GB2541405A

GB2541405A - Novel Methods

Info

Publication number: GB2541405A
Application number: GB1514605.3A
Authority: GB
Inventors: Suliman Al-Mogbel Mohammed; Alshaghdali Abdulrahman; Ashankyty Ibraheem; Elmouna Ahmed; Fazuldeen Mohammed; Mian Shahid; Nuglozeh Edem; Fairbairn Camilla; Malcolm Timothy; Moti Naushad; Turner Suzanne
Original assignee: University of Sheffield; University of Cambridge; Of Ha'il, University of
Current assignee: University of Sheffield; University of Cambridge; Of Ha'il, University of
Priority date: 2015-08-17
Filing date: 2015-08-17
Publication date: 2017-02-22
Also published as: GB201514605D0

Abstract

Claimed is an in vitro method for identifying a subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or at risk of developing the same, comprising testing to determine the function and/or expression and/or nucleotide sequence variation of the NUDT18 gene (Nucleoside diphosphate linked motif X motif 18, MutT human homolog 3, Nudix motif 18, MTH3). Also claimed is an associated method for identifying a lead candidate with efficacy in the treatment or prevention of an ALK-positive tumour. Alternatively claimed in the aforementioned methods are any one or more of the genes in Table 1, or identifying a subject with chromosomal instability by measuring any one or more of the genes in Table 1. Alternatively claimed are methods for prognosis of a subject with an ALK-positive tumour and methods for identifying a subject at risk of recurrence of an ALK-positive tumour. Preferably, the ALK-positive tumour is anaplastic large cell lymphoma (ALCL) and the testing involves detecting insertion/deletions and single nucleotide polymorphisms (SNPs). Preferably, testing DNA from the subject may comprise determining the presence of a variant sequence in any one or more of DIXDC1, EI24/PIG8, LTBP4 and ZNF516.

Description

NOVEL METHODS

Field of Invention

The present invention relates to methods for the diagnosis, prognosis and treatment of anaplastic lymphoma kinase (ALK)-positive tumours, such as anaplastic large cell lymphoma (ALCL).

Background

Tyrosine kinases are involved in the pathogenesis of most cancers. However, few tyrosine kinases have been shown to have a well-defined pathogenetic role in lymphomas. The anaplastic lymphoma kinase (ALK; also known as ALK tyrosine kinase receptor or CD246) is a receptor with tyrosine kinase activity, which regulates the development and maintenance of the nervous system. Mutations or amplification in ALK promote tumorogenesis and progression of diverse types of cancer, which makes it an attractive therapeutic target against cancer diseases. For example, ALK is the oncogene of most anaplastic large cell lymphomas, driving transformation through many molecular mechanisms.

Anaplastic large cell lymphoma (ALCL) is a form of non-Hodgkin lymphoma (NHL) predominantly diagnosed in children and young adults [1]. There are four established or provisional diagnostic entities of ALCL: cutaneous ALCL, systemic ALCL ALK+, systemic ALCL ALK-, and breast implant associated ALCL [2], ALK status represents aberrant, oncogenic expression of the Anaplastic Lymphoma Kinase (ALK), predominantly as a fusion protein consisting of the oligomerisation domain of Nucleophosmin 1 (NPM) fused to the kinase domain of ALK generating a hyperactive tyrosine kinase [3]. Many other ALK fusion partners also exist although these account for the minority of cases [4], Whilst ALCL has a relatively good prognosis (65-75% of patients achieving event free survival), many children suffer from the toxic effects of chemotherapy with as many as 30-40% undergoing disease relapse (for some children, on multiple occasions).

Currently there are no known biomarkers to predict which patients will undergo disease relapse and only one molecular marker to determine patient prognosis. In this latter regard, patients that have high levels of minimal residual disease (MRD) as determined by quantitative reverse transcriptase PCR for the ALK oncogene combined with low titres of circulating anti-ALK antibodies have an inferior prognosis [5-7]. In addition, patients with detectable levels of ALK fusion protein transcripts in blood or bone marrow (defined as minimal disseminated disease; MDD) as detected by qRT-PCR have a 50% risk of relapse6. Another parameter predicting risk is the histological subtype with those patients diagnosed with ALCL of a small cell or lymphohistiocytic variant having a higher risk of treatment failure [8],

At present, children are treated with a combination of chemotherapeutic agents including vinblastine and a regimen based on the Berlin-Frankfurt-Munster 90 (BFM90) protocol [9]. The largest ever clinical trial of paediatric ALCL patients was conducted over a 7 year period across Europe (ALCL99) comparing the BFM90 protocol to a protocol incorporating vinblastine and showed no difference in event free survival [9],

The mapping and characterisation of molecular changes within the genomes of ALCL patients could provide the foundation of identifying key genetic loci and their associated pathways that may be linked to phenotypes of clinical interest. Such alterations in DNA sequence may include for example single point mutations/polymorphisms (SNP), INDEL (insertion/deletion), inversions, copy number variations and chromosomal translocations as limited examples. It is essential therefore that characterisation studies exploring the biological changes occurring in tumour cells be systematically carried out in order to identify biological variation that may be integral to tumourigenesis, its progression towards more aggressive phenotypes and ultimately therapeutic response patterns.

There are several areas where novel genetic signatures (biomarkers) might be exploited in order to improve the clinical management of cancer patients: 1) Genetic variants indicative of aggressive disease phenotypes: Variant profiling could assist in the triaging of patients into groups that may or may not require aggressive therapeutic intervention and even the possibility that the disease is likely to exhibit recurrence. The ability to effectively prognosticate likely response patterns would provide the foundation for the development of companion diagnostic assays. Such tests may facilitate in the pre-selection of patients towards suitable treatments and appropriate forms of clinical management specifically predicated to a particular genetic profile. These patients would also be monitored as potentially high risk for disease recurrence. 2) Druqable targets: Identification of germline/somatic mutations that are linked to poor prognosis (e.g. pathways that may promote metastasis or increase the rate at which therapeutic resistance emerges) could provide a platform to identify targets linked with these detrimental phenotypes. They may also indicate patients at risk of MDD and thus would warrant from a clinical perspective that they remain under close observation. Characterising mutated genes and their associated biological pathways that are intrinsically linked with unfavourable outcomes would facilitate the development of strategies (and potentially novel pharmaceutical compounds) with the ability to control disease progression (e.g. circumventing drug resistance). 3) Molecular variation: Clinical differences at the intra- and inter-patient level is primarily caused by the genetic composition of an individual patient tumour. Complexity is increased further through genomic instability which leads to unregulated changes in the primary cancer genome promoting the creation of sub-clones with variable genomic (mutational) profiles. The consequence of such a dynamic genotype is to translate into a variable phenotype (e.g. metastatic potential; chemotherapeutic dosing levels). Common genetic changes that occur between different ALCL patients may identify important pathways necessary for tumour evolution in not only ALCL but also ALK +ve tumours (e.g. EML4-ALK translocations that have been identified in lung tumours). Concordant/discordant variants between ALCL cases may therefore provide information regarding genic loci that are important/less critical in the development and progression of ALK driven tumours respectively.

Genomic screening technologies have emerged that are capable of exploring variant signatures in patient tumour samples. These range from either a re-sequencing of specific gene combinations (e.g. panels of tumour suppressor genes) to whole genome sequencing. Exome sequencing represents another powerful category of these targeted re-sequencing methodologies and focuses upon the specific profiling of the exonic regions of genes. These include protein coding, 573’ UTR (un-translated regions) and even including micro-RNAs as part of epigenetic control mechanisms. Exome sequencing in conjunction with computational algorithms are now being used in discovery pipelines to identify variants of potential significance. In conjunction with gene annotation, filtering and in-silico prediction of variant effects on protein function, the methodology provides a powerful screening tool for the identification of candidates that may be of biological and/or clinical significance. Variants may for example occur in positions that result in the loss of a translational start/stop site, code for rare amino acids, introduce frame-shift mutations or impact in a detrimental manner splice donor/acceptor sites. Other variant impacts include synonymous amino acid changes which are believed to be low impact variant changes to those that can result in the production of in-frame deletions not affect catalytic domains or overall protein conformation but may modulate protein activity (moderate impact variants). Screening exercises play a pivotal role therefore in identifying variant changes and highlight the role of genes that are important molecular components in the development/progression and therapeutic response patterns of cancers.

The present invention seeks to provide new methods for the diagnosis and prognosis of anaplastic lymphoma kinase (ALK)-positive tumours, such as ALCL, based on genomic sequencing of the cells of such tumours.

Summary of Invention A first aspect of the invention provides an in vitro method for identifying a subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or at risk of developing the same, the method comprising the steps of: (a) providing a sample of cells (or protein and/or mRNA and/or DNA therefrom) from a subject to be tested; (b) testing the cells (or protein and/or mRNA and/or DNA therefrom) to determine therein the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 1 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 1 is indicative of the subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or being at risk of developing the same. TABLE 1

Genes modulated in ALK-positive tumours

In the above tables (and those below), genes are identified by reference to an abbreviation (‘Gene ID’); the full name of the gene can be obtained from recognised gene databases (such as GeneCards®).

By “anaplastic lymphoma kinase (ALK)-positive tumour” we include tumours in which the cells carry an ALK oncogene, resulting in aberrant expression of ALK. For example, elevated expression of ALK may arise as a consequence of gene amplification, i.e. with the tumour cells having multiple copies of the ALK gene (as occurs in some inflammatory breast cancers). Elevated expression of ALK may also arise through translocation and/or inversion events resulting in the creation of ALK gene fusions (such as NPM-ALK fusions in some anaplastic large cell lymphomas and EML4-ALK fusions in some lung adenocarcinomas). Aberrant expression of ALK may also occur as a consequence of mutations in the nucleotide sequence of the gene.

In one embodiment, the ALK-positive tumour is selected from the group consisting of anaplastic large cell lymphoma (ALCL), lung adenocarcinoma, breast cancer (for example, the inflammatory subtype thereof), neuroblastoma, inflammatory myofibroblastic tumours, renal cell carcinoma, oesophageal squamous cell carcinoma, colorectal adenocarcinoma, glioblastoma multiforme and anaplastic thyroid cancer.

For example, the ALK-positive tumour may be an anaplastic large cell lymphoma (ALCL).

By “modulation” of the genes identified in Table 1, we include that the function and/or expression of the gene(s) may be upregulated or inhibited relative to a reference value (e.g. in comparable cells from a healthy subject). Thus, by “reference value” we include comparable measures of the function and/or expression of said gene(s) in cells from a subject who does not have an ALK-positive tumour. Such measures of gene function and/or expression may include direct measurements of protein function and/or amount and/or mRNA amount, as well as indirect measures such as nucleotide sequence information for the mRNA and/or genomic DNA associated with said gene(s).

It will be appreciated by persons skilled in the art that the function and/or expression of the gene(s) may be determined at the level of protein function and/or mRNA expression and/or DNA sequence. For example, the function and/or expression of the gene(s) may be determined by analysis of the genomic DNA to identify sequence variants that impact negatively on the expression and/or function of the gene(s), e.g. the presence of insertions/deletions (INDELs) and/or single nucleotide polymorphisms (SNPs) that result in abrogation of gene expression.

It will be appreciated by persons skilled in the art that the subject to be tested may be any species of mammal; typically, however, the subject is human.

In the methods of the first aspect of the invention, the initial step comprises the provision of a sample of protein and/or mRNA and/or DNA from a subject to be tested; typically, the sample contains cells from the subject to be tested that are suspected of being tumour cells.

In one embodiment, the sample of cells in step (a) is from a tumour biopsy sample.

In an alternative embodiment, the sample in step (a) is from a blood sample.

In a further embodiment, the sample in step (a) is from urine or saliva.

Typically, the sample of cells from the subject to be tested is processed in some manner prior to undertaking step (b), with the nature of the processing being dependent upon the type of testing to be performed (e.g. genomic sequencing, proteome analysis, etc.).

In one embodiment, DNA is purified from the sample of cells for analysis in step (b). For example, genomic DNA may be prepared from the sample, as described in Youssif et al., 2009, Genes Chromosomes Cancer 48(100):1018-1026 (the disclosures of which are incorporated herein by reference). Sequencing of part or all of the genomic DNA {e.g. whole genome sequencing, exome sequencing, sequencing of a panel of selected genes, analysis of selected single nucleotide polymorphisms (SNPs), etc.) may then be conducted using methods known in the art, for example as described in Barnes, 2010, Methods Mol. Biol. 628:1-20 (the disclosures of which are incorporated herein by reference). For example, analysis of the DNA may comprise or consist of exome sequencing (e.g. using next-generation sequencers, such as those produced by lllumina and Life Technologies), PCR-based methods or hybridisation-based methods.

In an alternative embodiment, mRNA is purified from the sample of cells for analysis in step (b), e.g. as described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapters 2 to 4 therein) (the disclosures of which are incorporated herein by reference). For example, analysis of the mRNA may comprise or consist of sequencing (e.g. using next-generation sequencers, such as those produced by lllumina and Life Technologies), PCR-based methods or hybridisation-based methods.

In a further embodiment, proteins are purified from the sample of cells for analysis in step (b), e.g. as described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 5 therein) (the disclosures of which are incorporated herein by reference). For example, analysis of the protein(s) produced by the cells may comprise or consist of antibody-based methods to detect the presence of the protein products of the genes of Table 1.

One preferred embodiment of the methods of the invention comprises testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1, wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1 is indicative of the subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or being at risk of developing the same.

By "sequence variant” we include a difference in nucleotide sequence relative to a corresponding reference sequence (e.g. the corresponding sequence in normal, healthy cells). The nature of the sequence variation may be an insertion of one or more nucleotides at a given location within the gene, a deletion of one or more nucleotides at a given location within the gene, and/or a substitution of one or more nucleotides at a given location within the gene, wherein the sequence variation results in modulation of the function and/or expression of the gene. It will be appreciated by persons skilled in the art that such modulation of gene function/expression may arise from sequence variation in the coding region of the gene and/or in the promoter region of said gene.

In one embodiment, the variant sequence is detrimental to the function and/or expression of the gene. For example, the variant sequence may inhibit (in whole or in part) expression of the gene, such that level of its protein product within the tumour cells is reduced. Alternatively, the variant sequence may result in production of a modified protein product of the gene having reduced or no biological activity (for example, the protein may be a truncated version of the gene product with reduced or absent any biological activity).

In an alternative embodiment, the variant sequence may enhance the function and/or expression of the gene. For example, the variant sequence may be a deletion that removes a phosphorylation site from the gene product or an amino acid substitution that confers enhanced activity.

Advantageously, step (b) comprises exome sequencing (for example, using a MiSeq DNA sequencer from lllumina).

Alternatively, step (b) may comprise the use of a PCR-based assay to assess sequence variation within the genes.

In one embodiment, step (b) comprises testing for the presence of insertions or deletions (“INDELs”) in the coding region and/or the promoter region of one or more of the genes identified in Table 1(a).

Additionally, or alternatively, step (b) may comprise testing for the presence of single nucleotide polymorphisms (SNPs) in the coding region and/or the promoter region of one or more of the genes identified in Table 1(b).

Such analyses of sequence variation may be undertaken using a bioinformatics approach, such as those described in Bioinformatics and Computational Biology Solutions Using R and Bioconductor (2005), edited by Gentleman, Carey, Huber, Irizarry, Dudoit, Springer (the disclosures of which are incorporated herein by reference) and Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 7 therein) (the disclosures of which are incorporated herein by reference).

Thus, in one embodiment, step (b) comprises the use of an algorithm to detect sequence variation within the genes (for example, ‘SNPeffect' may be used for phenotyping human single nucleotide polymorphisms, insertions and deletions; see Baets et al., 2012, Nucleic Acids Res. 40(1):D935-9, the disclosures of which are incorporated herein by reference).

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in one or more genes selected from the group consisting of NEFL, DIXDC1, EI24/PIG8, LTBP4 and ZNF516 (see Example below).

In a related embodiment, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in at least two of the genes identified in Table 1, for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty, fifty-five or more of the genes identified in Table 1.

For example, step (b) may comprise testing DNA from the subject to determine the presence of an INDEL in the coding region and/or the promoter region of at least two of the genes identified in Table 1(a), for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five or more of the genes identified in Table 1(a). Thus, in one embodiment, step (b) comprises testing DNA from the subject to determine the presence of an INDEL in all of the genes identified in Table 1(a).

Preferably, the INDELs are classified as ‘high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact INDELs are identified in Table 2 below. TABLE 2

High Impact INDELs in concordant genes in ALK-positive tumours

* “exonic” - INDEL predicted to result in exonic frameshift ** “splicing" = INDEL predicted to result in splice acceptor and/or donor variant

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 2.

Additionally, or alternatively, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of at least two of the genes identified in Table 1(b), for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the genes identified in Table 1(b).

Preferably, the SNPs are classified as ‘high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact SNPs are identified in Table 3 below. TABLE 3

High Impact SNPs in concordant genes

* Predicted to result in loss of stop codon ** Predicted to result in gain of new stop codon *** Predicted to result in splice acceptor variant and intron variant + Predicted to result in splice donor variant and intron variant tt Predicted to result in loss of start codon

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 3.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 1. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELs identified in Table 2 and all of the SNPs identified in Table 3. A second, related aspect of the invention provides an in vitro method for identifying a subject with chromosomal instability, or at risk of the same, comprising (a) providing a sample of cells (or protein and/or mRNA and/or DNA therefrom) from a subject to be tested; and (b) testing the cells (or protein and/or mRNA and/or DNA therefrom) to determine the function of and/or expression of and/or nucleotide sequence variation within one or more of the genes identified in Table 1, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value (e.g. in comparable cells from a healthy subject) and/or the presence of a variant sequence in one or more of the genes identified in Table 1 is indicative of the subject having chromosomal instability, or being at risk of the same.

By “chromosomal instability” we include the susceptibility of the chromosomes within a cell to undergo structural changes during cell division. Such chromosomal instability is known to be associated with tumour formation and cancer (see Bakhoum & Compton, 2012, J. Clin. Invest. 122(4):1138-43, the disclosures of which are incorporated herein by reference).

In one embodiment, step (b) is performed as defined above in relation to the first aspect of the invention. A third, related aspect of the invention provides a method for treating a subject with an anaplastic lymphoma kinase (ALK)-positive tumour, or at risk of developing the same, comprising: (a) identifying the subject as having an ALK-positive tumour, or at risk of developing the same, using a method according to the first or second aspect of the invention; and (b) administering to the subject a medicament with efficacy in the treatment of the ALK-positive tumour and/or surgically removing the tumour from said subject, iln one embodiment, the ALK-positive tumour is selected from the group

It will be appreciated by persons skilled in the art that subjects identified as having an ALK-positive tumour may be treated with any medicament known to have efficacy in the treatment of the ALK-positive tumour, including conventional anti-cancer drugs (including ALK inhibitors, chemotherapeutic agents and radiotherapeutic agents). ALK inhibitors, such as crizotinib and ceritinib, are approved (e.g. by the FDA in the US) for the treatment of non-small cell lung carcinomas (NSCLC) and may thus represent a preferred medicament for subjects identified as having an ALK-positive tumour.

Cytotoxic chemotherapeutic agents useful as as anticancer agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfane; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2’-deoxycoformycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (ο,ρ’-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.

Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic medicament may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid.

The treatment regime will be selected based on a number of factors, notably the type of tumour, the stage of the disease and the age of the patient. For example, ALCL is usually treated with a combination of chemotherapy drugs (for example, cyclophosphamide, doxorubicin, vincristine and prednisolone).

Additionally, or alternatively, the subject may undergo surgery to remove the tumour(s).

The subject may also receive radiotherapy, often upon completion of a course of chemotherapy or surgical removal of the tumour(s).

It will be appreciated that the methods of the third aspect of the invention may further comprise step (c) of providing a second sample of cells (or protein and/or mRNA and/or DNA therefrom) obtained from the subject after the commencement of treatment (for example, after completion of an initial course of chemotherapy and/or radiotherapy), testing said cells (or protein and/or mRNA and/or DNA therefrom) to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 1, and comparing the results of said testing with the results obtained in step (a). Such follow-up testing allows the efficacy of the treatment regime to be assessed.

In one embodiment, the method further comprises maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

Advantageously, step (c) is repeated one or more times in order to monitor the therapeutic efficacy of the treatment received by the subject over a period of time, e.g. until the subject is considered no longer to be at risk from the tumour(s). A fourth aspect of the invention provides a method for identifying a lead candidate with efficacy in the treatment or prevention of an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 1, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of an ALK-positive tumour if it modulates (e.g. enhances) the function and/or expression of one or more of the genes identified in Table 1.

By “lead candidate” we include pharmaceutical and/or biopharmaceutical agents which have potential efficacy in the treatment of an ALK-positive tumour, and thus represent a promising candidate for drug development and optimisation.

Conveniently, the method comprises the use of a high-throughput screening assay.

Step (b) may be performed using methods well-known in the art for assessing the function and/or expression of genes and the products thereof.

In one embodiment, step (b) is performed as described above in relation to the methods of the first aspect of the invention.

In another embodiment, the method further comprises step (c) of testing the lead candidate for efficacy in an in vivo model of said ALK-positive tumour.

In addition to providing methods for the diagnosis and treatment of subjects with an ALK-positive tumour, the invention also provides methods for the prognosis of subjects with such tumours.

Thus, a fifth aspect of the invention provides an in vitro method for the prognosis of a subject with an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a sample of cells (or protein and/or mRNA and/or DNA therefrom) from a subject to be tested; (b) testing the cells (or protein and/or mRNA and/or DNA therefrom) to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 compared to a reference value (e.g. in comparable cells from a healthy subject) and/or the presence of a variant sequence in one or more of the genes identified in Table 4 is indicative of the subject having a poor prognosis. TABLE 4

Genes with prognostic value in ALK-positive tumours

By “prognosis” we include the prediction of treatment outcome (e.g. life expectancy) in a subject with an ALK-positive tumour. For example, the method may be used to identify subjects with an increased risk of death from the cancer within one year. Such knowledge can be invaluable in terms of deciding upon treatment strategies and the level of follow-up monitoring of patients during and after completion of treatment.

By “poor prognosis” we include that the patient is expected to live less than one year. Thus, the method allows the identification of patients in need of an aggressive treatment regime (for example, high doses and/or combination therapies) and/or regular monitoring of treatment efficacy/disease progression.

In the methods of the fifth aspect of the invention, the initial step comprises the provision of a sample of protein and/or mRNA and/or DNA from a subject to be tested; typically, the sample contains cells from the subject to be tested that are suspected of being tumour cells.

In an alternative embodiment, the sample in step (a) is from a blood sample.

In a further embodiment, the sample in step (a) is from urine or saliva.

In one embodiment, DNA is purified from the sample of cells for analysis in step (b).

In an alternative embodiment, mRNA is purified from the sample of cells for analysis in step (b).

In a further embodiment, proteins are purified from the sample of cells for analysis in step (b). A preferred embodiment of the method of the fifth aspect of the invention comprises testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4, wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4 is indicative of the subject having a poor prognosis.

By “sequence variant” we include a difference in nucleotide sequence relative to a corresponding reference sequence (e.g. the corresponding sequence in normal, healthy cells). The nature of the sequence variation may be an insertion of one or more nucleotides at a given location within the gene, a deletion of one or more nucleotides at a given location within the gene, and/or a substitution of one or more nucleotides at a given location within the gene, wherein the sequence variation results in modulation of the function or expression of the gene. It will be appreciated by persons skilled in the art that such modulation of gene function/expression may arise from sequence variation in the coding region of the gene and/or it promoter.

In one embodiment, the variant sequence is detrimental to the function and/or expression of the gene. For example, the variant sequence may inhibit (in whole or in part) expression of the gene, such that levels of its protein product within the tumour cells are reduced. Alternatively, the variant sequence may result in production of a modified protein product of the gene having reduced or no biological activity (for example, the protein may be truncated).

Alternatively, step (b) may comprise the use of a PCR-based assay.

In one embodiment, step (b) comprises testing for the presence of insertions or deletions (“INDELs”) in the coding region and/or the promoter region of one or more of the genes identified in Table 4(a).

Additionally, or alternatively, step (b) may comprise testing for the presence of SNPs in the coding region and/or the promoter region of one or more of the genes identified in Table 4(b).

Conveniently, step (b) comprises a bioinformatics approach, such as those described in Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls (2003), edited by Lorkowski &. Cullen, Wiley (see Chapter 7 therein) (the disclosures of which are incorporated herein by reference).

Thus, in one embodiment, step (b) comprises the use of an algorithm (for example, ‘SNPeffect’ for phenotyping human single nucleotide polymorphisms, insertions and deletions; see Baets et a/., 2012, Nucleic Acids Res. 40(1):D935-9, the disclosures of which are incorporated herein by reference).

One preferred embodiment of the methods of the fifth aspect of the invention comprises testing DNA from the subject to determine the presence of a variant sequence in at least two of the genes identified in Table 4.

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or promoter region of all of the genes identified in Table 4(a).

Preferably, the INDELs are classified as 'high impact', suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact INDELs are identified in Table 5 below. TABLE 5

High Impact INDELs associated with poor prognosis

* Predicted to result in exonic frameshift * Predicted to result in splice donor variant and intron variant

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 5.

In one embodiment, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in all of the genes identified in Table 4(b).

Preferably, the SNPs are classified as 'high impact’, suggestive of a greater likelihood of them having a functional effect on the gene.

Examples of high impact SNPs are identified in Table 6 below. TABLE 6

High Impact SNPs associated with poor prognosis

* Predicted to generate new stop codon ** Predicted to be a splice acceptor variant and intron variant

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 6.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 4. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 5 and all of the SNPs identified in Table 6.

In one preferred embodiment, step (b) further comprises testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 7, wherein modulation of the function and/or expression of the one or more of the genes identified in Table 7 compared to a reference value (e.g. in comparable cells from a healthy subject) and/or the presence of a variant sequence in one or more of the genes identified in Table 7 is indicative of the subject having a poor prognosis. TABLE 7

Additional genes with prognostic value in ALK-positive tumours

Thus, step (b) may comprise testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of at least two of the genes identified in Table 7.

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or promoter region of two or more of the genes identified in Table 7(a). For example, step (b) may comprise testing DNA from the subject to determine the presence of an INDEL in the coding region of all of the genes identified in Table 7(a).

Preferably, the INDELs are classified as 'moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact INDELs are identified in Table 8 below. TABLE 8

Moderate Impact INDELs associated with poor prognosis

* Predicted to result in disruptive inframe insertion ** Predicted to result in disruptive inframe deletion *** Predicted to result in in frame deletion

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELS identified in Table 8.

In one embodiment, step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of at least two of the genes identified in Table 7(b). For example, step (b) may comprise testing DNA from the subject to determine the presence of a SNP in all of the genes identified in Table 7(b).

Preferably, the SNPs are classified as ‘moderate impact’, suggestive of a possibility of them having a functional effect on the gene.

Examples of moderate impact SNPs are identified in Table 9 below (each of which is predicted to be a missense mutation). TABLE 9

Moderate Impact SNPs associated with poor prognosis

Thus, step (b) may comprise testing DNA from the subject to determine the presence of all of the SNPs identified in Table 9.

Advantageously, step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 7. For example, step (b) may comprise testing DNA from the subject to determine the presence of all of the INDELs identified in Table 8 and all of the SNPs identified in Table 9. A sixth, related aspect of the invention provides an in vitro method for identifying a subject at risk of recurrence of an anaplastic lymphoma kinase (ALK)-positive tumour comprising (a) providing a sample of cells (or protein and/or mRNA and/or DNA therefrom) from a subject to be tested; and (b) testing the cells (or protein and/or mRNA and/or DNA therefrom) to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 (and, optionally, in Table 7) wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 (and 7) compared to a reference value (e.g. in comparable cells from a healthy subject) and/or the presence of a variant sequence in one or more of the genes identified in Table 4 (and 7) is indicative of the subject being at risk of recurrence of the ALK-positive tumour.

In one embodiment, step (b) is performed as defined above in relation to the fifth aspect of the invention. A seventh, related aspect of the invention provides a method for treating a subject with an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) identifying the subject as having either a poor prognosis or a good prognosis using a method according to the fifth aspect of the invention; and (b) selecting an appropriate treatment regime for said subject.

Thus, where a subject is identified as having a poor prognosis, a more aggressive treatment regime may be selected (for example, with high doses and possibly also multiple therapeutic agents and/or surgical intervention).

Alternatively, where a subject is identified as having a good prognosis, a milder treatment regime may be selected (for example, with lower doses of single therapeutic agents).

In another embodiment of the seventh aspect of the invention, the method further comprises step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 and/or 7, and comparing the results of said testing with the results obtained in step (a).

Advantageously, step (c) is repeated in order to monitor the therapeutic efficacy of the treatment received by the subject.

An eighth aspect of the invention provides a method for identifying a lead candidate with efficacy in the treatment or prevention of an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 4 (and, optionally, Table 7), wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of an ALK-positive tumour if it modulates the function and/or expression of one or more of the genes identified in Table 4 (and Table 7).

In one embodiment, step (b) is performed as described above in relation to the methods of the fifth aspect of the invention.

Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figure:

Figure 1: General representation of the GATK pipeline used for variant discovery as recommended by the Broad Institute

EXAMPLE

Exome sequencing of a cohort of Anaplastic Large Cell Lymphoma tumour DNA: molecular identification of variants with prognostic, diagnostic and therapeutic potential

Introduction

In this study exome sequencing has been exploited in order to characterise the genomes of a cohort of ALCL tumours. Analyses were conducted in order to identify variants (SNP/INDEL) that were primarily located within the protein coding regions (although regions extending into the UTRs and exon/intron boundary junctions were also included). Exome sequencing can elicit many thousands of potential variants per analyses and consequently suitable strategies need to be implemented in order to select those with the highest potential of being relevant to the question(s) under investigation. To address this question this study exploited the use of bioinformatics to determine whether variants were present or absent for a specific chromosomal location. It was rationalised that such an approach may provide a method to analyse a subset of variants that are important by virtue of whether they remain concordant or discordant between samples. The presence or absence of particular variants may therefore provide evidence that a subset of genes have a key role in the process of ALCL and/or ALK-positive driven processes. Such an approach would not consider parochial issues such as molecular heterogeneity within a given patient, nor would the methodology identify a SNP/INDEL present in the same gene but occurring elsewhere in its exonic structure.

Two classes of genes were explored in this study of ALCL patients: 1) Genes potentially linked to ALCL and/or ALK +ve driven tumour formation: The first group of variants to be characterized were those that had either SNP or INDEL consistent (concordant) between samples. This analyses was intended to identify genes and their associated pathways that could be important in the development of ALCL tumours. Given that all patients analysed were NPM-ALK +ve there was also a possibility that genes identified as having potentially deleterious/modifying effects may also have applicability to other tumour types driven by the ALK tyrosine kinase (e.g. EML4-ALK translocations occurring in subsets of lung tumours). 2) Genes linked to poor prognosis: To identify variants linked to poor prognoses a comparative analyses was undertaken using ALCL samples from patients dying less than one year after initial diagnoses compared to patients in which their lifespan extended to a minimum of 4.5 years after initial diagnosis. Variants were identified that were concordant between the patients that died in less than one-year. Variants (occurring at the same genetic loci) that were present in any of the patients that had progressed to at least 4.5 years were filtered out and not utilised. Identification of candidate genetic sequences might ultimately be exploited as a method of stratifying patients into treatment arms, i.e. more aggressive therapeutic approaches might apply to those children at a higher risk of disease relapse and/or with a poor prognosis. These gene signatures may also provide a suitable mechanism for monitoring MDD/tumour recurrence and assist in the triage and clinical management of high risk patients.

Methods

Exome library preparation

Genomic DNA (gDNA) was prepared as described previously [10]. Exome libraries were constructed using the Nextera Rapid Capture kits (lllumina) as per manufacturer’s instructions. 50 ng of gDNA was utilised to construct DNA exomes for each sample. DNA quantitation was undertaken using a Qubit fluorimeter and size distribution of insert was assayed using a Bioanalyser 2100 with high sensitivity DNA kits. DNA sequencing A MiSeq DNA sequencing instrument (lllumina) was used for sequencing the exome libraries. Libraries were barcoded and multiplexed and run a minimum of 11x times in order to generate sufficient coverage. Sequencing reactions were carried out using 2 x 75 paired end cycles (+1 cycle to each forward and reverse read to allow for phasing/pre-phasing). Fastq files were generated and used for downstream variant analyses.

Bioinformatic analyses

Fastq files were unzipped and concatenated from multiple runs to produce a single fastq file. Adapters were automatically trimmed by the sequencing instrument. Pre-processing of sequences occurred as followed: base quality scores (less than Q30) and the first 14 base pairs of the 5’ were removed from forward and reverse reads using the Trim Galore wrapper and Cutadapt (v1.7.1) in order to minimise the risk of sequence bias. FastQC (vO.11.2) was used for general quality control of sequences and to confirm adapter removal/quality trimming. Alignments to the reference human genome (hg19) were conducted using BWA MEM (version 0.7.15) with 32 parallel threads. Optical duplicates were marked using Picard Tools (Broad Institute) and sequence metrics collected using the command “Collectmultiplemetrics”. Samtools “Flagstat” command was used to collect basic statistics regarding forward and reverse read alignment success as well as the percentage of reads properly paired.

The Genome Analysis Tool Kit (GATK version 3.0.0) was used for INDEL realignment, base quality score recalibration, raw variant calling and hard filtering to identify high quality variants (SNP/INDEL) for downstream analyses (refer to Figure 1 for the “Best Practice” set of guidelines recommended by the Broad Institute). INDEL and SNP were corrected for multi-allelic sites and left normalised using BCFTOOLS prior to annotation (e.g. Annovar (version June 2015)). Comparative analyses were carried out using GATK SelectVariant functions for concordance/discordance between vcf files of interest. SnpEff (v4.1) was exploited to determine predicted in silico impacts upon protein function of candidate genes.

Identification of concordant SNP and INDEL between ALCL patients 15 ALCL patient tumour samples were sequenced and processed computationally using the pipeline described above. To identify concordant SNP and INDEL amongst, sequential processing was undertaken and a single vcf file produced for each sample. These vcf files were then tested for concordance for all variants (SNP and INDEL were treated independently of each other). The product of the first comparison was then used as the baseline for concordance testing with the next sample. This process was iterated until all ALCL tumour samples had been tested for SNP and INDEL concordance.

Poor prognosis versus good prognosis

Patients were divided into two groups: 1) “Poor Prognosis” (patients dying within 0.7 years from initial presentation) 2) “Good Prognosis” (patients progressed to a minimum of 4.5 years after initial presentation (range 4.5 years - 8.7 years))

Poor Prognosis Group

Good Prognosis Group

The analyses were carried out as follows: 1) Concordance for SNP and INDEL were identified for both patients from the “Poor Prognosis Group” (i.e. both patients 4/268 and 14/125 had to have the same locus affected) 2) INDEL and SNP identified as concordant for the “Poor Prognosis Group” but also occurring in any of the “Good Prognosis Group” were filtered out. This left only INDEL and SNP that were unique to the “Poor Prognosis Group”.

Results

Concordant variants across ALCL patients (a) INDELs A total 39 distinct genes were identified as having concordant INDEL with potential high impact effect changes to protein structure (Table A). Several genes identified in this cohort had been previously associated as having differential expression in tumours other than ALCL or ALK +ve tumours and possessing potential tumour suppressor activity. The following represented selected examples: 1) NEFL: NEFL expression has been negatively correlated with primary breast with lymph node metastasis compared with primary breast tumours with negative lymph node involvement [11]. The suggestion is that low expression of NEFL is linked to poor prognosis and more aggressive phenotypes. The in-silico prediction of the NEFL INDEL was predicted to have high impact upon protein function and thus may therefore lead to an effective down-regulation of the gene through abrogated protein function. 2) DIXDC1: DIXDC1 is a scaffold protein involved in controlling the number and size of focal adhesions. It is a molecular component of the LKB1 tumour suppressor signaling pathway. DIXDC1 loss results in increased metastatic potential in lung cancer cells and is strongly associated with poor prognosis in murine models of metastasis [12]. 3) EI24/PIG8: p53 is a critical tumour suppressor gene that has been shown to be mutated in diverse types of cancers. Etoposide Induced 24 protein (EI24)/P53-Induced Gene 8 (PIG8) is a down-stream gene that is activated following p53 induction [13]. The gene was shown to be up-regulated following exposure to etoposide (a chemotherapeutic agent commonly used in the treatment of Non Hodgkin Lymphoma patients [14]). Inactivation of EI24 has been linked to the development of cervical tumours [15] lending further credence that this gene may therefore have a potential role in ALCL and tumours that have activated ALK mutations and chemotherapeutic response patterns. 4) LTBP4: Latent transforming growth factor-beta binding protein (LTBP4) is an extracellular matrix protein that is required for correct folding/functioning of TGF-β1. LTBP4 has been shown to be down-regulated in adenocarcinomas and squamous cell carcinomas of the esophagus [16]. Within this tissue system the protein was shown to impact metastatic potential without any noticeable effect on either proliferation rates or cell viability. It has also shown to be down-regulated in primary DCIS breast-tumours and invasive breast adenocarcinomas compared to normal tissue [17], There is a possibility that high impact INDEL could have a deleterious effect upon LTBP4 function in ALCL patients and ALK +ve tumours and serve to increase their metastatic potential to distal lymph node sites. This gene may therefore have potential as a prognosticator of poor prognosis/recurrence. 5) ZNF516: ZNF516 has been implicated as a cancer chromosomally instability (CIN) suppressor gene and has been associated with copy number loss in colorectal cancer cells. The discovery that this gene plays a pivotal role in reducing the molecular heterogeneity of tumours also gave rise to the possibility that ZNF516 could form the basis of a new therapeutic target [18j. ZNF516 may have a similar role in promoting cancer genome de-stabilization and increasing the difficulty of rationalizing treatment regimens [18]. Therapeutic targeting of ZNF516 may therefore provide new treatment options for ALCL and/or ALK +ve tumours.

Given that several tumour suppressor genes have been identified as being mutated in other cancer types there is strong a possibility that these same genes may have potential roles in suppressing ALCL tumours and/or those that are driven by ALK +ve signaling pathways. Several genes listed that have not historically been associated with tumourigenesis and thus may therefore represent novel candidates in ALCL/ALK-driven processes (Table A).

Table A: Concordant high impact effect INDEL between ALCL patients as determined by the in-silico modelling algorithm SnpEff

(b) SNPs A total 19 distinct genes were identified as having concordant SNP with potential high impact effect changes to protein structure (Table B). Several genes identified in this cohort had been previously associated as having differential expression in tumours other than ALCL or ALK +ve tumours with potential tumour suppressor/drug response modulating activity. The following represent selected examples: 1) CDC27: The anaphase-promoting complex or cyclosome (APC/C) is a multimeric protein complex that controls cell cycle progression. CDC27 is one the components of APC/C. A high number of mutations have been detected in CDC27 following sequencing of patients with testicular tumours [19]. Down-regulation of CDC27 has been linked to increased radiotherapy resistance in triple negative breast cancer cells [20], There is the implication therefore that CDC27 may have potential tumour suppressor activity. This gene is being explored as a potential chemotherapeutic target for curcumin. 2) PIGN; PIGN is a putative cancer chromosomal instability (CIN) suppressor gene and has been shown to be significantly down-regulated through copy-number loss in colorectal cancer patients [18]. This study has detected a SNP having a potential high impact effect on protein function which may therefore lead to CIN and increase the rate of molecular heterogeneity. Such heterogeneity may enhance biological aggressiveness (e.g. increased risk of recurrence/metastasis) and make selection of appropriate therapies challenging. The rationale would be to develop novel therapeutics targeting PIGN and minimise CIN. Treatment of low molecular heterogeneity tumours would therefore be potentially easier and thus produce favourable patient prognoses with respect to recurrence and long-term survival. 3) TLE4: A recent publication by Dayyani et al. has provided data to suggest that TLE4 has the ability to control the proliferative rate in acute myeloid leukaemia cells (AML). Downregulation of TLE4 resulted in an increased rate of proliferation of AML cells thus suggesting that TLE4 may have potential tumour suppressor activity [21]. TLE4 may have potential controlling activity in ALCL progression/evolution and ALK +ve tumours. 4) DSC3: Down-regulation of Desmocollin 3 (DSC3) RNA has been linked with Advanced Esophageal Adenocarcinomas and lymph node metastasis [22], Cui et al have shown that DSC3 is a target of p53 and that DSC3 can have gene expression controlled through epigenetic silencing (methylation of promoter regions) in human lung cancer cells. These authors postulate that DSC3 is a novel tumour suppressor gene and when down-regulated allows tumours to acquire aggressive phenotypes [23], DSC3 therefore may have potentially regulatory roles in tumour initiation, progression and therapeutic response patterns for in ALCL tumours and/or ALK-driven tumours.

These data presented would suggest that high impact SNP variants may potentially abrogate protein function for several proteins previously established as putative tumour suppressors in other tumour types. These genes may play concordant roles in ALCL tumours and/or ALK-driven oncogenic pathways. There are several genes listed (Table B) that may represent strong candidates as novel molecular components in ALCL/ALK-driven tumour processes and thus further characterisation is required in order to precisely map function to ALCL/ALK +ve biology.

Table B: Concordant high impact effect SIMP between ALCL patients as determined by the in-silico modelling algorithm SnpEff

Poor versus good prognosis (a) INDELs

HIGH IMPACT INDEL

Table C: Discordant high impact INDEL between poor and good prognoses patients as determined by the in-silico modelling algorithm SnpEff

This part of the study was focused upon potential prognostic variant markers indicative of poor life expectancy and may therefore provide genetic biomarkers for MDD/recurrence and pathways in which to create novel therapeutic compounds. A comparative analyses was undertaken between ALCL patients succumbing to disease 0.7 years after initial presentation. Concordant INDEL were identified between the poor prognosis samples and then INDEL filtered if concordant with any of the favourable prognosis samples. Two genes with high impact effect INDEL were identified. TYR03 has been shown to provide pro-survival signals in cells in which it is over-expressed [24], How this may potentially relate to the findings in this study where potential protein function is damaged remains unclear. ZFYVE19/ANCHR performs an integral role in delaying membrane abscission in order to avoid DNA damage and aneuploidy. Potential abrogation of this gene may therefore lead to increased risk of aneuploidy in ALCL tumours and facilitate greater diversification of the cancer genome [25]. This may therefore provide a novel role for ANCHR in ALCL/ALK +ve tumour biology.

MODERATE IMPACT INDEL

Table D: Discordant moderate impact INDEL between poor and good prognoses patients as determined by the in-silico modelling algorithm SnpEff

Three genes were identified as being concordant between the “Poor Prognosis Group” while absent from the samples present in the “Good Prognosis Group”. The moderate impact predictions may result in attenuated/altered protein function without necessarily preventing the protein from functioning. CTBP2 has been shown to be a transcriptional co-repressor but has co-activator properties essential for mediating the transactivation of RXR/RAR regulatory sequences through retinoic acid signalling pathways [26]. Retinoids are powerful agents for the differentiation of many tumour cells and thus may indicate a potential role of this protein in modulating nuclear receptor signalling pathways.

Myeloid-derived suppressor cells (MDSCs) and cancer stem cells (CSCs) play essential and dynamic roles with respect to the cancer microenvironment. Evidence suggests that the interplay can impact cancer phenotype and patient outcome. MDSCs have been shown to increase the expression of miRNA101 in ovarian cancer cells which is a direct inhibitor of CTBP2. CTBP2 is a transcriptional co-repressor of CSC genes and thus down-regulation of CTPB2 resulted in alleviation of repression of CSC-associated genes promoting the expression of stem cell phenotypes i.e. less differentiated [27], Recent evidence suggests that a cancer stem cell population may exist within ALCL tumours [28] and therefore analogous pathways may be at play within ALCL tumours/ALK +ve tumours as witnessed within ovarian tumour biology. A gene linked to haemopoietic stem cell (HSC) homeostasis is ITPKB (Inositol -triphosphate 3-kinase), which is responsible for synthesising IP4. IP4 causes suppression of the AKT/mTOR pathway thereby promoting HSC quiescence. ITPKB loss has been shown to result in HSC activation [29]. The possibility exists that modification of ITPKB (through an in-frame deletion) may potentially alter the activity of ITPKB in ALCL thus promoting a proliferative cellular phenotype. Recent data suggests that a HSC subpopulation may exist within ALCL that is persistent and gives rise to ALCL tumours [28]. This may open the possibility that similar gene pathways may be at work in ALCL analogous to those in HSCs and ALK-driven tumours. (b) SNPs

HIGH IMPACT SNP

Table E: Discordant high impact SNP between poor and good prognosis patients as determined by the in-silico modelling algorithm SnpEff

Two genes with high impact SNP were detected. The first was noted in olfactory receptor OR4C16 while the second occurred quite interestingly in TYR03 (the same gene in which a high-impact INDEL was observed - see above).

MODERATE IMPACT SNP

Table F: Discordant moderate impact SNP between poor and good prognosis patients as determined by the in-silico modelling algorithm SnpEff

112 moderate impact SNP were identified within the poor prognosis patient samples. Two genes of interest were FANCA (linked to the Fanconi Anaemia pathway) in which over expression of a trans-dominant negative version was shown to sensitise lymphoblastoid cells to cisplatin. Pathways involving other gene members of the FANC pathway including FANCD2 ubiquitination, were impacted directly - suggesting possible cross-talk between FANCA and FANCD2 [30], Interestingly a Leu153Ser mutation in the FANCD2 gene was strongly associated with both clonal progression of a T-lineage acute lymphoblastic leukaemia and severe chemotherapy toxicity. This may open up the possibility that FANCA and FANCD2 pathways are associated with chemotherapy sensitisation and drug toxicity [31]. The gene DFNB31 (Whirlin) was also detected with a moderate impact SNP capable of potentially affecting its protein activity. A GG genotype at DFNB31 (rs2274159) was associated with greater sensitivity to cetuximab suggesting that modification of protein structure in this gene may have the possibility to attenuate response profiles to biological mediated therapeutics [32].

With respect to the other 110 moderate impact SNP there may be novel pathways and loci critical to the development, biological progression and selection of more aggressive phenotypes in ALCL. As discussed in the previous sections the pathways identified do not necessarily have to be restricted to ALCL tumours given that the transformative event involving constitutive activation of the ALK tyrosine kinase and that this type of transformative event is also common in other tumour types (e.g. non-small cell lung cancer; glioblastoma; neuroblastoma).

Discussion

Personalised molecular medicine is predicated upon tailoring specific genomic signatures to clinical management protocols and therapeutic modalities capable of translating into maximum patient benefit. High throughput DNA sequencing is providing a powerful screening technology for human variants of clinical importance. The data presented within this study has explored exonic variation intrinsically common to a cohort of ALCL tumour samples. The methodology has exploited the use of computational algorithms to identify SNP/INDEL that are discordant to the reference human genome hg19. Through a series of filtering steps a panel of high quality SNP and INDEL were then identified for downstream analyses of biological/clinical relevance.

In the first series of comparisons concordance of SNP and INDEL were explored between ALCL tumour samples in order to identify variants that may be clinically and/or biological relevant. Variants identified included tumour suppressor genes, genes intrinsically linked to chemotherapy response, chromosomal stability, cell cycle control, focal adhesion points and histone protein modifiers impacting gene expression at the epigenetic level. Many of these loci have been previously reported as key molecular players in other tumour types but this is the first time that a link has been established between these genes and ALCL tumour biology. These genes may thus form the basis of diagnostic/prognostic companion diagnostic assays for ALCL (e.g. MDD/tumour recurrence) and with provide the foundation for developing novel therapeutic agents.

This study also undertook to examine variants that were directly linked to poor prognosis which may facilitate the triaging of patients into distinct treatment groups, for example distinguishing between those patients with a poor prognosis that may require aggressive treatment regimens from other patient with a better prognosis that may benefit from reduced levels of chemotherapy (with fewer toxic side-effects). Genes were identified that had been previously linked to controlling genetic pathways linked to stem cell related plasticity, chemotherapy response and potential intersection to pathways linked to drug toxicity.

While the genetic material used for these studies originated in ALCL tumours, several of the genes with sequence variants have been linked to aggressive phenotypes in other tumour types. Consequently, we believe the data presented in this study to be of relevance to ALK-positive tumours in general.

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Claims

1. An in vitro method for identifying a subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or at risk of developing the same, the method comprising the steps of: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine therein the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 1 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 1 is indicative of the subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or being at risk of developing the same.

2. An in vitro method according to Claim 1 wherein the ALK-positive tumour is selected from the group consisting of anaplastic large cell lymphoma (ALCL), lung adenocarcinoma, breast cancer (for example, the inflammatory subtype thereof), neuroblastoma, inflammatory myofibroblastic tumours, renal cell carcinoma, oesophageal squamous cell carcinoma, colorectal adenocarcinoma, glioblastoma multiforme and anaplastic thyroid cancer.

3. An in vitro method according to Claim 2 wherein the ALK-positive tumour is an anaplastic large cell lymphoma (ALCL).

4. An in vitro method according to any one of the preceding claims wherein the sample in step (a) is from a tumour biopsy sample.

5. An in vitro method according to any one of Claims 1 to 3 wherein the sample in step (a) is from a blood sample.

6. An in vitro method according to any one of Claims 1 to wherein the sample in step (a) is from a urine or saliva sample,

7. An in vitro method according to any one of the preceding claims wherein DNA is purified from the sample of cells for analysis in step (b).

8. An in vitro method according to any one of the preceding claims wherein mRNA is purified from the sample of cells for analysis in step (b).

9. An in vitro method according to any one of the preceding claims wherein proteins are purified from the sample of cells for analysis in step (b).

10. An in vitro method according to any one of the preceding claims comprising testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1, wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 1 is indicative of the subject having an anaplastic lymphoma kinase (ALK)-positive tumour, or being at risk of developing the same.

11. An in vitro method according to Claim 10 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

12. An in vitro method according to any one of the preceding claims wherein step (b) comprises exome sequencing.

13. An in vitro method according to any one of the preceding claims wherein step (b) comprises the use of a PCR assay.

14. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing for the presence of INDELs in the coding region and/or the promoter region of one or more of the genes identified in Table 1(a).

15. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing for the presence of SNPs in the coding region and/or the promoter region of one or more of the genes identified in Table 1 (b).

16. An in vitro method according to any one of the preceding claims wherein step (b) comprises a bioinformatics analysis.

17. An in vitro method according to any one of the preceding claims wherein step (b) comprises the use of an algorithm.

18. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in one or more genes selected from the groups consisting of NEFL, DIXDC1, EI24/PIG8, LTBP4 and ZNF516.

19. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in at least two of the genes identified in Table 1, for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty, fifty-five or more of the genes identified in Table 1.

20. An in vitro method according to Claim 19 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or the promoter region of at least two of the genes identified in Table 1 (a), for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five or more of the genes identified in Table 1(a).

21. An in vitro method according to Claim 20 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in all of the genes identified in Table 1(a).

22. An in vitro method according to Claim 20 or 21 wherein the INDEL is classified as high-impact.

23. An in vitro method according to Claim 22 wherein the INDEL is selected from the group of INDELS identified in Table 2.

24. An in vitro method according to Claim 23 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELS identified in Table 2.

25. An in vitro method according to any one of Claims 18 to 24 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of at least two of the genes identified in Table 1(b), for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the genes identified in Table 3.

26. An in vitro method according to Claim 25 wherein step (b) comprises testing DNA from the subject to determine the presence of a SNP in the coding region and/or the promoter region of all the genes identified in Table 1(b).

27. An in vitro method according to Claim 25 or 26 wherein the SNP is classified as high-impact.

28. An in vitro method according to Claim 27 wherein the SNP is selected from the group of SNPs identified in Table 3.

29. An in vitro method according to Claim 28 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 3.

30. An in vitro method according to any one of the preceding claims wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 1.

31. An in vitro method according to Claim 30 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 2 and all of the SNPs identified in Table 3.

32. An in vitro method for identifying a subject with chromosomal instability comprising (a) providing a sample of cells from a subject to be tested; and (b) testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 1 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 1 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 1 is indicative of the subject having chromosomal instability.

33. An in vitro method according to Claim 32 wherein step (b) is performed as defined in any one of Claims 10 to 31.

34. A method for treating a subject with an anaplastic lymphoma kinase (ALK)-positive tumour, or at risk of developing the same, comprising: (a) identifying the subject as having an ALK-positive tumour, or at risk of developing the same, using a method according to any one of Claims 1 to 31; and (b) administering to the subject a medicament with efficacy in the treatment of the ALK-positive tumour and/or surgically removing the tumour from said subject.

35. A method according to Claim 34 wherein the ALK-positive tumour is selected from the group consisting of anaplastic large cell lymphoma (ALCL), lung adenocarcinoma, breast cancer (for example, the inflammatory subtype thereof), neuroblastoma, inflammatory myofibroblastic tumours, renal cell carcinoma, oesophageal squamous cell carcinoma, colorectal adenocarcinoma, glioblastoma multiforme and anaplastic thyroid cancer.

36. A method according to Claim 34 or 35 further comprising step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 1 and/or 3, and comparing the results of said testing with the results obtained in step (a).

37. A method according to Claim 36 further comprising maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

38. A method according to Claim 36 or 37 comprising repeating step (c) in order to monitor the therapeutic efficacy of the treatment received by the subject.

39. A method for identifying a lead candidate with efficacy in the treatment or prevention of an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 1, wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of an ALK-positive tumour if it modulates the function and/or expression of one or more of the genes identified in Table 1.

40. A method according to Claim 39 wherein step (b) is performed as defined in any one of Claims 10 to 31.

41. A method according to Claim 39 or 40 further comprising step (c) of testing the lead candidate for efficacy in an in vivo model of said ALK-positive tumour.

42. An in vitro method for the prognosis of a subject with an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a sample of cells from a subject to be tested; (b) testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 4 is indicative of the subject having a poor prognosis.

43. An in vitro method according to Claim 42 wherein the ALK-positive tumour is selected from the group consisting of anaplastic large cell lymphoma (ALCL), lung adenocarcinoma, breast cancer (for example, the inflammatory subtype thereof), neuroblastoma, inflammatory myofibroblastic tumours, renal cell carcinoma, oesophageal squamous cell carcinoma, colorectal adenocarcinoma, glioblastoma multiforme and anaplastic thyroid cancer.

44. An in vitro method according to Claim 43 wherein the ALK-positive tumour is an anaplastic large cell lymphoma (ALCL).

45. An in vitro method according to any one of Claims 42 to 44 wherein the sample of cells in step (a) is from a tumour biopsy sample.

46. An in vitro method according to any one of Claims 42 to 44 wherein the sample in step (a) is from a blood sample.

47. An in vitro method according to any one of Claims 42 to 44 wherein the sample in step (a) is from a urine or saliva sample.

48. An in vitro method according to any one of Claims 42 to 47 wherein DNA is purified from the sample of cells for analysis in step (b).

49. An in vitro method according to any one of Claims 42 to 48 wherein mRNA is purified from the sample of cells for analysis in step (b).

50. An in vitro method according to any one of Claims 42 to 49 wherein proteins are purified from the sample of cells for analysis in step (b).

51. An in vitro method according to any one of Claims 42 to 50 comprising testing a DNA sample from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4 wherein the presence of a variant sequence in the coding region and/or promoter region of one or more of the genes identified in Table 4 is indicative of the subject having of the subject having a poor prognosis.

52. An in vitro method according to Claim 51 wherein the variant sequence is detrimental to the function of the gene or the product thereof.

53. An in vitro method according to any one of Claims 42 to 52 wherein step (b) comprises exome sequencing.

54. An in vitro method according to any one of Claims 42 to 53 wherein step (b) comprises the use of a PCR assay.

55. An in vitro method according to any one of Claims 42 to 54 wherein step (b) comprises testing for the presence of INDELs in the coding region and/or the promoter region of one or more of the genes identified in Table 4(a).

56. An in vitro method according to any one of Claims 42 to 55 wherein step (b) comprises testing for the presence of SNPs in the coding region and/or the promoter region of one or more of the genes identified in Table 4(b).

57. An in vitro method according to any one of Claims 42 to 56 wherein step (b) comprises a bioinformatics analysis.

58. An in vitro method according to any one of Claims 42 to 57 wherein step (b) comprises the use of an algorithm.

59. An in vitro method according to any one of Claims 42 to 58 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in at least two of the genes identified in Table 4.

60. An in vitro method according to Claim 59 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or promoter region of all of the genes identified in Table 4(a).

61. An in vitro method according to Claim 55 wherein the INDEL is classified as high-impact.

62. An in vitro method according to Claim 61 wherein the INDEL is selected from the group of INDELS identified in Table 5.

63. An in vitro method according to Claim 62 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 5.

64. An in vitro method according to any one of Claims 59 to 63 wherein step (b) comprises testing DNA from the subject to determine the presence of an SNP in the coding region and/or the promoter region of at least two of the genes identified in Table 4(b).

65. An in vitro method according to Claim 64 wherein step (b) comprises testing DNA from the subject to determine the presence of an SNP in the coding region and/or the promoter region of all the genes identified in Table 4(b).

66. An in vitro method according to Claim 64 or 65 wherein the SNP is classified as high-impact.

67. An in vitro method according to Claim 66 wherein the SNP is selected from the group of SNPs identified in Table 6.

68. An in vitro method according to Claim 67 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 6.

69. An in vitro method according to any one of Claims 59 to 68 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in all of the genes identified in Table 4.

70. An in vitro method according to Claim 69 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 5 and all of the SNPs identified in Table 6.

71. An in vitro method according to any one of Claims 59 to 71 wherein step (b) further comprises testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 7 wherein modulation of the function and/or expression of the one or more of the genes identified in Table 7 compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 7 is indicative of the subject having a poor prognosis.

72. An in vitro method according to Claim 71 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in the coding region and/or promoter region of at least two of the genes identified in Table 7.

73. An in vitro method according to Claim 72 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region and/or promoter region of two or more of the genes identified in Table 7(a).

74. An in vitro method according to Claim 73 wherein step (b) comprises testing DNA from the subject to determine the presence of an INDEL in the coding region of all of the genes identified in Table 7(a).

75. An in vitro method according to Claim 73 or 74 wherein the INDEL is classified as moderate-impact.

76. An in vitro method according to Claim 75 wherein the INDEL is selected from the group of INDELS identified in Table 8.

77. An in vitro method according to Claim 76 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 8.

78. An in vitro method according to any one of Claims 71 to 77 wherein step (b) comprises testing DNA from the subject to determine the presence of an SNP in the coding region and/or the promoter region of at least two of the genes identified in Table 7(b), for example at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty, sixty, seventy, eighty, ninety, one hundred, one hundred and ten or more of the genes identified in Table 7(b).

79. An in vitro method according to Claim 78 wherein step (b) comprises testing DNA from the subject to determine the presence of an SNP in the coding region and/or the promoter region of FANCA and/or DFNB31(Whirlin).

80. An in vitro method according to Claim 78 or 79 wherein step (b) comprises testing DNA from the subject to determine the presence of an SNP in the coding region and/or the promoter region of all the genes identified in Table 7(b).

81. An in vitro method according to any one of Claims 78 to 80 wherein the SNP is classified as moderate-impact.

82. An in vitro method according to Claim 81 wherein the SNP is selected from the group of SNPs identified in Table 9.

83. An in vitro method according to Claim 82 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the SNPs identified in Table 9.

84. An in vitro method according to any one of Claims 71 to 83 wherein step (b) comprises testing DNA from the subject to determine the presence of a variant sequence in the coding region of all of the genes identified in Table 7.

85. An in vitro method according to Claim 84 wherein step (b) comprises testing DNA from the subject to determine the presence of all of the INDELs identified in Table 8 and all of the SNPs identified in Table 9.

86. An in vitro method for identifying a subject at risk of recurrence of an anaplastic lymphoma kinase (ALK)-positive tumour comprising (a) providing a sample of cells from a subject to be tested; and (b) testing the cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 (and, optionally, in Table 7) wherein modulation of the function and/or expression of the one or more of the genes identified in Table 4 (and 7) compared to a reference value and/or the presence of a variant sequence in one or more of the genes identified in Table 4 (and 7) is indicative of the subject being at risk of recurrence of the ALK-positive tumour.

87. An in vitro method according to Claim 86 wherein step (b) is performed as defined in any one of Claims 51 to 85.

88. A method for treating a subject with an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) identifying the subject as having either a poor prognosis or a good prognosis using a method according to any one of Claims 51 to 85; and (b) selecting an appropriate treatment regime for said subject.

89. A method according to Claim 88 wherein the ALK-positive tumour is selected from the group consisting of anaplastic large cell lymphoma (ALCL), lung adenocarcinoma, breast cancer (for example, the inflammatory subtype thereof), neuroblastoma, inflammatory myofibroblastic tumours, renal cell carcinoma, oesophageal squamous cell carcinoma, colorectal adenocarcinoma, glioblastoma multiforme and anaplastic thyroid cancer.

90. A method according to Claim 88 or 89 further comprising step (c) of providing a second sample of cells obtained from the subject after the commencement of treatment, testing said cells to determine the function and/or expression and/or nucleotide sequence variation of one or more of the genes identified in Table 4 and/or 7, and comparing the results of said testing with the results obtained in step (a).

91. A method according to Claim 90 further comprising maintaining or modifying the treatment of the subject in order to optimise its therapeutic efficacy.

92. A method according to Claim 90 or 91 comprising repeating step (c) in order to monitor the therapeutic efficacy of the treatment received by the subject.

93. A method for identifying a lead candidate with efficacy in the treatment or prevention of an anaplastic lymphoma kinase (ALK)-positive tumour comprising: (a) providing a compound to be tested; and (b) determining in vitro whether said test compound modulates the function and/or expression of one or more of the genes identified in Table 4 (and, optionally, Table 7), wherein the test compound is identified as a lead candidate with efficacy in the treatment or prevention of an ALK-positive tumour if it modulates the function and/or expression of one or more of the genes identified in Table 4 (and Table 7).

94. A method according to Claim 93 wherein step (b) is performed as defined in any one of Claims 51 to 85.

95. A method according to Claim 93 or 94 further comprising step (c) of testing the lead candidate for efficacy in an in vivo model of said ALK-positive tumour.

96. A method for the diagnosis, prognosis or treatment of a subject with an ALK-positive tumour substantially as described herein with reference to the Examples.