or optionally substituted C 1~9 It is a heteroaryl, or two R 8 These, together with the atoms to which they are bonded, form an arbitrarily substituted C 2~9 Forming heterocyclines, R 9 is hydrogen or halogen, Q is C which is arbitrarily substituted. 1~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 Alkynylene, optionally substituted C 3~8 Cycloalkylene, optionally substituted C 3~8 Cycloalkenylene, optionally substituted C 6~10 Arrine, optionally substituted C 2~9 Heterocyclylene, or optionally substituted C 1~9 (It may be a heteroarylene.)

[0157] In some embodiments, one X group is N and the remaining X groups are C. In some embodiments, all Y groups are CR. 2In some embodiments, one Y group is N and the remaining Y group is CR. 2 In some embodiments, two Y groups are N, and the remaining Y group is CR. 2 That is the case.

[0158] In some embodiments, R 1 OH is R 3 is hydrogen, optionally substituted C 1~6 It is alkyl or halogen. In some embodiments, R 1 OH is R 3 is an arbitrarily substituted C 1~6 It is alkyl or halogen. In some embodiments, R 1 and R 3 Together, -CR 9 =N-NH- forms (for example, R 9 (It is hydrogen).

[0159] In some embodiments, the Myt1 inhibitor is of formula (V).

[0160] [ka]

[0161] In some embodiments, Myt1 inhibitors are

[0162] [ka]

[0163] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (Vi).

[0164] [ka]

[0165] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0166] [ka]

[0167] In some embodiments, the Myt1 inhibitor is of formula (VA).

[0168] [ka]

[0169] In some embodiments, Myt1 inhibitors are

[0170] [ka]

[0171] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VA-i).

[0172] [ka]

[0173] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0174] [ka]

[0175] In some embodiments, the Myt1 inhibitor is of formula (VB).

[0176] [ka]

[0177] In some embodiments, Myt1 inhibitors are

[0178] [ka]

[0179] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VB-i).

[0180] [ka]

[0181] In some embodiments, the compound is concentrated with respect to the following atropisomers.

[0182] [ka]

[0183] In some embodiments, the Myt1 inhibitor is of formula (VC).

[0184] [ka]

[0185] In some embodiments, Myt1 inhibitors are

[0186] [ka]

[0187] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VC-i).

[0188] [ka]

[0189] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0190] [ka]

[0191] In some embodiments, the Myt1 inhibitor is of formula (VD).

[0192] [ka]

[0193] In some embodiments, Myt1 inhibitors are

[0194] [ka]

[0195] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VD-i).

[0196] [ka]

[0197] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0198] [ka]

[0199] In some embodiments, the Myt1 inhibitor is of formula (VI).

[0200] [ka]

[0201] In some embodiments, Myt1 inhibitors are

[0202] [ka]

[0203] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VI-i).

[0204] [ka]

[0205] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0206] [ka]

[0207] In some embodiments, the Myt1 inhibitor is of formula (VII).

[0208] [ka]

[0209] In some embodiments, Myt1 inhibitors are

[0210] [ka]

[0211] That is the case. In some embodiments, the compound is concentrated for the atropisomer of (VII-i).

[0212] [ka]

[0213] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0214] [ka]

[0215] In some embodiments, the Myt1 inhibitor is of formula (VIII).

[0216] [ka]

[0217] In some embodiments, Myt1 inhibitors are

[0218] [ka]

[0219] That is the case. In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of (VIII-i).

[0220] [ka]

[0221] In some embodiments, the Myt1 inhibitor has been enriched with respect to the following atrop isomers.

[0222] [ka]

[0223] In some embodiments, the Myt1 inhibitor is of formula (IX).

[0224] [ka]

[0225] Myt1 inhibitors include, for example, compounds of formula (XA):

[0226] [ka]

[0227] or its pharmaceutically acceptable salt, (In the formula, each

[0228] [ka]

[0229] These are single or double bonds, One, two, or three X groups are N, and the remaining X groups are C. Each Y is independently either N or C. Each Z is independently either N or CH. R 1 OH is R 3 is hydrogen, optionally substituted with C 1~6 Alkyl, halogen, or optionally substituted C 3~8 It is cycloalkyl, or R 1 and R 3 Together, -CR 9 =N-NH- forms Each R 2 These are, independently, non-existent, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, arbitrarily substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, cyano, -N(R) 7 )2, -OR 7 ,-C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -QR 7B And R 2A and R 2B These, together with the atoms to which they are bonded, form ring A, or R 2 and R 2A Together with the atoms to which they are bonded, they form ring A, R 2B It either does not exist or is hydrogen. R 4 is hydrogen, optionally substituted C 1~6 Alkyl, halogen, or optionally substituted C 3~8 It is a cycloalkyl, R 5 is hydrogen, halogen, or -N(R) 7 )2, R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO2R 7A And, Each R 7 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, or -SO2R 7A and, or two R 7 The group, together with the atom to which it is bonded, is an optionally substituted C 2~9 Forming heterocyclines, Each R 7A This is an independently and arbitrarily substituted C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl or optionally substituted C 6~10 It is Ariel, Each R 7B These are independently hydroxyl, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, -N(R 7 )2, -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or optionally substituted alkoxy, Each R 8 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkoxyalkyl groups, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 3~8 Cycloalkyl or optionally substituted C 1~9 It is a heteroaryl, or two R 8 These, together with the atoms to which they are bonded, form an arbitrarily substituted C 2~9 Forming heterocyclines, R 9 is hydrogen or halogen, Ring A is a 5-membered or 6-membered carbon ring or a 5-membered or 6-membered heteroring, where A has 1, 2, 3, or 4 non-hydrogen R 2 It is arbitrarily substituted in the base, Q is C which is arbitrarily substituted. 1~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 Alkynylene, optionally substituted C 3~8 Cycloalkylene, optionally substituted C 3~8 Cycloalkenylene, optionally substituted C 6~10 Arrine, optionally substituted C 2~9 Heterocyclylene, or optionally substituted C 1~9 It can be a heteroarrene, In the formula, if N is bonded to Y, then each R 2 (It may not exist.)

[0230] Myt1 inhibitors include, for example, compounds of formula (X):

[0231] [ka]

[0232] or its pharmaceutically acceptable salt, (In the formula, each

[0233] [ka]

[0234] These are single or double bonds, A is a 5-membered or 6-membered carbon ring or a 5-membered or 6-membered heterocyclic ring, and A has 1, 2, 3, or 4 non-hydrogen R 2 It is arbitrarily substituted in the base, One, two, or three X groups are N, and the remaining X groups are C. Each Y is independently either N or C. Each Z is independently either N or CH. R 1OH is R 3 is hydrogen, optionally substituted with C 1~6 Alkyl, halogen, or optionally substituted C 3~8 It is cycloalkyl, or R 1 and R 3 Together, -CR 9 =N-NH- forms Each R 2 These are, independently, non-existent, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkinyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkenyl, optionally substituted C 2~9 Heterocyclyl, arbitrarily substituted C 2~9 Heterocyclyl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, halogen, cyano, -N(R) 7 )2, -OR 7 ,-C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or -QR 7B And, R 4 is hydrogen, optionally substituted C 1~6 Alkyl, halogen, or optionally substituted C 3~8 It is a cycloalkyl, R 5 is hydrogen, halogen, or -N(R) 7 )2, R 6 is -C(O)NH(R 8 ), -C(O)R 7A , or -SO2R 7A And, Each R 7 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C6~10 Aryl C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, optionally substituted C 1~9 Heteroaryl C 1~6 Alkyl, or -SO2R 7A and, or two R 7 The group, together with the atom to which it is bonded, is an optionally substituted C 2~9 Forming heterocyclines, Each R 7A This is an independently and arbitrarily substituted C 1~6 Alkyl, optionally substituted C 3~8 Cycloalkyl or optionally substituted C 6~10 It is Ariel, Each R 7B These are independently hydroxyl, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 2~9 Heterocyclyl, arbitrarily substituted C 1~9 Heteroaryl, -N(R 7 )2, -C(O)N(R 8 )2, -SO2N(R 8 )2, -SO2R 7A , or optionally substituted alkoxy, Each R 8 These are, independently, hydrogen, and optionally substituted C. 1~6 Alkyl, optionally substituted C 2~6 Alkoxyalkyl groups, optionally substituted C 6~10 Aryl C 1~6 Alkyl, optionally substituted C 6~10 Aryl, arbitrarily substituted C 3~8 Cycloalkyl or optionally substituted C 1~9 It is a heteroaryl, or two R 8 These, together with the atoms to which they are bonded, form an arbitrarily substituted C 2~9 Forming heterocyclines, R 9 is hydrogen or halogen, Q is C which is arbitrarily substituted. 1~6 Alkylene, optionally substituted C 2~6 Alkenylene, optionally substituted C 2~6 Alkynylene, optionally substituted C 3~8 Cycloalkylene, optionally substituted C 3~8 Cycloalkenylene, optionally substituted C 6~10 Arrine, optionally substituted C 2~9 Heterocyclylene, or optionally substituted C 1~9 It can be a heteroarrene, In the formula, if N is bonded to Y, then each R 2 (It may not exist.)

[0235] In some embodiments, one X group is N and the remaining X groups are C. In some embodiments, all Y groups are C. In some embodiments, one Y group is N and the remaining Y groups are C. In some embodiments, two Y groups are N and the remaining Y groups are C.

[0236] In some embodiments, the Myt1 inhibitor is of formula (XI).

[0237] [ka]

[0238] In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XI-i).

[0239] [ka]

[0240] In some embodiments, the Myt1 inhibitor is of formula (XIA).

[0241] [ka]

[0242] In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIA-i).

[0243] [ka]

[0244] In some embodiments, the Myt1 inhibitor is expressed by formula (XIA-ii):

[0245] [ka]

[0246] (wherein m is 0, 1, 2, 3, or 4) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIA-iii).

[0247] [ka]

[0248] In some embodiments, the Myt1 inhibitor is expressed by formula (XIA-iv):

[0249] [ka]

[0250] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIA-v).

[0251] [ka]

[0252] In some embodiments, the Myt1 inhibitor is expressed by formula (XIA-vi):

[0253] [ka]

[0254] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIA-vii).

[0255] [ka]

[0256] In some embodiments, the Myt1 inhibitor is of formula (XIB).

[0257] [ka]

[0258] In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIB-i).

[0259] [ka]

[0260] In some embodiments, the Myt1 inhibitor is expressed by formula (XIB-ii):

[0261] [ka]

[0262] (wherein m is 0, 1, 2, 3, or 4) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIB-iii).

[0263] [ka]

[0264] In some embodiments, the Myt1 inhibitor is expressed by formula (XIB-iv):

[0265] [ka]

[0266] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIB-v).

[0267] [ka]

[0268] In some embodiments, the Myt1 inhibitor is expressed by formula (XIB-vi):

[0269] [ka]

[0270] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIB-vii).

[0271] [ka]

[0272] In some embodiments, the Myt1 inhibitor is of formula (XIC).

[0273] [ka]

[0274] In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIC-i).

[0275] [ka]

[0276] In some embodiments, the Myt1 inhibitor is expressed by formula (XIC-ii):

[0277] [ka]

[0278] (wherein m is 0, 1, 2, 3, or 4) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIC-iii).

[0279] [ka]

[0280] In some embodiments, the Myt1 inhibitor is expressed by formula (XIC-iv):

[0281] [ka]

[0282] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIC-v).

[0283] [ka]

[0284] In some embodiments, the Myt1 inhibitor is expressed by formula (XIC-vi):

[0285] [ka]

[0286] (wherein m is 0, 1, 2, or 3) In some embodiments, the Myt1 inhibitor is enriched for the atrop isomer of formula (XIC-vii).

[0287] [ka]

[0288] In some embodiments, the Myt1 inhibitor is of formula (XIC-viii).

[0289] [ka]

[0290] In some embodiments, R 2 and R 2A Together with the atoms to which they are bonded, they form ring A, R 2B It either does not exist or is hydrogen. In some embodiments, the Myt1 inhibitor is of formula (XII).

[0291] [ka]

[0292] Myt1 inhibitors may be, for example, the compounds listed in Table 1 below or their pharmaceutically acceptable salts.

[0293] [Table 1-1]

[0294] [Table 1-2]

[0295] [Table 1-3]

[0296] [Table 1-4]

[0297] [Table 1-5]

[0298] [Table 1-6]

[0299] [Table 1-7]

[0300] [Table 1-8]

[0301] [Table 1-9]

[0302] Table 1-10

[0303] Table 1-11

[0304] Table 1-12

[0305] Table 1-13

[0306] Table 1-14

[0307] Table 1-15

[0308] Table 1-16

[0309] Table 1-17

[0310] Table 1-18

[0311] Table 1-19

[0312] Table 1-20

[0313] [Table 1-21]

[0314] [Table 1-22]

[0315] [Table 1-23] [Examples]

[0316] Example 1. Identification of a novel control locus for FISH. Increased gene expression is often associated with carcinoma and can be the result of gene duplication events, chromosomal (chr) duplication events, or both. For example, amplification of the cyclin E1 (CCNE1) gene is associated with many types of cancer (see, e.g., Figure 1). A standard method for determining gene amplification is to quantify such cytogenetic abnormalities using a pair of fluorescent in situ hybridization FISH nucleic acid probes (e.g., fluorescent probes) (e.g., Ayse et al., (2017) Mod Path., 20:297-303; Kuhn et al., (2013) Mod Path., 27:1014-1019; Karst et al., (2014) Cancer Res., 74(4):1141-1152; and Aziz et al., (2018) Gynecol Oncol., 151(2):327-336; these references are incorporated herein by reference). Using the CCNE1 gene as an example, a first FISH probe (e.g., a target probe) is designed to bind to all or part of CCNE1 (see, for example, Figure 4), while a second FISH probe (e.g., a control probe) is designed to bind to a stable control locus on the same chromosome (e.g., a chromosomal band), but distinct from the CCNE1 locus. The ratio of the fluorescence signals produced by the two FISH probes indicates the amplification state of the CCNE1 gene and, consequently, can inform the likelihood of cancer cells.

[0317] In this example, we identified several chromosomal bands within chromosome 19 (chr19) (see, e.g., Table 2) that exhibit substantially consistent ploidy in tumor cells across targeted cancer indications (e.g., patients with ovarian cancer and uterine malignancies). These chromosomal bands can then serve as stable control loci for FISH assays used to determine amplification of target genes (e.g., target genomic loci).

[0318] To identify these bands, the inventors first analyzed copy number variation (i.e., ploidy) data from The Cancer Genome Atlas (TCGA). Tumor copy number information from TCGA was downloaded from the NIH Genomic Data Commons Data Portal (portal.gdc.cancer.gov / ) on June 6, 2020. Chromosomal band names and genomic locations were downloaded from Ensembl's BioMart (useast.ensembl.org / info / data / biomart / index) on October 16, 2020. Band-level copy numbers were determined by cross-referencing ASCAT2 allele-specific copy number segment files with the genomic locations of the bands using the bedr package in the R working environment. Ploidy was calculated for chromosomal bands where chromosomal segments overlapped using a weighted average (e.g., median). This resulted in the ploidy status of these bands no longer being integers. For each tumor indication tested, the inventors calculated the percentage of patients whose chromosome band had a weighted average copy number of 1.8–2.2 copies and considered these to be diploid (i.e., unamplified). For each indication, the inventors ranked the chromosome bands according to the percentage of patients with a diploid state. For each band, the average rank across indications was calculated. The average rank was then used to identify the chromosome band that was most consistently diploid across the indications in question (see, for example, Table 2). The top-ranked (upper quartile) loci shown in Table 2 (e.g., q13.43, q13.41, q13.42, p13.3, q13.32, and q13.33) represent an expanded repertoire of control loci that can be used in the FISH method provided herein to accurately determine CCNE1 amplification in cells and, consequently, to inform about the likelihood of cancer cells.

[0319] [Table 2]

[0320] The bands shown are relative to the Homo sapiens genome assembly NCBI36. TCGA=The Cancer Genome Atlas, OV=ovary, UCEC=uterine corpus endometrial cancer, UCS=uterine carcinosarcoma Table 2 shows the calculated metrics for the following tumor indications: TCGA-OV (ovarian cancer), TCGA-UCEC (uterine cancer, e.g., endometrial cancer of the uterine body), and TCGA-UCS (uterine carcinosarcoma). The first set of metrics (e.g., "percentage of patients with band ploidy = 2") corresponds to the values ​​shown on the y-axis of the figures shown in Figures 2, 3, 5, and 6. This represents the percentage of patients whose band copy number is approximately 2 (e.g., 1.8–2.2, e.g., 1.8, 1.9, 2.0, 2.1, or 2.2). The second set of metrics (e.g., "ploidy rank within chromosome 19") is calculated for each indication based on the percentage of patients with band ploidy approximately 2. In other words, this metric ranks the bands so that a higher percentage of diploid status is ranked higher. In case of a tie, the mean rank is assigned to both bands. The final metric (mean rank) is calculated by averaging the ranks of each band across the selected tumor indications. This metric is used to sort the table so that the band with the highest mean rank (q13.43) is in the first row. Cross-referencing the control locus information in Table 2 with the control locus of prior art shown in Figure 4 demonstrates that the previously used chromosomal bands p13.3 (column 4), p13.2 (column 11), and p13.11 (columns 15 and 16) have a higher frequency of deviation from the diploid state than band q13.43.

[0321] Example 2. Optimization of the FISH assay for CCNE1 FISH was performed on two gastric cancer tissues (CCNE1 amplified vs. unamplified) and two cell lines (OVCAR3 (CCNE1 amplified), MCF-7 (unamplified)) using a CCNE-targeted probe to determine ideal assay conditions. Two hybridization times (overnight vs. 2 hours) were compared. Overnight hybridization was found to provide optimal staining. Titration of the CCNE1 probe and subtelomere control probe was tested for optimal staining. A 1:1 dilution of the probe was selected as the best titration. Pretreatment conditions were tested on the same tissue set by varying the time and temperature. A 15-minute pretreatment step at 98°C was selected as the optimal condition for tissue samples, while a 5-minute treatment at 80°C was selected for the cell lines. Finally, protease treatment conditions were investigated by varying the treatment time between 15 and 25 minutes. Pepsin solution treatment at 37°C for 15 minutes provided the best signal-to-noise ratio. Figure 7 shows an optimized CCNE1 FISH assay for staining FFPE cell line pellets and FFPE tumor tissue specimens. While pretreatment conditions differ between these two types of samples, protease and hybridization conditions are common. Representative images of cell pellet and tissue staining are provided in Figure 8. As outlined in Figure 9A, the optimal assay conditions were further tested with n=6 cancer tissues (n=2 ovarian, n=2 uterine, n=1 colon, and n=1 breast), as well as n=6 cell lines with various CCNE1 amplification states to confirm assay performance. Counting of 20 cells was completed for all optimization runs. Figure 8 shows representative images of non-amplified endometrial tumors (Figure 8, upper left panel), endometrial and ovarian tumors with low levels of amplification (Figure 8, upper right and lower left panels), and ovarian tumors with high levels of amplification (Figure 8, lower right panel). The corresponding CCNE1 amplification state estimated by SNIPDx®, the cyclin E protein H score, and the median control / CCNE1 signal per cell estimated by FISH are shown below each panel.

[0322] Example 3. Verification of sensitivity, specificity, and accuracy of the FISH assay. After optimizing the staining protocol, assay sensitivity and specificity were determined as shown in Figure 9A. A total of 51 tissue samples, including 30 uterine cancers, 3 ovarian cancers, 2 gastrointestinal tumors, 5 colorectal cancers, and 1 triple-negative breast cancer, were used to validate the sensitivity and specificity of the CCNE1 FISH assay (see, for example, the right side of Figure 9B). A total of 10 normal tissue samples from the ovaries (n=5) and colon (n=5) were also included. Tumor samples were profiled using the targeted next-generation sequencing assay SNiPDx® as a baseline for calculating the amplified state CCNE1 copy number. Figure 9B shows a total of 16 samples from the uterine cancer cohort, 3 samples from the ovarian cancer cohort, and 1 sample from the gastrointestinal cohort, analyzed by SNiPDx®. [CCNE1 total copy count - plicability] ≥ 4 This indicates that the amplification was assumed using a threshold value.

[0323] Conversely, 14 uterine tumor samples, 1 gastrointestinal tumor, 1 triple-negative breast cancer, and 5 colorectal cancer tissues were deemed not to have been amplified by SNIPDx®. Furthermore, of the 41 tumor samples included in the sensitivity / specificity test, 19 were determined to have been amplified by both assays, and 20 were reported not to have been amplified, resulting in an overall agreement of 95.1% (39 / 41 samples) between the two assays (see, e.g., Figure 10 and Table 3). Among the discrepant samples, one sample determined to have been amplified by SNIPDx® was reported not to have been amplified by FISH, and conversely, one tumor sample deemed not to have been amplified by SNIPDx® was reported to have been amplified by FISH (sensitivity: 95.0% (19 / 20) and specificity: 95.2% (20 / 21)). Amplification by SNiPDx® was determined using a threshold of [CCNE1 total copy number - ploidy] ≥ 2. The threshold for reporting amplification by FISH is CCNE1 probe / control probe ≥ 2.

[0324] [Table 3]

[0325] Next, as outlined in Figure 9A, assay-day precision was performed using five tumor specimens (one tissue with a high copy number, one non-amplified tissue, and three specimens with amplification levels close to the threshold criteria described above). The samples were assayed over five days. The mean coefficient of variation (CV) for the five samples over five days was 14.78% (see, for example, Figure 11).

[0326] Finally, as outlined in Figure 9A, the intraday precision of the assay was determined by assaying five tumor samples, selected using the same criteria as in the interday precision experiment, as five replicates on the same day. The mean coefficient of variation (CV) across the five samples was 4.47% (see, for example, Figure 12).

[0327] Example 4. Verification of CCNE1 copy number by optimized FISH assay. Next, the ability of the optimized FISH assay to determine the CCNE1 copy number was validated using the next-generation sequencing (NGS) assay, SNIPDx® (see, for example, Figure 13). As shown in the upper panel of Figure 13, the mean CCNE1 signal per cell estimated by FISH (Y-axis) was significantly higher in tumor specimens considered to contain CCNE1 amplified by the SNIPDx® NGS assay (median 8.7, range 3.25–21.9) compared with non-amplified tumor tissue (median 2.75, range 1.85–5.95). In normal tissue, the mean number of CCNE1 copies per cell is 2, as expected for diploid and genetically stable specimens.

[0328] As shown in the lower panel of Figure 13, correlation analysis between total CCNE1 copy numbers estimated by FISH (X-axis) and SNIPDx® NGS assay (Y-axis) shows strong consistency between the two methodologies (Pearson ρ = 0.923; mp value = 9.8e-18).

[0329] Example 5. Amplification calculation in CCNE1 (Cyclone E1) Amplification of CCNE1 in biological samples (e.g., tumors) can be evaluated using the methods described herein. For example, the sample may be a tumor sample isolated from a human subject and may be evaluated by a standard fluorescence in situ hybridization (FISH) technique utilizing two fluorescently labeled nucleic acid probes. The first probe targets CCNE1. Such probes are standard in the art and commercially available (e.g., Empire Genomics, Inc., catalog RP11-345J21-OR). The second probe may target one of the following chromosomal bands: q13.43, q13.41, q13.42, p13.3, q13.32, and q13.33. FISH can be performed on the tumor sample using these two probes, thereby generating two signals. The number of signals (e.g., foci) generated by the first probe (e.g., the CCNE1 probe) is counted in the cells. The number of signals (e.g., foci) generated by the second probe is counted in the cells. The signals can be determined by fluorescence microscopy. The signal ratio of the first probe to the second probe is calculated by dividing the number of signals from the first probe by the number of signals from the second probe. It is recommended that the signal ratio of at least 50 cells per tissue sample (e.g., 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or more) be counted, although fewer cells per tissue sample (e.g., 20, 25, 30, 35, 40, or 45) may also be counted. The mean (e.g., median) signal ratio of all cells is then calculated. If this mean signal ratio is greater than or equal to 2 (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, or more), the CCNE1 gene in the tumor sample is considered amplified. Amplification of CCNE1 in tumor samples is consistent with the malignancy of the tumor.

[0330] To calculate the total copy number (TCN) of CCNE1, the number of signals from the first probe is divided by the number of cells counted and averaged in the tissue sample.

Claims

1. A method for identifying a stable control locus for use in a fluorescence in situ hybridization (FISH) assay, wherein the method is: (a) Determining the ploidy of multiple chromosomal bands within a chromosome obtained from the biological sample of the subject, (b) Repeating step (a) multiple times, wherein the biological sample is obtained from a different subject each time, (c) Determining the weighted average ploidy of each chromosome band, such that the weighted average ploidy of 1.8 copies to 2.2 copies is considered diploid, (d) Determine the percentage of individuals considered to be diploid for each chromosome band, (e) Ranking each chromosome band from high to low according to the percentage of subjects considered to be diploid, (f) Select a chromosome band within the upper quartile of step (e), thereby identifying the stable control locus, Methods that include...

2. The method according to claim 1, wherein the biological sample includes cancer cells.

3. The method according to claim 1 or 2, wherein the subject is a patient diagnosed with cancer.

4. A method for determining whether cyclin E1 (CCNE1) is amplified in a cell, wherein the method is: (a) A population of cells that have chromosomal DNA, (i) Multiple target probes capable of emitting a first detectable signal and complementary to CCNE1, and (ii) Multiple control probes capable of emitting a second detectable signal and complementary to the CCNE1 control locus. The means of bringing into contact with the CCNE1 control gene locus and the CCNE1 itself, where the CCNE1 is located on the same chromosomal DNA molecule. (b) To quantify the total number of first and second detectable signals in the cells, (c) Determining the signal ratio in the cells of step (b), (d) Repeating steps (b) and (c) multiple times, wherein the cells are different each time, (e) Determine the average signal ratio of process (d), (f) Determining the amplification of CCNE1 from the average signal ratio, wherein an average signal ratio ≥ 2 indicates that CCNE1 is amplified. Methods that include...

5. The method according to claim 4, wherein the plurality of target probes bind to the Homo sapiens genome assembly NCBI36 within the q12 band (chr19q12) of chromosome (chr)19.

6. The method according to claim 4 or 5, wherein the plurality of control probes bind to the Homo sapiens genome assembly NCBI36 at chr19 q13.43, chr19 q13.41, chr19 q13.42, chr19 p13.3, chr19 q13.32, chr19 q13.33, chr19 q13.31, chr19 p13.13, or within chr19 q13.

13.

7. The method according to claim 6, wherein the plurality of control probes bind to the Homo sapiens genome assembly NCBI36 within chr19 q13.

43.

8. The method according to claim 7, wherein the plurality of control probes bind to the Homo sapiens genome assembly NCBI36 at nucleotide positions 29,644,930 to 29,841,126 of chr19 q13.

43.

9. A method for determining whether a target genomic locus is amplified in a cell, wherein the method is: (a) A population of cells that have chromosomal DNA, (i) A plurality of target probes capable of emitting a first detectable signal and complementary to the target genomic locus, and (ii) Multiple control probes that can emit a second detectable signal and are complementary to a stable control locus, The means of bringing into contact with the stable control locus and the target genomic locus, where the stable control locus and the target genomic locus are located on the same chromosomal DNA molecule. (b) To quantify the total number of first and second detectable signals in the cells, (c) Determining the signal ratio in the cells of step (b), (d) Repeating steps (b) and (c) multiple times, wherein the cells are different each time, (e) Determine the average signal ratio of process (d), (f) Determining amplification of the target genome locus from the average signal ratio, wherein an average signal ratio ≥ 2 indicates that the target genome locus is amplified. Methods that include...

10. The aforementioned stable control locus is (a) Determining the ploidy of multiple chromosomal bands within a chromosome obtained from the biological sample of the subject, (b) Repeating step (a) multiple times, wherein the biological sample is obtained from a different subject each time, (c) Determining the weighted average ploidy of each chromosome band, such that the weighted average ploidy of 1.8 copies to 2.2 copies is considered diploid, (d) Determine the percentage of individuals considered to be diploid for each chromosome band, (e) Ranking each chromosome band from high to low according to the percentage of subjects considered to be diploid, (f) Select a chromosome band within the upper quartile of step (e), thereby identifying the stable control locus, The method according to claim 9, as identified by [the specified method].

11. The method according to claim 10, wherein the biological sample includes cancer cells.

12. The method according to any one of claims 4 to 11, wherein the contact is performed in vitro.

13. The method according to any one of claims 4 to 12, wherein the population of cells is derived from a human tumor sample.

14. The method according to any one of claims 4 to 13, wherein the population of cells is derived from a human subject diagnosed with cancer.

15. The method according to any one of claims 4 to 13, wherein the population of cells is derived from a human subject at risk of developing cancer.

16. The method according to any one of claims 4 to 13, wherein the population of cells is derived from a human subject suspected of having cancer.

17. The method according to any one of claims 4 to 16, wherein the average signal ratio is determined from about 30 to about 70 cells.

18. The method according to claim 17, wherein the average signal ratio is determined from approximately 50 cells.

19. The method according to any one of claims 4 to 18, wherein each of the plurality of target probes and / or control probes comprises a polynucleotide having modifications to a base, sugar, and / or backbone.

20. The method according to claim 19, wherein the modification is selected from a 2'-O-methyl (2'-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2'-F) modified nucleoside.

21. A method for identifying patients who are at risk of having cancer or who are suspected of having cancer, (a) Obtaining a cell sample from the subject, (b) the method described in any one of claims 4 to 8, A method for identifying a subject as a patient if it is determined that the CCNE1 gene is amplified in the sample.

22. A method for treating a patient according to claim 3 or 21, comprising administering an effective amount of membrane-bound tyrosine and a threonine-specific cdc2 inhibitory kinase (Myt1) inhibitor.

23. The Myt1 inhibitor is a compound of formula (I): 【Chemistry 1】 or its pharmaceutically acceptable salt, (In the formula, Each of X, Y, and Z is independently N or CR ２ And, R １ and each R ２ is, independently, hydrogen, optionally substituted C １～６ alkyl, optionally substituted C ２～６ alkenyl, optionally substituted C ２～６ alkynyl, optionally substituted C ３～８ cycloalkyl, optionally substituted C ３～８ cycloalkenyl, optionally substituted C ２～９ heterocyclyl, optionally substituted C ２～９ heterocyclyl C １～６ alkyl, optionally substituted C ６～１０ aryl, optionally substituted C １～９ heteroaryl, optionally substituted C １～９ heteroaryl C １～６ alkyl, halogen, cyano, -N(R ７ ), -OR ２ ), -C(O)N(R ７ ), -SO ８ N(R ２ ), -SO ２ R ８ ), or -Q-R ２ ), or R ２ R ７Ａ is, together with one R ７Ｂ adjacent to R １ forms an optionally substituted C １ alkylene, ２ and ３～６ ​ R ３ and R ４ Each of these is independently and arbitrarily substituted C １～６ It is an alkyl or halogen, R ５ is H or -N (R ７ ) ２ And, R ６ is -C(O)NH(R ８ ), -C(O)R ７Ａ , or -SO ２ R ７Ａ And, Each R ７ These are, independently, hydrogen, and optionally substituted C. １～６ Alkyl, optionally substituted C ６～１０ Aryl C １～６ Alkyl, optionally substituted C ３～８ Cycloalkyl, optionally substituted C ６～１０ Aryl, arbitrarily substituted C ２～９ Heterocyclyl, arbitrarily substituted C １～９ Heteroaryl, arbitrarily substituted C １～９ Heteroaryl C １～６ Alkyl, or -SO ２ R ７Ａ or two R ７ The group, together with the atom to which it is bonded, is an arbitrarily substituted C ２～９ Forming heterocyclines, Each R ７Ａ This is an independently and arbitrarily substituted C １～６ Alkyl, optionally substituted C ３～８ Cycloalkyl or optionally substituted C ６～１０ It is Ariel, Each R ７Ｂ These are independently hydroxyl, optionally substituted C １～６ Alkyl, optionally substituted C ６～１０ Aryl, arbitrarily substituted C ２～９ Heterocyclyl, arbitrarily substituted C １～９ Heteroaryl, -N(R ７ ) ２ , -C(O)N(R ８ ) ２ , -SO ２ N(R) ８ ) ２ , -SO ２ R ７Ａ , or optionally substituted alkoxy, Each R ８ These are, independently, hydrogen, and optionally substituted C. １～６ Alkyl, optionally substituted C ２～６ Alkoxyalkyl groups, optionally substituted C ６～１０ Aryl C １～６ Alkyl, optionally substituted C ６～１０ Aryl, arbitrarily substituted C ３～８ Cycloalkyl or optionally substituted C １～９ It is a heteroaryl or two R ８ These, together with the atoms to which they are bonded, form arbitrarily substituted C ２～９ Forming heterocyclines, Q is optionally substituted C １～６ alkylene, optionally substituted C ２～６ alkenylene, optionally substituted C ２～６ alkynylene, optionally substituted C ３～８ cycloalkylene, optionally substituted C ３～８ cycloalkenylene, optionally substituted C ６～１０ arylene, optionally substituted C ２～９ heterocyclylene, or optionally substituted C １～９ heteroarylene), the method according to claim 22.

24. The method according to claim 23, or a pharmaceutically acceptable salt thereof, wherein the compound is concentrated with respect to the atrop isomer of formula (IA). 【Chemistry 2】

25. X is CR ２ The method according to claim 23 or 24, or a pharmaceutically acceptable salt thereof.

26. The method according to claim 23, wherein the compound is of formula (II). 【Transformation 3】

27. The method according to claim 26, wherein the compound is concentrated with respect to the atrop isomer of formula (IIA). 【Chemistry 4】

28. The aforementioned compound is of formula (III): 【Transformation 5】 (wherein R ２Ａ is hydrogen, optionally substituted C １～６ alkyl, optionally substituted C ２～６ alkenyl, optionally substituted C ２～６ alkynyl, optionally substituted C ３～８ cycloalkyl, optionally substituted C ３～８ cycloalkenyl, optionally substituted C ２～９ heterocyclyl, optionally substituted C ２～９ heterocyclyl C １～６ alkyl, optionally substituted C ６～１０ aryl, optionally substituted C １～９ heteroaryl, optionally substituted C １～９ heteroaryl C １～６ alkyl, halogen, -N(R ７ ) ２ , -OR ７ , -C(O)N(R ８ ) ２ , -SO ２ N(R ８ ) ２ , -SO ２ R ７Ａ , or -Q-R ７Ｂ is), the method according to claim 23.

29. The method according to claim 28, wherein the compound is concentrated with respect to the atrop isomer of formula (IIIA). 【Transformation 6】

30. The method according to claim 22, wherein the Myt1 inhibitor is selected from the group consisting of compounds 1 to 328 and pharmaceutically acceptable salts thereof.

31. The method according to claim 22, wherein the Myt1 inhibitor is compound 182 or a pharmaceutically acceptable salt thereof.

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