The present invention relates to use of c-Myc as a biomarker for predicting the response of a brain neoplasm to a compound of formula I as described below, such as 3-(4-{1-[2-(4-amino-phenyl)-2-oxo-ethyl]-1 H-benzoimidazol-2-yl}-furazan-3-ylarnino)- propionitri le (BAL27862) and prodrugs thereof. In other aspects, it relates to methods of treatment and kits.

Microtubules are one of the components of the cell cytoskeleton and are composed of heterodimers of alpha and beta tubulin. Microtubule targeting agents (MTAs) are among the most effective cytotoxic chemotherapeutic agents having a broad spectrum of activity. They are used for example in the treatment of hematologic malignancies and solid tumors, such as lung cancer, breast and prostate cancer. However, the use for treatment of glioblastoma multiforme (GBM) is restricted due to the blood-brain barrier, which does not allow most of the clinically relevant MTAs to cross into the brain where the tumor is located.

Resistance to MTAs can be either inherent or can be acquired after exposure to such agents. Such resistance therefore impacts patient survival rates, as well as choices of treatment regimes. Several potential mechanisms of resistance have been identified. This can include defects in the microtubule targets itself or in microtubule- associated proteins known to alter its biological properties and therefore responsiveness towards MTAs. Furthermore, defects in other cell proteins have been suggested to be associated with resistance to certain microtubule targeting agents, such as overexpression of efflux transporters, which actively pump the drug out of the tumor cells. The standard oncology treatments have very often incomplete and temporary effects that only shrink the bulky tumor and the residual tumor tends to relapse.

GBM tumors are the most common and most aggressive brain tumors among gliomas in adults. Despite progress, effective therapies are limited, especially in the recurrent setting. GBM tumors cause death due to rapid growth and a highly invasive behavior within the whole brain.

The MYC oncogene family consists of three members, C-MYC, MYCN and MYCL, which encode the proteins c-Myc, N-Myc and L-Myc, respectively. The Myc oncoproteins are gene transcription factors. The major downstream effectors of Myc include those involved in ribosome biogenesis, protein translation, cell-cycle progression and metabolism which are relevant for a broad range of biological functions, such as cell proliferation, differentiation, survival and immune surveillance (Chen H. Targeting oncogenic Myc as strategy for cancer treatment, Sig Transduct Target Ther 2018, 3:5).

In cancer, the activity of MYC is frequently deregulated, contributing to the initiation and maintenance of cancer disease. Deregulation often leads to constitutive overexpression of MYC, which has been associated with poor prognosis in various cancer types. MYC overexpression has been reported to be frequent in a range of cancer type (such as breast cancer) but has been reported with relatively low frequency (i.e. <3%) in GBM (Kalkat M. MYC deregulation in primary human cancers, Genes 2017, 8:6, 151 ). The human c-Myc protein sequence and corresponding nucleic acid sequence is available via the National Center for Biotechnology Information (NCBI) reference number NM_001354870 (Figure 2, SEQ ID NO: 3 and 4).

Microtubule end-binding protein 1 (EB1 ) was originally discovered as a binding partner of the APC (adenomatous polyposis coli) tumor suppressor. EB1 is encoded by the MAPRE1 gene and is a member of the RP/EB family involved in the regulation of microtubule dynamics, cell polarity and chromosomal stability during mitosis (Dong X. Oncogenic function of microtubule end-binding protein 1 in breast cancer, J Pathol 2010, 220:3, 361-9; Wang Y. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway, Oncogene 2005, 24:44, 6637-45). EB1 IMAPRE1 has been assigned Human Gene Nomenclature Committee Identification number HGNC ID:6890 and Entrez Gene ID 22919. A sequence corresponding to human EB1 protein and nucleic acid is available via the National Center for Biotechnology Information (NCBI) reference number NM_012325 (Figure 1 , SEQ ID NO: 1 and 2). Despite being an important binding partner of APC, the general role of EB1 in tumor formation has not been well established. However, recent reports suggest that EB1 itself has oncogenic functions. Expression studies have demonstrated that EB1 is overexpressed in breast cancer (Dong X.) gastric cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma (Wang Y.) and its expression level correlates with poor outcome including reduced progression-free survival (PFS) and overall survival (OS). This has also been shown in a more recently performed study in a cohort of 109 primary GBM patients (Berges R. End-binding 1 protein overexpression correlates with GBM progression and sensitizes to Y/nca-alkaloids in vitro and in vivo, Oncotarget 2014, 5:24, 12769-87). High EB1 expression was correlated to poor OS (p<0.001 ) and poor PFS (p<0.001 ) in the investigated GBM patients. BAL27862 is described in WO 2004/103994 for the treatment of neoplastic diseases and autoimmune diseases. Prodrugs thereof (e.g. BAL101553) are described in WO 2011/012577. Use of EB1 as a biomarker for compounds such as BAL27862 and BAL101553 for brain neoplasms, in particular GBM, is described in WO 2017/068182, in Berges R. EB1 -dependent long survival of glioblastoma-grafted mice with the oral tubulin-binder BAL101553 is associated with inhibition of tumor angiogenesis, Oncotarget 2020, 11 :8, 759-774 and in Berges R. The novel tubulin-binding, checkpoint activator BAL101553 inhibits EB1 -dependent migration and invasion and promotes differentiation of GBM stem-like cells, Mol Cancer Ther 2016, 15:11 , 2740-49.

It has now surprisingly been found that high levels of c-Myc are associated with increased sensitivity of GBM to BAL101553 treatment.

In a first aspect the invention provides use of c-Myc as a biomarker for predicting the response of a brain neoplasm to a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

Although compounds of formula I are microtubule destabilizers, they have a different effect on the phenotype of cells compared to other microtubule targeting agents, including other microtubule destabilizers. In addition, they affect microtubule biology in a different manner than conventional microtubule targeting agents and consequently have potent activity in tumor models which are resistant to conventional microtubule-targeting agents. See the Examples of WO 2012/098208, WO 2012/098207, WO 2012/098203, WO 2012/113802 and WO 2012/130887. Thus, predictions cannot be made from information about conventional microtubule targeting agents concerning if, or how, expression of particular genes is involved in the action of compounds of formula I. In a further aspect the invention provides a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use in the treatment of a brain neoplasm in a subject, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

In a further aspect the invention provides use of a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof in the manufacture of a medicament for the treatment of a brain neoplasm in a subject, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

In a further aspect the invention provides a method of treating a brain neoplasm in a subject in need thereof, comprising treating the subject with a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

In a further aspect the invention provides a method for predicting the response to treatment of a brain neoplasm in a subject by administration of a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof said method comprising the steps of: a) determining the level of c-Myc in a sample or samples obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values of the levels from step a) with a standard value or a set of standard values which comparison is predictive of responsiveness to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

In a further aspect the invention provides a kit for predicting the response of a brain neoplasm, preferably GBM, to a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof comprising reagents necessary for measuring the level of c-Myc in a sample.

In some embodiments the subject has a level of c-Myc and a level of EB1 measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values. Embodiments of the present invention will now be described by way of example with reference to the accompanying figures. The invention however is not to be understood as limited to these embodiments.

Brief Description of the Figures

Figure 1 shows the amino acid sequence (Figure 1A) (GenBank AAC09471 - SEQ ID NO:3) and the nucleic acid sequence (Figure 1 B) (GenBank 1124166 - SEQ ID NO: 4) of human EB1 .

Figure 2 shows the amino acid sequence (Figure 2A) (GenBank NP_002458 SEQ ID NO:3) and the nucleic acid sequence (Figure 2B) (Genbank NG_007161 SEQ ID NO: 4) of human c-Myc in which the start and stop codons are shown in bold and underlined.

Figure 3 shows EB1 immunohistochemistry staining in one patient with a long- lasting partial response (top panel, 80% moderate to strong EB1 staining) compared to patient with stable disease after 7 cycles (middle panel, 0% moderate to strong EB1 staining) and patient with progressive disease as best objective response (bottom panel, 0% moderate to strong EB1 staining).

Detailed Description

Compound of formula I

The compound of formula I may be administered as the free base or in the form of a pharmaceutically acceptable salt. Such salts are preferably acid addition salts. Salts are formed, preferably with organic or inorganic acids, with a basic nitrogen atom. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2- hydroxyethanesulfonic acid, ethane-1 ,2-disulfonic acid, benzenesulfonic acid, 2- naphthalenesulfonic acid, 1 ,5-naphthalene-disulfonic acid, 2-, 3- or 4-methyl- benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N- cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid. Unless stated otherwise, reference herein to compounds of formula I includes reference to pharmaceutically acceptable salts thereof.

The compound according to the invention may be administered in the form of a prodrug which is broken down in the human or animal body to give a compound of the formula I. Examples of prodrugs include in vivo hydrolysable amides of a compound of the formula I. Particular prodrugs considered are amides of naturally occurring amino acids and amides of small peptides, in particular small peptides consisting of up to five, preferably two or three amino acids as well as amides of pegylated hydroxy acids, preferably hydroxy acetic acid and lactic acid. Prodrug amides are formed from the amino function of the compound of formula I and the acid function of the amino acid or the C terminal of the peptide. Preferably the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine.

In some embodiments the compound of formula I is in the form of a prodrug selected from the compounds of formulae:

In an especially preferred embodiment the compound of formula I according to the invention is in the form of the prodrug BAL101553: or pharmaceutically acceptable salt thereof, preferably a hydrochloride salt, most preferably a dihydrochloride salt.

The prodrugs of the invention may be prepared as described for example in WO 2011/012577 and WO 2018/197475. Crystalline forms thereof may be prepared as described in WO 2018/197475.

As for the compound of formula I, prodrugs may be provided in the form of a pharmaceutically acceptable salt, as described above. Unless stated otherwise, reference to prodrugs of the compound of formula I includes reference to pharmaceutically acceptable salts thereof.

Brain neoplasms

Brain neoplasms, e.g. brain tumors, include but are not limited to glial- and non- glial-tumors, astrocytomas (including GBM and unspecified gliomas), oligodendrogliomas, ependydomas, menigiomas, hemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNET’s), medullablastomas, hemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically-driven brain neoplasms including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis. Preferably, brain neoplasm refers to gliomas, more preferably GBM.

Samples

The measurement of the level of c-Myc or the level of c-Myc and the level of EB1 is performed ex vivo on a sample of biological tissue derived from the subject. The sample may be any biological material separated from the body such as, for example, normal tissue, tumor tissue, cell lines, blood (including serum and/or plasma), e.g. peripheral blood (including circulating DNA or RNA, circulating tumor cells (CTCs) or cancer stem cells (CSCs)), cerebrospinal fluid (including CTCs or CSCs), lymph fluid, cell lysate, tissue lysate, urine and aspirates. Preferably the sample is derived from normal tissue, tumor tissue, cell lines, blood (including serum and/or plasma), e.g. peripheral blood (including circulating DNA, RNA, CTCs or CSCs) or cerebrospinal fluid (including CTCs or CSCs). More preferably the sample is derived from tumor tissue, DNA, RNA, CTCs or CSCs derived from peripheral blood or cerebrospinal fluid. Even more preferably the sample is derived from tumor tissue, DNA, RNA, CTCs or CSCs derived from peripheral blood. In one particularly preferred embodiment the sample is derived from tumor tissue. For example, the level of c-Myc or the level of c-Myc and the level of EB1 in cancer cells and/or CSCs may be measured in a fresh, frozen or formalin fixed/paraffin embedded tumor tissue sample. When the sample is derived from a body fluid, for example blood (e.g. peripheral blood), serum and plasma, urine, cerebrospinal fluid or saliva the level of c-Myc or the level of c-Myc and the level of EB1 may be measured in the extracellular vesicles in the sample.

The sample is pre-obtained from the subject before the sample is subjected to method steps involving measuring the level of the biomarker. Methods for taking a sample from a subject are well known in the art. Methods of removing a sample from a tumor may involve for example tumor resection or biopsy, for example by punch biopsy, core biopsy or aspiration fine needle biopsy, endoscopic biopsy, or surface biopsy.

Brain CSCs very often spread to different sites in the brain and form secondary lesions and metastases. To do so, they may invade their surroundings and thereby may also destroy structures supporting the blood-brain-bamer. As a consequence of this invasion process, they may gain access to the blood circulation system and therefore can be identified/purified from peripheral blood (Watkins S. Disruption of astrocytes-vascular coupling and the blood-brain barrier by invading glioma cells, Nature Communications 2014, 5, 4196; Diaz, M. Transmigration of Neural Stem Cells across the blood-brain barrier induced by glioma cells. PLoS One 2013, 8(4): e60655; Beauchesne P. Extra- Neural Metastases of Malignant Gliomas: Myth or Reality, Cancers 2011 , 3:1 , 461 -477).

A blood sample may be collected by venipuncture and further processed according to standard techniques. Circulating tumor cells and/or circulating CSCs may also be obtained from blood or cerebrospinal fluid based on, for example, size (e.g. ISET - Isolation by Size of Epithelial Tumor cells; by Cluster-chip based on the size of preformed cell clusters) or immunomagnetic cell enrichment (e.g. CellSearch®, Veridex, Raritan, NJ; MACS®, Miltenyi Biotec, Germany; RosetteSep™, EasySep™ and RoboSep™, STEMCELL Technologies, France) or fluorescence activated cell sorting (FACS) (e.g. BD Stemflow Kit, BD-Biosciences, USA). FACS sorters are available from a number of commercial sources (e.g. Bio-Rad [Hercules, CA, USA] Benchtop S3™ cell sorter; Beckman Coulter [Brea, CA, USA] MoFlo™ cell sorter; BD Biosciences FACSDiva™ cell sorter). Cell separation based on dielectric properties (ApoStream®, APOCELL, USA) can also be performed.

Brain tumor material, e.g. GBM material, may be obtained from patients undergoing surgery or tumor biopsy. Resected GBM tumor material may then be further processed by methods known in the art including, but not restricted to: formalin fixation and paraffin embedding; snap freezing and storage at -80C; treatment RNA preservation solution (e.g. RNA/ater, ThermoFisher Scientific; RNAprotect, QIAGEN) and storage according to manufacturers’ instructions; tumor dissociation according to a number of methods including, but not restricted to placing the tumor material into a tube on ice containing neural stem cell (NSC) basal medium supplemented with 10-15% antibiotics (Penicilin/Streptomycin). The following procedure is given as an example. The GBM tumor samples are washed 2-3 times with sterile PBS/NSC basal medium to remove blood and debris. In a sterile Petri dish the washed tumor tissue is cut into small pieces and minced with a scalpel blade, transferred into a Falcon tube and trypsinized in a few mL’s of pre-warmed 0.05% trypsin-EDTA for 10-15 minutes in a water bath at 37°C. After the incubation period, an equal volume of soybean trypsin inhibitor is added to stop the enzymatic trypsin reaction. The tumor cell suspension is pelleted down by centrifugation at 800 rpm (110g) for 5 minutes. The supernatant is discarded and the pellet is resuspended in 1 mL of sterile NSC basal medium. The clumps are dissociated by gently pipetting up and down and the cell suspension is passed through a 40 micron cell strainer to remove small cell debris (Azari, H. Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay. J. Vis.

Exp.2011 ;Oct 30(56):e3633). As an alternative approach, dissociation of the tumor tissue into a tumor cell suspension may also be performed using a commercially available kit including, but not restricted to, “The Brain Tumor Dissociation Kits” available from Miltenyi Biotec Inc., Auburn, CA 95602, USA.

It is known that brain tumors such as those associated with GBM release extracellular vesicles into the blood which may then circulate around the body (Arscott et al. Ionizing Radiation and Glioblastoma Exosomes: Implications in Tumor Biology and Cell Migration, Translational Oncology, 2006;6:638-648). Extracellular vesicles include microvesicles and exosomes and are generally nanometer-sized membrane-derived vesicles. Extracellular vesicles contain molecular information (including protein and RNA) reflecting the cell of origin including hallmarks of tumor biology (Redzik J. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers, Pharmgenomics Pers Med 2014, 7, 65-77; Capello F. Exosome levels in human body fluids: A tumor marker by themselves?, Eur J Pharm Sci 2016, 96, 93-98) and are accessible in body fluids such as blood, including serum and plasma, urine, cerebrospinal fluid and saliva. This provides an alternative method of determining whether c-Myc and/or EB1 is present at relevant levels, in particular in GBM tumors. Extracellular vesicles can be isolated using a variety of protocols and commercially available kits including: a) differential ultracentrifugation (Thery C. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protec Cell Biol 2006, Chapter 3 Unit 3.22); b) chemical precipitation (e.g. Total Exosome Isolation Kits from Invitrogen, ThermoFischer, Waltham, MA USA; ExoQuick™ Exosome Precipitation Solution from SBI System Biosciences, Palo Alto, USA; Exo-Prep from HansaBioMed, Tallinn, Estonia); and c) immunoaffinity capture (Zarovni N. Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods 2015, 87, 46-58) using for example ELISA immunoplates (e.g. from HansaBioMed), Immunobeads (e.g. from HansaBioMed) or ExoCap™ from JSR Life Sciences, Leuven, Belgium. The level of c- Myc and/or EB1 nucleic acid and/or protein in the extracellular vesicles can be measured by standard techniques as described below.

When the level of c-Myc and the level of EB1 are both measured, the respective levels may be measured in the same sample or in different samples.

Sample comparison

The subject according to the invention may be a human or animal in need of treatment. Preferably the subject is human. As mentioned above, the biomarker c-Myc and/or EB1 is measured ex vivo in a sample or samples taken from the human or animal body, preferably taken from the human body. The sample or samples are preobtained from the human or animal body, e.g. pre-obtained from the human body before the sample is subjected to the method steps involving measuring the level of the biomarker.

A biomarker is in general a substance that is used as an indicator of a biological response, preferably as an indicator of the susceptibility to a given treatment, which in the present application is treatment with a compound of formula I or prodrug thereof. It has been found herein that higher c-Myc levels predict responsiveness to the compounds of the invention, or put another way, lower levels of c-Myc predict resistance to the compounds of the invention.

In one preferred embodiment, higher c-Myc levels in the sample or samples relative to a standard value or set of standard values predicts responsiveness. As used herein, an increase or relatively high or high or higher levels relative to a standard level or set of standard levels means the amount or concentration of the biomarker in a sample is detectably more in the sample relative to the standard level or set of standard levels. This encompasses at least an increase of, or higher level of, about 1 % relative to the standard, e.g. at least an increase of about 5% relative to the standard. For example it is an increase of, or higher level of, at least about 10% relative to the standard. For example it is an increase of, or higher level of, at least about 20% relative to the standard. For example, such an increase of, or higher level of, may include, but is not limited to, at least about 1 %, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90% or at least about 100% or > 100% increase relative to the standard.

In another preferred embodiment, lower c-Myc levels in the sample or samples relative to a standard value or set of standard values predicts resistance. As used herein, a decrease or relatively low or low or lower levels relative to a standard level or set of standard levels means the amount or concentration of the biomarker in a sample is detectably less in the sample relative to the standard level or set of standard levels. This encompasses at least a decrease of, or lower level of, about 1 % relative to the standard, e.g. at least a decrease of about 5% relative to the standard. For example it is a decrease of, or lower level of, at least about 10% relative to the standard. For example it is a decrease of, or lower level of, at least about 20% relative to the standard. For example, such a decrease of, or lower level of, may include, but is not limited to, at least about 1 %, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90% or at least about 100% decrease relative to the standard. A decrease also includes the absence of detectable c-Myc in the sample.

Preferably a higher level of c-Myc in the sample i) relative to a standard value or set of standard values from subjects with the same tumor histotype; ii) taken after treatment initiation and compared to the sample taken from the same subject before treatment initiation; or iii) relative to a standard value or set of standard values from normal cells, tissue or body fluid; predicts sensitivity of the brain neoplasm to the compound of formula I or a prodrug thereof.

More preferably a higher level of c-Myc in the sample relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or a prodrug thereof.

In another preferred embodiment, higher c-Myc levels and higher EB1 levels in the sample or samples relative to a standard value or set of standard values predicts responsiveness. For example the level of EB1 may show at least an increase of, or higher level of, about 1 % relative to the standard, e.g. at least an increase of about 5% relative to the standard. For example it is an increase of, or higher level of, at least about 10% relative to the standard. For example it is an increase of, or higher level of, at least about 20% relative to the standard. For example, such an increase of, or higher level of, may include, but is not limited to, at least about 1 %, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90% or at least about 100% or > 100% increase relative to the standard.

In another preferred embodiment, lower c-Myc levels and lower EB1 levels in the sample relative to a standard value or set of standard values predicts resistance. For example the level of EB1 may show at least a decrease of, or lower level of, about 1 % relative to the standard, preferably at least a decrease of about 5% relative to the standard. For example, it is a decrease of, or lower level of, at least about 10% relative to the standard. For example it is a decrease of, or lower level of, at least about 20% relative to the standard. For example, such a decrease of, or lower level of, may include, but is not limited to, at least about 1 %, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90% or at least about 100% decrease relative to the standard. Thus a decrease also includes the absence of detectable EB1 in the sample.

Preferably a higher c-Myc level in the sample and a higher EB1 level in the sample i) relative to a standard value or set of standard values from subjects with the same tumor histotype; or ii) taken after treatment initiation and compared to the sample taken from the same subject before treatment initiation; or iii) relative to a standard value or set of standard values from normal cells, tissue or body fluid; predicts sensitivity of the brain neoplasm to the compound of formula I or prodrug thereof.

More preferably a higher c-Myc level and a higher EB1 level in the sample relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or a prodrug thereof. In one preferred embodiment the standard value or set of standard values are established from samples from a population of subjects with that cancer type. The samples from these standard subjects may for example be derived from tumor tissue or from circulating DNA, RNA, CTCs, CSCs or extracellular vesicles, as long as the origin of the sample is consistent between the standard and the sample to be compared.

The standard value or set of standard values are established ex-vivo from preobtained samples which may be from cell lines or animal tumor models, or preferably biological material from at least one human subject and more preferably from an average of subjects (e.g., n=2 to 1000 or more). The standard value or set of standard values may then be correlated with the response data of the same cell lines, or same subjects, to treatment with a compound of formula I or a prodrug thereof. From this correlation a comparator module, for example in the form of a relative scale or scoring system, optionally including cut-off or threshold values, can be established which indicates the levels of biomarker associated with a spectrum of response levels to the compound of formula I or a prodrug thereof. The spectrum of response levels may comprise relative sensitivity to the therapeutic activity of the compound, (e.g. high sensitivity to low sensitivity), as well as resistance to the therapeutic activity. In a preferred embodiment this comparator module comprises a cut-off value or set of values which predicts sensitivity to treatment.

For example, if an immunohistochemical method is used to measure the level of c-Myc and/or EB1 in a sample, standard values may be in the form of a scoring system. Such a system might take into account the percentage of cells in which staining for c- Myc and/or EB1 is present. The system may also take into account the relative intensity of staining in the individual cells. The standard values or set of standard values of the level of c-Myc and/or EB1 may then be correlated with data indicating the response, especially sensitivity, of the subject or tissue or cell line to the therapeutic activity of a compound of formula I or a prodrug thereof. Such data may then form part of a comparator module.

Response is the reaction of the cell lines, or preferably of the subject, or more preferably of the brain neoplasm in a subject, to the activity, preferably therapeutic activity, of a compound of formula I or a prodrug thereof. The spectrum of response levels may comprise relative sensitivity to the activity, preferably therapeutic activity, of the compound, (e.g. high sensitivity to low sensitivity), as well as resistance to the activity, preferably therapeutic activity. The response data may for example be monitored in terms of: objective response rates, time to disease progression, progression free survival, overall survival and/or quality of life.

The response of a brain neoplasm may be evaluated by using criteria well known to a person in the field of cancer treatment, for example but not restricted to RANG Criteria for High-Grade Gliomas, see Wen P.Y. Updated response assessment criteria for high-grade gliomas: response assessment in neuro-oncology working group, J Clin Oncol 2010, 28(11 ), 1963-72.

Sensitivity is associated with there being an observable and/or measurable reduction in, or presence of, one or more of the following: reduction in the number of abnormal cells, preferably cancerous cells or absence of the abnormal cells, preferably cancerous cells; reduction in tumor size; inhibition (i.e. , slowed to some extent and preferably stopped) of further tumor growth; reduction in the levels of cancer cell infiltration into other organs (including the spread of cancer); inhibition (i.e., slowed to some extent and preferably stopped) of tumor metastasis; alleviation of one or more of the symptoms associated with the specific cancer; and reduced morbidity and mortality or improved quality of life.

In a preferred embodiment sensitivity means there is an observable and/or measurable reduction in, or absence of, one or more of the following criteria: reduction in tumor size; inhibition of further tumor growth, inhibition of cancer cell infiltration into other organs; and inhibition of tumor metastasis. In a more preferred embodiment sensitivity refers to one or more of the following criteria: a reduction in tumor size; inhibition of further tumor growth, inhibition of cancer cell infiltration into other organs; and inhibition of tumor metastasis. Measurement of the aforementioned sensitivity criteria is according to clinical guidelines well known to a person in the field of cancer treatment, such as those listed above for measuring the response of a cancerous disease.

Response may also be established in vitro by assessing cell proliferation and/or cell death. For example, effects on cell death or proliferation may be assessed in vitro by one or more of the following well established assays: A) Nuclear staining with Hoechst 33342 dye providing information about nuclear morphology and DNA fragmentation which are hallmarks of apoptosis. B) AnnexinV binding assay which reflects the phosphatidylserine content of the outer lipid bilayer of the plasma membrane. This event is considered an early hallmark of apoptosis. C) TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay), a fluorescence method for evaluating cells undergoing apoptosis or necrosis by measuring DNA fragmentation by labeling the terminal end of nucleic acids. D) MTS proliferation assay measuring the metabolic activity of cells. Viable cells are metabolically active whereas cells with a compromised respiratory chain show a reduced activity in this test. E) Crystal violet staining assay, where effects on cell number are monitored through direct staining of cellular components. F) Proliferation assay monitoring DNA synthesis through incorporation of bromodeoxyuridine (Brdll). Inhibitory effects on growth/proliferation can be directly determined. G) YO-PRO assay which involves a membrane impermeable, fluorescent, monomeric cyanine, nucleic acid stain, which permits analysis of dying (e.g. apoptotic) cells without interfering with cell viability. Overall effects on cell number can also be analyzed after cell permeabilisation. H) Propidium iodide staining for cell cycle distribution which shows alterations in distribution among the different phases of the cell cycle. Cell cycle arresting points can be determined. I) Anchorage-independent growth assays, such as colony outgrowth assays which assess the ability of single cell suspensions to grow into colonies in soft agar.

In one preferred embodiment relating to determination in vitro, sensitivity means there is a decrease in the proliferation rate of abnormal cells and/or reduction in the number of abnormal cells. More preferably sensitivity means there is a decrease in the proliferation rate of cancerous cells and/or a reduction in the number of cancerous cells. The reduction in the number of abnormal, preferably cancerous, cells may occur through a variety of programmed and non-programmed cell death mechanisms. Apoptosis, caspase-independent programmed cell death and autophagic cell death are examples of programmed cell death. However, the cell death criteria involved in embodiments of the invention is not to be taken as limited to any one cell death mechanism.

In a preferred embodiment relating to determination of resistance in vitro, resistance means there is no decrease in the proliferation rate of abnormal cells and/or reduction in the number of abnormal cells. More preferably resistance means there is no decrease in the proliferation rate of cancerous cells and/or no reduction in the number of cancerous cells. The reduction in the number of abnormal, preferably cancerous, cells may occur through a variety of programmed and non-programmed cell death mechanisms. Apoptosis, caspase-independent programmed cell death and autophagic cell death are examples of programmed cell death. However, the cell death criteria involved in embodiments of the invention is not to be taken as limited to any one cell death mechanism. c-Myc

The term c-Myc is used herein to encompass all the previously mentioned synonyms and refers to this entity on both the nucleic acid and protein levels as appropriate. Nucleic acid levels refer to for example mRNA, cDNA or DNA and the term protein includes the translated polypeptide or protein sequence and post-translationally modified forms thereof.

A preferred example of the protein sequence of c-Myc (human c-Myc) is listed in SEQ. ID NO: 3. However, the term c-Myc also encompasses homologues, mutant forms, allelic variants, isotypes, splice variants and equivalents of this sequence. Preferably also it encompasses human homologues, mutant forms, allelic variants, isotypes, splice variants and equivalents of this sequence. More preferably it encompasses sequences having at least about 75% identity, especially preferably at least about 85% identity, particularly preferably at least about 95% identity, and more particularly preferably about 99% identity, to said sequence.

In an especially preferred embodiment, c-Myc is the entity on the nucleic acid or protein levels, which is represented on the protein level by SEQ ID NO: 3 or sequences having at least about 95% identity with this sequence, preferably at least about 99% identity. In a particularly preferred embodiment, c-Myc is represented by SEQ. ID. NO: 3.

A preferred example of the cDNA nucleic acid sequence of c-Myc (Human c-Myc) is represented by SEQ ID NO: 4. However, the term c-Myc also encompasses modifications, more degenerate variants of said sequence, complements of said sequence, and oligonucleotides that hybridize to one of said sequences. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. More preferably it encompasses sequences having at least about 75% identity to said sequence, especially preferably at least about 85% identity, particularly preferably at least about 95% identity and more particularly preferably about 99% identity.

In yet another preferred embodiment, c-Myc is the entity on the nucleic acid or protein levels, which is represented on the nucleic acid level by SEQ ID NO. 4 or sequences having at least about 95% identity with this sequence, preferably at least about 99% identity. In a particularly preferred embodiment, c-Myc is represented by SEQ. ID. NO:. 4. Percent sequence identity is calculated using the National Institute of Health Basica Local Alignment Search Tool (BLAST) using the default parameters, available at https://blast.ncbi.nlm.nih.gov/Blast.cgi.

EB1

The term EB1 is used herein to encompass all the previously mentioned synonyms and refers to this entity on both the nucleic acid and protein levels as appropriate. Nucleic acid levels refer to for example mRNA, cDNA or DNA and the term protein includes the translated polypeptide or protein sequence and post-translationally modified forms thereof.

A preferred example of the protein sequence of EB1 (human EB1 ) is listed in SEQ. ID NO: 1. However, the term EB1 also encompasses homologues, mutant forms, allelic variants, isotypes, splice variants and equivalents of this sequence. Preferably also it encompasses human homologues, mutant forms, allelic variants, isotypes, splice variants and equivalents of this sequence. More preferably it encompasses sequences having at least about 75% identity, especially preferably at least about 85% identity, particularly preferably at least about 95% identity, and more particularly preferably about 99% identity, to said sequence.

In an especially preferred embodiment, EB1 is the entity on the nucleic acid or protein levels, which is represented on the protein level by SEQ ID NO: 1 or sequences having at least about 95% identity with this sequence, preferably at least about 99% identity. In a particularly preferred embodiment, EB1 is represented by SEQ. ID. NO: 1.

A preferred example of the cDNA nucleic acid sequence of EB1 (Human EB1 ) is accessible via NCBI Reference Sequence U24166 represented by SEQ ID NO: 2. However, the term EB1 also encompasses modifications, more degenerate variants of said sequence, complements of said sequence, and oligonucleotides that hybridize to one of said sequences. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. More preferably it encompasses sequences having at least about 75% identity to said sequence, especially preferably at least about 85% identity, particularly preferably at least about 95% identity and more particularly preferably about 99% identity.

In yet another preferred embodiment, EB1 is the entity on the nucleic acid or protein levels, which is represented on the nucleic acid level by SEQ ID NO. 2 or sequences having at least about 95% identity with this sequence, preferably at least about 99% identity. In a particularly preferred embodiment, EB1 is represented by SEQ.

ID. NO:. 2.

Level of c-Myc

Levels of c-Myc may be detected either on the protein level or on the nucleic acid level, e.g. RNA or DNA. The level of c-Myc may be assayed in the sample by technical means well known to a skilled person. It may be assayed at the transcriptional or translational level.

In one preferred embodiment the level of c-Myc nucleic acid is measured. The level of nucleic acid may be e.g. expression or amplification, e.g. mRNA expression and/or DNA amplification. The level of c-Myc amplification may be determined by the mean average number of gene copies of c-Myc per cell in the sample. A mean average of two gene copies per cell represents the normal, non-amplified status, i.e. the standard value for amplification status may be considered as a mean average of 2 gene copies of c-Myc per cell in the sample. An amplified status may be considered as a mean average of more than two gene copies of c-Myc per cell in the sample, e.g. a mean average of at least 3, at least 4, at least 5 or at least 6 gene copies of c-Myc per cell in the sample. The usual appearance of a gene amplification, sensu strictu, is that of densely clustered >6 gene signals.

Examples of methods of gene amplification I expression analysis known in the art which are suitable to measure the level of c-Myc at the nucleic acid level include, but are not limited to, i) using a labelled probe that is capable of hybridizing to mRNA (e.g. fluorescence in situ hybridization (FISH); ii) using PCR involving one or more primers based on the c-Myc gene sequence, for example using quantitative PCR methods using labelled probes, e.g. fluorogenic probes, such as quantitative real-time PCR; iii) microarrays; iv) northern blotting v) serial analysis of gene expression (SAGE), READS (restriction enzyme amplification of digested cDNAs), differential display and measuring microRNA. Measuring the level of c-Myc at the nucleic acid level by FISH is of particular interest and is a well-known tool for detecting specific biomarkers in neoplasms, see for example Hu L. Fluorescence in situ hybridization (FISH): an increasing demanded tool for biomarker research and personalized medicine, Biomarker Research 2014, 2:1 , 3.

In another preferred embodiment the level of c-Myc at the protein level is measured. Examples of methods of protein expression analysis known in the art which are suitable to measure the level of c-Myc at the protein level include, but are not limited to, i) immunohistochemistry (IHC) analysis, ii) western blotting iii) immunoprecipitation iv) enzyme linked immunosorbent assay (ELISA) v) radioimmunoassay vi) Fluorescence activated cell sorting (FACS) vii) mass spectrometry, including matrix assisted laser desorption/ionization (MALDI, e.g. MALDI-TOF) and surface enhanced laser desorption/ionization (SELDI, e.g. SELDI-TOF).

The antibodies involved in some of the above methods may be monoclonal or polyclonal antibodies, antibody fragments, and/or various types of synthetic antibodies, including chimeric antibodies, DARPS (designed Ankyrin repeat proteins) or DNA/RNA aptamers. The antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result. Antibodies specific to c-Myc are available commercially, for example, from Cell Signaling Technology, Inc., or can be prepared via conventional antibody generation methods well known to a skilled person.

Preferred methods of protein analysis are flow cytometry (FACS), ELISA, fluorescence microscopy, mass spectrometry techniques, immunohistochemistry and western blotting, more preferably FACS, western blotting and immunohistochemistry. In FACS, fluorescently labelled antibodies or probes are used to bind to specific cellular proteins or antigens on whole cells (either fixed or native) in suspension where the intensity of the signal from the detectable label bound to the cellular antigen corresponds to the amount of protein expressed in the single cell and can be quantified. In fluorescence microscopy, fluorescently labelled antibodies or probes are used to bind to specific cellular proteins (antigens) where the protein amount and the positions can be detected and measured by the signal from the detectable label. In western blotting, also known as immunoblotting, labelled antibodies may be used to assess levels of protein, where the intensity of the signal from the detectable label corresponds to the amount of protein, and can be quantified for example by densitometry. Immunohistochemistry again uses labelled antibodies or probes to detect the presence and relative amount of the biomarker. It can be used to assess the percentage of cells for which the biomarker is present. It can also be used to assess the localization or relative amount of the biomarker in individual cells; the latter is seen as a function of the intensity of staining. ELISA stands for enzyme linked immunosorbant assay, since it uses an enzyme linked to an antibody or antigen for the detection of a specific protein. ELISA is typically performed as follows (although other variations in methodology exist): a solid substrate such as a 96 well plate is coated with a primary antibody, which recognizes the biomarker. The bound biomarker is then recognized by a secondary antibody specific for the biomarker. This may be directly joined to an enzyme or a third anti-immunoglobulin antibody may be used which is joined to an enzyme. A substrate is added and the enzyme catalyzes a reaction, yielding a specific color. By measuring the optical density of this color, the presence and amount of the biomarker can be determined.

Level of EB1

Levels of EB1 may be detected either on the protein level or on the nucleic acid level, e.g. RNA or DNA. The level of EB1 may be assayed in the sample by technical means well known to a skilled person. It may be assayed at the transcriptional or translational level.

In one preferred embodiment the level of EB1 nucleic acid, preferably EB1 mRNA, in a sample is measured. The level of nucleic acid may be e.g. expression or amplification, e.g. mRNA expression and/or DNA amplification. Examples of methods of gene amplification I expression analysis known in the art which are suitable to measure the level of EB1 at the nucleic acid level include, but are not limited to, i) using a labelled probe that is capable of hybridizing to mRNA (e.g. FISH); ii) using PCR involving one or more primers based on the EB1 gene sequence, for example using quantitative PCR methods using labelled probes, e.g. fluorogenic probes, such as quantitative real-time PCR; iii) micro-arrays; IV) northern blotting V) serial analysis of gene expression (SAGE), READS (restriction enzyme amplification of digested cDNAs), differential display and measuring microRNA.

In a preferred embodiment the level of EB1 at the protein level is measured. Examples of methods of protein expression analysis known in the art which are suitable to measure the level of EB1 at the protein level include, but are not limited to, i) immunohistochemistry (IHC) analysis, ii) western blotting iii) immunoprecipitation iv) enzyme linked immunosorbent assay (ELISA) v) radioimmunoassay vi) Fluorescence activated cell sorting (FACS) vii) mass spectrometry, including matrix assisted laser desorption/ionization (MALDI, e.g. MALDI-TOF) and surface enhanced laser desorption/ionization (SELDI, e.g. SELDI-TOF).

The antibodies involved in some of the above methods may be monoclonal or polyclonal antibodies, antibody fragments, and/or various types of synthetic antibodies, including chimeric antibodies, DARPS (designed Ankyrin repeat proteins) or DNA/RNA aptamers. The antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result. Antibodies specific to EB1 are available commercially, for example, from BD Biosciences and Cell Signaling Technology, Inc., or can be prepared via conventional antibody generation methods well known to a skilled person.

Preferred methods of protein analysis are flow cytometry (FACS), ELISA, fluorescence microscopy, mass spectrometry techniques, immunohistochemistry and western blotting, more preferably FACS, western blotting and immunohistochemistry. In FACS, fluorescently labelled antibodies or probes are used to bind to specific cellular proteins or antigens on whole cells (either fixed or native) in suspension where the intensity of the signal from the detectable label bound to the cellular antigen corresponds to the amount of protein expressed in the single cell and can be quantified. In fluorescence microscopy, fluorescently labelled antibodies or probes are used to bind to specific cellular proteins (antigens) where the protein amount and the positions can be detected and measured by the signal from the detectable label. In western blotting, also known as immunoblotting, labelled antibodies may be used to assess levels of protein, where the intensity of the signal from the detectable label corresponds to the amount of protein, and can be quantified for example by densitometry. Immunohistochemistry again uses labelled antibodies or probes to detect the presence and relative amount of the biomarker. It can be used to assess the percentage of cells for which the biomarker is present. It can also be used to assess the localization or relative amount of the biomarker in individual cells; the latter is seen as a function of the intensity of staining. For example the level of EB1 may be considered positive when at least 50% of tumor cells (e.g. at least 60%, e.g. at least 70%) in the sample have at least a moderate to strong staining intensity e.g. according to a pathologist’s judgement (see e.g. Figure 3 for reference). ELISA stands for enzyme linked immunosorbent assay, since it uses an enzyme linked to an antibody or antigen for the detection of a specific protein. ELISA is typically performed as follows (although other variations in methodology exist): a solid substrate such as a 96 well plate is coated with a primary antibody, which recognizes the biomarker. The bound biomarker is then recognized by a secondary antibody specific for the biomarker. This may be directly joined to an enzyme or a third anti-immunoglobulin antibody may be used which is joined to an enzyme. A substrate is added and the enzyme catalyzes a reaction, yielding a specific color. By measuring the optical density of this color, the presence and amount of the biomarker can be determined. For the avoidance of doubt, the levels of c-Myc and EB1 may be measured using independent methodologies. For example the level of c-Myc may be measured at the nucleic acid level and the level of EB1 may be measured at the protein level, or vice versa.

Uses of biomarker

The biomarker may be c-Myc alone or c-Myc in combination with EB1 . In one preferred embodiment, the biomarker is used to predict inherent sensitivity of the brain neoplasm in a subject to the compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above. In another preferred embodiment, the biomarker is used to predict acquired resistance of the brain neoplasm in a subject to the compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above.

The biomarker may be used to select subjects suffering or predisposed to suffering from a brain neoplasm, for treatment with a compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above. The levels of such a biomarker may be used to identify patients likely to respond or to not respond or to continue to respond or to not continue to respond to treatment with such agents. Stratification of patients may be made in order to avoid unnecessary treatment regimes. In particular the biomarker may be used to identify subjects from whom a sample or samples display a higher level of c-Myc or a higher level of c-Myc and a higher level of EB1 , relative to a standard level or set of standard levels, whereupon such subjects may then be selected for treatment with the compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above.

The biomarker may also be used to assist in the determination of treatment regimes, regarding amounts and schedules of dosing. Additionally, the biomarker may be used to assist in the selection of a combination of drugs to be given to a subject, including a compound or compounds of formula I or a prodrug thereof, and another chemotherapeutic (cytotoxic) agent or agents. Furthermore, the biomarker may be used to assist in the determination of therapy strategies in a subject including whether a compound of formula I or a prodrug thereof (e.g. BAL101553) is to be administered in combination with targeted therapy, endocrine therapy, biologies, radiotherapy, immunotherapy or surgical intervention, or a combination of these. c-Myc or c-Myc and EB1 may also be used in combination with other biomarkers to predict the response to a compound of formula I or a prodrug thereof (e.g. BAL101553) and to determine treatment regimes. It may furthermore be used in combination with chemo-sensitivity testing to predict sensitivity and to determine treatment regimes. Chemo-sensitivity testing involves directly applying a compound of formula I to cells taken from the subject to determine the response of the cells to the compound.

Method of treatment

The invention also involves in some aspects a method of treatment and c-Myc or c-Myc and EB1 for use in a method of treatment, wherein the level of c-Myc or the level of c-Myc and EB1 is first established relative to a standard level or set of standard levels or pre-treatment initiation levels and then a compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above, is administered if the level of c-Myc or the level of c-Myc and the level of EB1 i is higher than a standard value or set of standard values or has not decreased relative to pre-treatment initiation levels respectively. After a patient has been determined to have a higher level of c-Myc or c-Myc and EB1 the patient may continue to receive treatment with the compound of formula I or a prodrug thereof (e.g. BAL101553) without further determinations of the level of c-Myc or c-Myc and EB1 .

The compound of formula I or a prodrug thereof (e.g. BAL101553) may be administered in a pharmaceutical composition, as is well known to a person skilled in the art. Suitable compositions and dosages are for example disclosed in WO 2004/103994 pages 35-39, which are specifically incorporated by reference herein. Compositions for intravenous or oral administration are generally preferred. Examples of intravenous administration with reference to prodrugs of the compound of formula I are described in WO 2015/173341 and WO 2018/210868.

The compound of formula I or a prodrug thereof (e.g. BAL101553) is administered as a pharmaceutically effective amount, i.e. an amount sufficient to provide an observable or clinically significant improvement over the baseline. Generally the compound of formula I or a prodrug thereof (e.g. BAL101553) may be administered orally or intravenously and will be administered at dosages which do not exceed the maximum tolerated dose (MTD) for a particular mode of administration and indication, as determined in a clinical dose escalation study. In a GBM phase I clinical trial the MTD of BAL101553 administered as a daily oral formulation was found to be 30 mg per day (see Basilea Pharmaceutica Ltd press release dated 27 August 2019). In a phase 1/2a clinical trial the MTD of BAL101553 administered as a 48-hour infusion on days 1 , 8 and 15 of a 28 day cycle was found to be 70 mg/m ² (see Joerger M. Journal of Clinical Oncology 2018, 36:15_suppl, 2529-2529). In a further phase 1/2a clinical trial the MTD of BAL101553 administered as a 2-hour infusion days 1 , 8 and 15 of a 28 day cycle was found to be 30 mg/m ² (see Lopez J. Journal of Clinical Oncology 2016 34:15_suppl.).

When administered orally the dosage of BAL101553 as the dihydrochloride salt per day on days when administered may be e.g. in the range of about 1 mg to about 35 mg (e.g. 1 mg to 35 mg), e.g. in the range of about 1 mg to about 30 mg (e.g. 1 mg to 30 mg), e.g. in the range of about 2 mg to about 30 mg (e.g. 2 mg to 30 mg), e.g. in the range of about 4 mg to about 30 mg (e.g. 4 mg to 30 mg), e.g. in the range of about 8 mg to about 30 mg (e.g. 8 mg to 30 mg), or in any single amount within these ranges (e.g. 4mg, 8 mg, 12 mg, 16 mg, 20mg, 25mg, 28mg, 30mg or 35mg, in particular 30mg). For example the dosage per day on days when administered may be at least about 1 mg, e.g. at least about 2 mg, e.g. at least about 4 mg, e.g. at least about 8 mg, e.g. up to about 50 mg, e.g. up to about 30 mg, e.g. up to about 25 mg, e.g. up to about 20 mg. When the compound of formula I or pharmaceutically acceptable salt thereof is administered, or a different prodrug or pharmaceutically acceptable salt thereof, then the corresponding dosages amounts to give the same number of moles are administered based on the respective molecular weights.

When administered orally the compound of formula I or a prodrug thereof (e.g. BAL101553) may be administered according to a continuous treatment schedule or a cyclic treatment schedule. Administration may be more than once per day (e.g. twice per day) if needed or desired and the dosage per administration is reduced accordingly so that the dosage on a given day remains within the specified limits. In one embodiment administration is according to a continuous treatment schedule with one dose per day for as long as needed. In one embodiment administration is according to a continuous treatment schedule with two doses per day for as long as needed.

For example, when administered intravenously the dose of BAL101553 as the dihydrochloride salt per week during weeks when administered may be e.g. in the range of about 1 mg/m ² to about 160 mg/m ² (e.g. 1 mg/m ² to 160 mg/m ² ), e.g. in the range of about 15 mg/m ² to about 100 mg/m ² (e.g. 15 mg/m ² to 100 mg/m ² ), e.g. in the range of about 30 mg/m ² to about 100 mg/m ² (e.g. 30 mg/m ² to 100 mg/m ² ), e.g. in the range of about 30 mg/m ² to about 70 mg/m ² (e.g. 30 mg/m ² to 70 mg/m ² ), or in any single amount within these ranges (e.g.30 mg/m ² , 45 mg/m ² , 70 mg/m ² or 90 mg/m ² , in particular 70 mg/m ² ). For example the dose per week during weeks when administered may be e.g. at least 1 mg/m ² , e.g. at least about 10 mg/m ² , e.g. at least about 15 mg/m ² , e.g. at least about 30 mg/m ² , e.g. up to about 160 mg/m ² , e.g. up to about 100 mg/m ² , e.g. up to about 70 mg/m ² Likewise as above, when the compound of formula I or pharmaceutically acceptable salt thereof is administered, or a different prodrug or pharmaceutically acceptable salt thereof, then the corresponding dosages amounts to give the same number of moles are administered based on the respective molecular weights.

When administered intravenously the dose of the compound of formula I or a prodrug thereof (e.g. BAL101553) may be administered once per week or more than once per week, e.g. twice or three times per week. The intravenous dose may be over a period as long as needed, e.g. over a period of about 1 to about 96 hours, e.g. about 40 to about 80 hours, e.g. about 72 hours, e.g. about 40 hours to about 60 hours. In one embodiment the dose may be over a period of about 48 hours. In another embodiment the dose may be over a period of about 60 hours. In another embodiment the dose may be over a period of about 72 hours. Such intravenous administration may utilize a continuous infusion pump or other intravenous administration device. WO 2018/210868 describes intravenous infusion of the compound of formula I in more detail. In another embodiment the duration of administration may be e.g. a period of about 1 to about 4 hours, e.g. about 2 hours. In one embodiment the compound of formula I or a prodrug thereof (e.g. BAL101553) is administered according to a 21 -day treatment cycle with two days of dosing, e.g. initiated on days 1 and 8. In another embodiment the compound of formula I or a prodrug thereof (e.g. BAL101553) is administered according to a 28-day treatment cycle with three days of dosing, e.g. initiated on days 1 , 8 and 15. In another embodiment the compound of formula I or a prodrug thereof (e.g. BAL101553) is administered according to a 28-day treatment cycle with two days of dosing, e.g. initiated on days 1 and 15.

The pharmaceutical compositions of the invention comprise the active ingredient and a pharmaceutically acceptable carrier. An example of an oral composition includes, but is not limited to, hard capsules (e.g. HPMC capsules) containing 1 mg active ingredient, 98mg of mannitol and 1 mg magnesium stearate, or 5mg active ingredient, 94mg mannitol and 1 mg magnesium stearate. For intravenous administration of the compound of formula I or a prodrug thereof (e.g. BAL101553, in particular in the form of its dihydrochloride salt) may be provided in powder (e.g. lyophilized) form e.g. with 0.1 N hydrochloric acid as pH agent, and reconstituted with a suitable diluent, e.g. saline solution or Ringer lactate solution, immediately prior to administration. The active ingredient may be initially reconstituted with saline solution or Ringer lactate solution and then diluted to the required concentration with Ringer lactate solution. The pharmaceutical composition may contain, from about 0.1 percent to about 99.9 percent, preferably from about 1 percent to about 60 percent, of the therapeutic agent(s).

A compound of formula I or a prodrug thereof (e.g. BAL101553) can be administered alone or in combination with one or more other therapeutic agents. Possible combination therapy may take the form of fixed combinations, or the administration of a compound of the invention and one or more other therapeutic agents which are staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic agents.

A compound of formula I or a prodrug thereof (e.g. BAL101553) can, besides or in addition, be administered especially for tumor therapy in combination with chemotherapy (cytotoxic therapy), targeted therapy, endocrine therapy, biologies, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Longterm therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumor regression, or even chemo-preventive therapy, for example in patients at risk. Combination of BAL27862/BAL101553 with eribulin and gemcitabine is described in WO 2019/097073 and WO 2020/058405 respectively.

Kit and device

In one aspect the invention relates to a kit, and in another aspect to a device, for predicting the response of a brain neoplasm in a subject, to a compound of formula I or a prodrug thereof (e.g. BAL101553) as defined above, comprising reagents necessary for measuring the level of c-Myc or the level of c-Myc and the level of EB1 in a sample. Preferably, the reagents comprise a capture reagent comprising a detector for c-Myc and/or EB1 and a detector reagent.

The kit and device may also preferably comprise a comparator module which comprises a standard value or set of standard values to which the level of c-Myc or the level of c-Myc and the level of EB1 in the sample is compared. In a preferred embodiment, the comparator module is included in instructions for use of the kit. In another preferred embodiment the comparator module is in the form of a display device, for example a strip of color or numerically coded material which is designed to be placed next to the readout of the sample measurement to indicate resistance levels. The standard value or set of standard values may be determined as described above.

In one preferred embodiment the reagents are those that are capable of measuring the level of c-Myc and/or EB1 at the protein level. In this case the reagents are preferably antibodies or antibody fragments which selectively bind to c-Myc or EB1 respectively. Suitable samples are tissue, tumor tissue, CTC, CSC or extracellular vesicle samples, sections of fixed and paraffin-embedded or frozen tissue, tumor tissue or CSC specimens, and blood, cerebrospinal fluid and other body liquid-derived samples (including CTCs, CSCs and extracellular vesicles). Preferably, the reagents are in the form of one specific primary antibody which binds to c-Myc or EB1 respectively and a secondary antibody which binds to the primary antibody, and which is itself labelled for detection. The primary antibody may also be labelled for direct detection. The kits or devices may optionally also contain a wash solution(s) that selectively allows retention of the bound biomarker to the capture reagent as compared with other biomarkers after washing. Such kits can then be used in ELISA, western blotting, flow cytometry (FACS), immunofluorescence microscopy, immunohistochemical or other immunochemical methods to detect the level of the biomarker. Non-antibody based specific probes may also be used to detect the level of c-Myc or the level of c-Myc and the level of EB1 , e.g. drug affinity responsive target stability (DARTS) or aptamers.

The reagents may also in another preferred embodiment be those that are capable of measuring the level of c-Myc and/or EB1 nucleic acids in a sample. Suitable samples are tissue, tumor tissue, circulating DNA and RNA, CTC, CSC or extracellular vesicle samples, sections of fixed and paraffin-embedded or frozen tissue, tumor tissue or CSC specimens, and blood, cerebrospinal fluid and other body liquid-derived samples (including CTCs, CSCs and extracellular vesicles). Preferably, the reagents comprise a labelled probe or primers for hybridization to c-Myc or EB1 nucleic acid respectively in the sample. Suitable detection systems, either based on PCR amplification techniques or detection of labelled probes, allow quantification of c-Myc or EB1 nucleic acid in the sample respectively. This can be done i) in-situ on the specimen itself, preferably in sections from paraffin-embedded or frozen specimens, ii) in extracts from tumor, tissue or blood-and cerebrospinal fluid-derived specimens (including CTC, CSCs and extracellular vesicles), where suitable reagents selectively enrich for nucleic acids. The kits or devices enable the measurement and quantification of i) the amount of hybridized labelled probes to the specimens in-situ or ii) the amount of primer-based amplification products by methods based on specific physico-chemical properties of the probes itself or the reporters attached to the primers. Similarly, the kit and device may contain reagents which selectively bind to CTCs and CSCs. Such reagents may target CTC- and CSC-specific markers. Furthermore the device may comprise imaging devices or measurement devices (for example, but not restricted to, measurement of fluorescence) which further process the measured signals and transfer them into a scale in a comparator module.

The following numbered paragraphs further explain the invention:

A1 . Use of c-Myc as a biomarker for predicting the response of a brain neoplasm to a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A2. Use according to paragraph A1 , wherein the level of c-Myc is measured ex vivo in a sample or samples taken from a subject.

A3. Use according to paragraph A1 or paragraph A2, wherein a higher level of c-Myc relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A4. Use according to paragraph A3, wherein the level of c-Myc is the level of c-Myc RNA or DNA.

A5. Use according to paragraph A4, wherein the level of c-Myc is the level of c-Myc DNA amplification. A6. Use according to paragraph A5, wherein the level of c-Myc DNA amplification predicts sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 3 or more per tumor cell.

A7. Use according to paragraph A5, wherein the level of c-Myc DNA amplification predicts sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 6 or more per tumor cell.

A8. Use according to paragraph A3, wherein the level of c-Myc is the level of c-Myc protein.

A9. Use according to any one of paragraphs A2 to A8, wherein a higher level of c-Myc in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A10. Use according to any one of paragraphs A1 to A9, wherein c-Myc and EB1 are used as a biomarker for predicting the response of a brain neoplasm to a compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A11 . Use according to paragraph A10, wherein the level of c-Myc and EB1 is measured ex vivo in a sample or samples taken from a subject and wherein a higher level of c-Myc and a higher level of EB1 relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A12. Use according to paragraph A11 , wherein the level of EB1 is the level of EB1 RNA or DNA.

A13. Use according to paragraph A11 , wherein the level of EB1 is the level of EB1 protein. A14. Use according to paragraph A13, wherein the level of EB1 protein predicts sensitivity when at least 50% of the tumor cells in the sample or samples show moderate to strong staining by immunihistochemistry.

A15. Use according to paragraph A14, wherein the level of EB1 protein predicts sensitivity when at least 70% of the tumor cells in the sample or samples show moderate to strong staining by immunihistochemistry.

A16. Use according to any one of paragraphs A11 to A15, wherein a higher level of c-Myc and EB1 in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

A17. Use according to claim any one of paragraphs A2 to A16, wherein the subject is a human.

A18. Use according to any one of paragraphs A2 to A17, wherein the sample is derived from tumor tissue, cell lines, blood, cancer stem cells, circulating tumor cells or extracellular vesicles.

A19. Use according to paragraph A18, wherein the sample is derived from tumor tissue.

A20. Use according to any one of paragraphs A1 to A19, wherein the brain neoplasm is selected from glial- and non-glial-tumors, astrocytomas, oligodendrogliomas, ependydomas, menigiomas, haemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNETs), medullablastomas, haemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically driven brain neoplasms, including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis.

A21 . Use according to any one of paragraphs A1 to A19, wherein the brain neoplasm is glioblastoma multiforme. A22. Use according to any one of paragraphs A1 to A21 , wherein the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine.

A23. Use according to any one of paragraphs A1 to A22, wherein the compound is BAL101553 or pharmaceutically acceptable salt thereof.

A24. Use according to paragraph A23, wherein the compound is a hydrochloride salt of BAL101553.

A25. Use according to A23, wherein the compound is a dihydrochloride salt of BAL101553.

A26. Use of c-Myc as a biomarker for predicting the response of a glioblastoma multiforme to a compound of formula I (see A1 for structure) or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof; wherein the level of c-Myc DNA amplification is measured ex vivo in a sample or samples taken from a subject; wherein a higher level of c-Myc DNA amplification relative to a standard value or set of standard values predicts sensitivity of the glioblastoma multiforme to the compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof.

A27. Use of c-Myc as a biomarker for predicting the response of glioblastoma multiforme to a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof; wherein the level of c-Myc DNA amplification is measured ex vivo in a sample or samples taken from a subject; and wherein the level of c-Myc DNA amplification predicts sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 3 or more per tumor cell.

A28. Use of c-Myc and EB1 as a biomarker for predicting the response of glioblastoma multiforme to a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof; wherein the level of c-Myc DNA amplification and the level of EB1 protein is measured ex vivo in a sample or samples taken from a subject; and wherein a higher level of c-Myc DNA amplification and a higher level of EB1 protein relative to a standard value or set of standard values predicts sensitivity of the glioblastoma multiforme to the compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof.

A29. Use of c-Myc and EB1 as a biomarker for predicting the response of glioblastoma multiforme to a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof; wherein the level of c-Myc DNA amplification and the level of EB1 protein is measured ex vivo in a sample or samples taken from a subject; and wherein the level of c-Myc DNA amplification and level of EB1 protein predict sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 3 or more per tumor cell and at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

B1 . A compound of formula I (see A1 for structure) or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use in the treatment of a brain neoplasm in a subject, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

B2. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B1 , wherein the treatment comprises measuring ex vivo the level of c-Myc in the sample or samples taken from the subject.

B3. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B1 or paragraph B2, wherein a higher level of c-Myc relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof. B4. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B3, wherein the level of c-Myc is the level of c-Myc RNA or DNA.

B5. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B3, wherein the level of c-Myc is the level of c-Myc DNA amplification.

B6. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B5, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 3 or more per tumor cell.

B7. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B5, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 6 or more per tumor cell.

B8. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B3, wherein the level of c-Myc is the level of c-Myc protein.

B9. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B8, wherein a higher level of c-Myc in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof. B10. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B9, wherein the subject has a level of c-Myc and a level of EB1 measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

B11 . A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B10, wherein the treatment comprises measuring ex vivo the level of c-Myc and the level of EB1 in the sample or samples taken from the subject to obtain a value or values representing these levels.

B12. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B11 , wherein a higher level of c-Myc and a higher level of EB1 relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

B13. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B10 to B12, wherein the level of EB1 is the level of EB1 RNA or DNA.

B14. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B10 to B12, wherein the level of EB1 is the level of EB1 protein.

B15. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B14, wherein the subject has a level of EB1 protein in which at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry. B16. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B14, wherein the subject has a level of EB1 protein in which at least 70% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

B17. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B10 to B17, wherein a higher level of c-Myc and EB1 in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

B18. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to claim any one of paragraphs B1 to B17, wherein the subject is a human.

B19. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B18, wherein the sample is derived from tumor tissue, cell lines, blood, cancer stem cells, circulating tumor cells or extracellular vesicles.

B20. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B19, wherein the sample is derived from tumor tissue.

B21 . A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B20, wherein the brain neoplasm is selected from glialand non-glial-tumors, astrocytomas, oligodendrogliomas, ependydomas, menigiomas, haemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNETs), medullablastomas, haemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically driven brain neoplasms, including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis.

B22. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B20, wherein the brain neoplasm is glioblastoma multiforme.

B23. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B22, wherein the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine.

B24. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to any one of paragraphs B1 to B23, wherein the compound is BAL101553 or pharmaceutically acceptable salt thereof.

B25. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B24, wherein the compound is a hydrochloride salt of BAL101553.

B26. A compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof for use according to paragraph B20 or B25, wherein the compound is a dihydrochloride salt of BAL101553.

B27. A compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof for use in the treatment of glioblastoma multiforme in a subject, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

B28. A compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof for use in the treatment of glioblastoma multiforme in a subject, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 3 or more per tumor cell.

B29. A compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof for use in the treatment of glioblastoma multiforme in a subject, wherein the subject has a level of c-Myc DNA amplification and a level of EB1 protein measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

B30. A compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof for use in the treatment of glioblastoma multiforme in a subject, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 3 or more per tumor cell and a level of EB1 protein in which at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

Paragraphs B1 to B30 may be additionally written in the format use of a compound of formula or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof in the manufacture of a medicament for the treatment of a brain neoplasm in a subject, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

C1. A kit for predicting the response of a brain neoplasm, preferably glioblastoma multiforme, to a compound of formula I (see A1 for structure) or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof comprising reagents necessary for measuring the level of c- Myc in a sample.

C2. A kit according to paragraph C1 , wherein the kit comprises a comparator module which comprises a standard value or set of standard values to which the level of c-Myc in the sample is compared. C3. A kit according to paragraph C1 or paragraph C2, wherein the reagents comprise a capture reagent comprising a detector for c-Myc.

C4. A kit according to paragraph C3, wherein the capture reagent is an antibody.

C5. A kit according to paragraph C3, wherein the capture reagent is a nucleic acid probe or primer.

C6. A kit according to any one of paragraphs C1 to C6, wherein the kit comprising reagents necessary for measuring the level of c-Myc and the level of EB1 in a sample.

C7. A kit according to paragraph C6, wherein the kit comprises a comparator module which comprises a standard value or set of standard values to which the level of c-Myc and the level of EB1 in the sample is compared.

C8. A kit according to paragraph C6 or paragraph C7, wherein the reagents comprise a capture reagent comprising a detector for EB1 .

C9. A kit according to paragraph C8, wherein the capture reagent comprising a detector for EB1 is an antibody.

C10. A kit according to paragraph C8, wherein the capture reagent comprising a detector for EB1 is a nucleic acid probe or primer.

C11 . A kit according to any one of paragraphs C1 to C10, wherein the kit comprises the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

C12. A kit according to any one of paragraphs C1 to C11 , wherein the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine.

C13. A kit according to any one of paragraphs C1 to C12, wherein the compound is BAL101553 or pharmaceutically acceptable salt thereof.

C14. A kit according to paragraph C13, wherein the compound is a hydrochloride salt of BAL101553.

C15. A kit according to paragraph C13 or paragraph C14, wherein the compound is a dihydrochloride salt of BAL101553.

D1 . A method of treating a brain neoplasm in a subject in need thereof, comprising treating the subject with a pharmaceutically effective amount of a compound of formula I (see A1 for structure) or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

D2. A method according to paragraph D1 , wherein the treatment comprises measuring ex vivo the level of c-Myc in the sample or samples from the subject.

D3. A method according to paragraph D1 or paragraph D2, wherein a higher level of c-Myc relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

D4. A method according to any one of paragraphs D1 to D3, wherein the level of c-Myc is the level of c-Myc RNA or DNA.

D5. A method according to paragraph D4, wherein the level of c-Myc is the level of c-Myc DNA amplification.

D6. A method according to paragraph D5, wherein the subject has a level of c- Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is a mean average gene copy number of at least 3 or more per tumor cell.

D7. A method according to paragraph D5, wherein the subject has a level of c- Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is a mean average gene copy number of at least 6 or more per tumor cell. D8. A method according to any one of paragraphs D1 to D3, wherein the level of c-Myc is the level of c-Myc protein.

D9. A method according to any one of paragraphs D1 to D8, wherein a higher level of c-Myc in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

D10. A method according to any one of paragraphs D1 to D9, wherein the subject has a level of c-Myc and a level of EB1 measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

D11 . A method according to paragraph D10, wherein the method comprises measuring the level of c-Myc and the level of EB1 in a sample or samples from a subject to obtain a value or values representing these levels.

D12. A method according to paragraph D10 or paragraph D11 , wherein a higher level of c-Myc and a higher level of EB1 relative to a standard value or set of standard values predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

D13. A method according to any one of paragraphs D10 to D12, wherein the level of EB1 is the level of EB1 RNA or DNA.

D14. A method according to any one of paragraphs D10 to D12, wherein the level of EB1 is the level of EB1 protein.

D15. A method according to paragraph D14, wherein the subject has a level of EB1 protein in which at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry. D16. A method according to paragraph D14, wherein the subject has a level of EB1 protein in which at least 70% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

D17. A method according to any one of paragraphs D10 to D16, wherein a higher level of c-Myc and EB1 in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts sensitivity of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

D18. A method according to any one of paragraphs D1 to D17, wherein the subject is a human.

D19. A method according to any one of paragraphs D1 to D18, wherein the sample is derived from tumor tissue, cell lines, blood, cancer stem cells, circulating tumor cells or extracellular vesicles.

D20. A method according to paragraph D19, wherein the sample is derived from tumor tissue.

D21 . A method according to any one of paragraphs D1 to D20, wherein the brain neoplasm is selected from glial- and non-glial-tumors, astrocytomas, oligodendrogliomas, ependydomas, menigiomas, hemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNETs), medullablastomas, hemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically driven brain neoplasms, including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis.

D22. A method according to any one of paragraphs D1 to D20, wherein the brain neoplasm is glioblastoma multiforme.

D23. A method according to any one of paragraphs D1 to D22, wherein the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine. D24. A method according to any one of paragraphs D1 to D23, wherein the compound is BAL101553 or pharmaceutically acceptable salt thereof.

D25. A method according to paragraph D24, wherein the compound is a hydrochloride salt of BAL101553.

D26. A method according to paragraph D24 or paragraph D25, wherein the compound is a dihydrochloride salt of BAL101553.

D27. A method of treating glioblastoma multiforme in a subject in need thereof, comprising treating the subject with a pharmaceutically effective amount of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

D28. A method of treating glioblastoma multiforme in a subject in need thereof, comprising treating the subject with a pharmaceutically effective amount of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 3 or more per tumor cell.

D29. A method of treating glioblastoma multiforme in a subject in need thereof, comprising treating the subject with a pharmaceutically effective amount of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc DNA amplification and a level of EB1 protein measured ex vivo in a sample or samples taken from the subject which is higher than a standard value or set of standard values.

D30. A method of treating glioblastoma multiforme in a subject in need thereof, comprising treating the subject with a pharmaceutically effective amount of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt of a prodrug thereof, wherein the subject has a level of c-Myc DNA amplification measured ex vivo in a sample or samples taken from the subject which is at least a mean average gene copy number of 3 or more per tumor cell and a level of EB1 protein in which at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

E1. A method for predicting the response to treatment of a brain neoplasm, preferably glioblastoma multiforme, in a subject by administration of a compound of formula I (see A1 for structure) or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof said method comprising the steps of: a) determining the level of c-Myc in a sample or samples obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values of the levels from step a) with a standard value or a set of standard values which comparison is predictive of responsiveness to the compound of formula I or prodrug thereof.

E2. A method according to paragraph E1 , wherein step a) comprises measuring the level of c-Myc a sample or samples to obtain a value or values representing this level.

E3. A method according to paragraph E1 or paragraph E2, wherein a higher level of c-Myc relative to a standard value or set of standard values predicts responsiveness of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

E4. A method according to any one of paragraphs E1 to E3, wherein the level of c-Myc is the level of c-Myc RNA or DNA.

E5. A method according to any one of paragraphs E1 to E3, wherein the level of c-Myc is the level of c-Myc DNA amplification.

E6. A method according to paragraph E5, wherein the level of c-Myc DNA amplification predicts responsiveness when the mean average gene copy number of c- Myc in the sample or samples is at least 3 or more per tumor cell. E7. A method according to paragraph E5, wherein the level of c-Myc DNA amplification predicts responsiveness when the mean average gene copy number of c- Myc in the sample or samples is at least 6 or more per tumor cell.

E8. A method according any one of paragraphs E1 to E3, wherein the level of c-Myc is the level of c-Myc protein.

E9. A method according to any one of paragraphs E1 to E8, wherein a higher level of c-Myc in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts responsiveness of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

E10. A method according to any one of paragraphs E1 to E9, wherein the method comprises the steps of: a) determining the level of c-Myc and EB1 in a sample or samples obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values of the levels of c-Myc and EB1 from step a) with a standard value or a set of standard values which comparison is predictive of responsiveness to the compound of formula I or prodrug thereof.

E11 . A method according to paragraph E10, wherein step a) comprises measuring the level of c-Myc and the level of EB1 in a sample or samples from a subject to obtain a value or values representing these levels.

E12. A method according to paragraph E10 or paragraph E11 , wherein a higher level of c-Myc and a higher level of EB1 relative to a standard value or set of standard values predicts responsiveness of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

E13. A method according to any one of paragraphs E10 to E12, wherein the level of EB1 is the level of EB1 RNA or DNA. E14. A method according to any one of paragraphs E10 to E12, wherein the level of EB1 is the level of EB1 protein.

E15. A method according to paragraph E14, wherein the level of EB1 protein predicts sensitivity when at least 50% of the tumor cells in the sample or samples show moderate to strong staining by immunihistochemistry.

E16. A method according to paragraph E14, wherein the level of EB1 protein predicts sensitivity when at least 70% of the tumor cells in the sample or samples show moderate to strong staining by immunihistochemistry.

E17. A method according to any one of paragraphs E10 to E16, wherein a higher level of c-Myc and EB1 in the sample or samples relative to a standard value or set of standard values from subjects with the same tumor histotype predicts responsiveness of the brain neoplasm to the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug or pharmaceutically acceptable salt of a prodrug thereof.

E18. A method according to any one of paragraphs E1 to E17, wherein the subject is a human.

E19. A method according to any one of paragraphs E1 to E18, wherein the sample is derived from tumor tissue, cell lines, blood, cancer stem cells, circulating tumor cells or extracellular vesicles.

E20. A method according to paragraph E19, wherein the sample is derived from tumor tissue.

E21 . A method according to any one of paragraphs E1 to E20, wherein the brain neoplasm is selected from glial- and non-glial-tumors, astrocytomas, oligodendrogliomas, ependydomas, menigiomas, hemangioblastomas, acoustic neuromas, craniopharyngiomas, primary central nervous system lymphoma, germ cell tumors, pituitary tumors, pineal region tumors, primitive neuroectodermal tumors (PNETs), medullablastomas, hemangiopericytomas, spinal cord tumors including meningiomas, chordomas and genetically driven brain neoplasms, including neurofibromatosis, peripheral nerve sheath tumors and tuberous sclerosis.

E22. A method according to any one of paragraphs E1 to E20, wherein the brain neoplasm is glioblastoma multiforme.

E23. A method according to any one of paragraphs E1 to E22, wherein the prodrug is an amide formed from the amino group of the compound of formula I and the carboxy group of glycine, alanine or lysine.

E24. A method according to any one of paragraphs E1 to E23, wherein the compound is BAL101553 or pharmaceutically acceptable salt thereof.

E25. A method according to paragraph E24, wherein the compound is a hydrochloride salt of BAL101553.

E26. A method according to paragraph E24 or paragraph E25, wherein the compound is a dihydrochloride salt of BAL101553.

E27. A method for predicting the response to treatment of glioblastoma multiforme in a subject by administration of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof said method comprising the steps of: a) determining the level of c-Myc DNA amplification in a sample or samples obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values of the levels from step a) with a standard value or a set of standard values which comparison is predictive of responsiveness to the compound of formula I or prodrug thereof.

E28. A method for predicting the response to treatment of glioblastoma multiforme in a subject by administration of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof said method comprising the step of determining the level of c-Myc DNA amplification in a sample or samples obtained from the subject; wherein the level of c-Myc DNA amplification predicts sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 3 or more per tumor cell.

E29. A method for predicting the response to treatment of glioblastoma multiforme in a subject by administration of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof said method comprising the steps of: a) determining the level of c-Myc DNA amplification and the level of EB1 protein in a sample or samples obtained from the subject to obtain a value or values representing this level; and b) comparing the value or values of the levels from step a) with a standard value or a set of standard values which comparison is predictive of responsiveness to the compound of formula I or prodrug thereof.

E30. A method for predicting the response to treatment of glioblastoma multiforme in a subject by administration of a compound of formula I or pharmaceutically acceptable salt thereof or BAL101553 or pharmaceutically acceptable salt thereof said method comprising the step of determining the level of c-Myc DNA amplification and the level of EB1 protein in a sample or samples obtained from the subject; wherein the level of c-Myc DNA amplification predicts sensitivity when the mean average gene copy number of c-Myc in the sample or samples is at least 3 or more per tumor cell and at least 50% of the tumor cells in the sample or samples show moderate to strong staining of EB1 by immunohistochemistry.

E31 . A method according to any one of paragraphs E1 to E30, wherein the method includes administering the compound of formula I or pharmaceutically acceptable salt thereof or a prodrug (e.g. BA101553) or pharmaceutically acceptable salt of a prodrug thereof to the subject determined to be responsive.

All aspects and embodiments of the invention described herein may be combined in any combination where possible.

The words “comprise” or “comprises” or “comprising” are to be understood as to imply the inclusion of a stated item or group of items, but not the exclusion of any other item or group of items. The term “about” means a variation of no more than 10% of the relevant figure, preferably no more than 5%.

For the avoidance of doubt, where ranges are mentioned (e.g. “in the range of...”) the end points of the range are also included in the range.

A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Particular embodiments of the invention are described in the following Examples, which serve to illustrate the invention in more detail and should not be construed as limiting the invention in any way.

Experimental methodology

Tumor tissue micro-array staining

Patient GBM samples were obtained from surgical resections and processed by formalin fixation and paraffin embedding according to standard clinical practice. Tumor tissue micro-arrays (TMAs) were prepared from formalin fixed/paraffin-em bedded blocks using the approach described in Zlobec I. A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies, J. Vis. Exp. (91 ), e51893, doi: 10.3791/51893, 2014. In this approach histological slides for each patient are scanned and uploaded onto a web-based digital platform. There they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. Staining is assessed visually to determine whether positive for EB1 , with a positive result being moderate to strong EB1 staining in at least 70% of tumor cells. Whole genome sequencing

For whole genome sequencing, library preparation was performed using 50-200 ng of genomic DNA input. Libraries were constructed using Kapa HyperPlus kit (Roche). DNA was fragmented, end-repaired, A-tailed and adapter-ligated. Dual size selection was used to retain 300-400bp fragments, and this was followed by DNA amplification. Such prepared libraries were then whole genome sequenced on Illumina NovaSeq, tumors to 150x and germline samples to 50x.

The sequencing data (BCL files) were demultiplexed using bcl2fastq2 v2.20.0 to generate FASTQs. The FASTQ data was aligned to BAM using bwa vO.7.15, quality metrics were generated with Picard tools 2.8.1 , CNV analysis was performed using CNVkit vO.9.4, structural variant analysis with Manta 1 .2.2, variant calling with GATK v.4.0.5.1 to produce VCF files and finally the variant calling data was annotated using Oncotator v.1 .9.9.0 with a downloaded database from April 5, 2016.

FISH methodology

FISH for the investigation of C-MYC gene status was performed with the ZytoLight MYC Dual Color Break Apart Probe (ZytoVision Bremerhaven, Germany) on 2pm sections mounted on positively charged slides. The method was performed according to the manufacturer’s protocol. Properly fixated vital areas containing at least 50 tumor cells were evaluated by interphase FISH looking: A) for separated (broken- apart) green and red signals that are located at least twice the distance of the fused signals, and that would correspond to C-MYC gene rearrangements due to translocations, and B) for the total count of fused C-MYC gene signals/cell and presence of tight clusters of >6 fused C-MYC gene signals that would correspond to gene amplifications.

Detailed examples

In a clinical study with BAL101553, one patient with progressive GBM showed a profound and long-lasting clinical and radiological response. The GBM tissue of this patient was EB1 -positive and a whole-genome-analysis from the GBM tissue of this patient showed an amplification of the C-MYC gene (i.e. six or more copies). In this study, out of 28 patients dosed with BAL101553, this patient was the only patient showing an objective response. GBM tissue samples from three of the non-responding patients were subjected to whole-genome-analysis, none of which showed an amplification of the C-MYC gene (no more than two copies). In addition, GBM tissue samples from eight of the non-responding patients were subject to EB1 -staining and all showed weak or completely negative EB1 -staining.

Subsequently, available archival GBM tissue samples obtained from the University of Basel with known C-MYC amplification (one sample) or the absence thereof (i.e. C-MYC wildtype, 6 samples) were stained for EB1 . This investigation revealed that the GBM sample with the C-MYC amplification showed EB1 -positive staining while all 6 samples that had no C-MYC amplification (i.e. were C-MYC wildtype) were EB1 -negative which is summarized in Table 1 below.

Table 1

Further investigations using TMAs suggest that EB1 -positivity occurs in 2-5% of GBM. Considering that C-MYC amplifications in GBM are also rare (i.e. <3%, Kalkat M. MYC deregulation in primary human cancers. Genes 2017, 8, 151 , the probability of a combined finding of EB1 -positivity and C-MYC amplification by chance is estimated to be below 0.15% (i.e.< 5% x 3%). Therefore, considering the rarity of either EB1- positivity or C-MYC amplifications in GBM, the finding of EB1 -positivity in a C-MYC amplified GBM sample shown in the Table above, together with the finding in the clinical study of a responding patient whose GBM tissue was EB1 -positive and whose whole- genome-sequencing analysis showed a C-MYC amplification strongly indicates an association between c-Myc and EB1 .

Such association was further assessed in a series of archival tissue samples obtained from the University of Bern. In this assessment, a C-MYC amplification was defined by the presence of at least 6 gene copies per tumor cell. As shown in the Table below, in the archival tissue samples from 73 GBM patients there were three samples with C-MYC amplification with one also being EB1 -positive, i.e. 33% (1 out of 3) of C- MYC amplified samples were EB1 -positive compared to 17% (12 out of 70) of C-MYC non-amplified samples. See Table 2 below. Table 2

An association between EB1 -positivity and C-MYC is further more supported through an assessment by the number of C-MYC gene copies. While in the 73 GBM tissue samples only three samples showed a C-MYC amplification (defined as at least 6 gene copies per cell, with 2 gene copies being “normal”), 4 or more gene copies were present in 9 GBM tissue samples. The distribution of these samples show that samples with tumor cells that show an increase in gene copies are more frequently EB1 -positive (i.e. 3 out of 9 = 33%) than tumor cells with a normal number of gene copies or with less than 4 gene copies (i.e. 2N) of C-MYC gene copies (i.e. 10 out of 64 = 16%). See Table 3 below.

Table 3