METHOD FOR PREDICTING RESPONSE OR PROGNOSIS OF LUNG ADENOCARCINOMA WITH EGFR-ACTIVATING MUTATIONS

FIELD OF THE INVENTION

The invention provides a method for predicting the response of an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and a method for predicting prognosis in an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with EGFR-TKI. Particularly, clustered genomic alterations in specific chromosomes are determined as a tool for predicting the response or prognosis in the methods.

BACKGROUND OF THE INVENTION

Lung adenocarcinoma is the predominant type of lung cancer and is the most common cause of cancer deaths worldwide. Among all histological types of lung cancer, adenocarcinoma is the most common and has the greatest heterogeneity.

Treatment of lung adenocarcinoma (such as Non-small-cell lung cancer; NSCLC) has been relatively poor. Chemotherapy, the mainstay treatment of advanced cancers, is only marginally effective, with the exception of localized cancers. While surgery is the most potentially curative therapeutic option for lung adenocarcinoma, it is not always possible depending on the stage of the cancer. Recent approaches for developing anti-cancer drugs to treat the lung adenocarcinoma patients focus on reducing or eliminating the cancer cells' ability to grow and divide. These anti-cancer drugs are used to disrupt the signals which tell the cells to grow or die. Normally, cell growth is tightly controlled by the signals that the cells receive. In cancer, however, this signaling goes wrong and the cells continue to grow and divide in an uncontrollable fashion, thereby forming a tumor. One of these signaling pathways begins when a protein, called epidermal growth factor (EGF), binds to a receptor that is found on the surface of many cells.

EGFR is a member of the type 1 tyrosine kinase family of growth factor receptors, which play a critical role in cellular growth, differentiation and survival. Activation of these receptors typically occurs via specific ligand binding, resulting in hetero- or homodimerization between receptor family members, with subsequent autophosphorylation of the tyrosine kinase domain. Mutations of EGFR are present in a subpopulation of NSCLC patients. EGFR mutation rate is higher in East Asian patients (19-26%) than in those of European or US descent (8-17%). EGFR-mutation mediated phosphorylation can activate downstream anti-apoptotic signal transduction via Akt pathway or proliferative signals via MAPK/ERK pathway. Strikingly, patients with NSCLC harboring these genetic alterations revealed a remarkable response to EGFR-Tyrosine Kinase Inhibitors (TKIs) and the treatment efficacy was confirmed in clinical trials (Maemondo M, et al: Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 362:2380-8, 2010; Lynch T J, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-39, 2004; Paez J G, et al: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497-500, 2004; Mitsudomi T, et al: Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 11:121-8, 2010; Mok T S, et al: Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. N Engl J Med 361:947-957, 2009). High response rate may be due to EGFR mutations within critical residues of the catalytic domain, causing physical structure alteration in drug binding (Yun C H, et al: Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11:217-27, 2007). U.S. Pat. No. 7,932,026 teaches mutations in EGFR and methods of detecting such mutations as well as prognostic methods for identifying a tumor that is susceptible to anticancer therapy such as chemotherapy and/or kinase inhibitor treatment.

Although several studies have established that the EGFR-TKIs are in general more effective for patients with EGFR-activating mutations than EGFR wild-type, the responses are quite heterogeneous even among the EGFR mutant patients (Mok T S, et al: Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. N Engl J Med 361:947-957, 2009). The IPASS study reported that only 71% of patients with EGFR activating mutation responded well to EFGR-TKIs (Mok T S, et al: Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. N Engl J Med 361:947-957, 2009). To identify non-responsive patients, U.S. Pat. No. 7,858,389 provides methods using mass spectral data analysis and a classification algorithm provide an ability to determine whether a non-small-cell lung cancer (NSCLC) patient is likely to benefit from a monoclonal antibody drug targeting an epidermal growth factor receptor pathway. U.S. Pat. No. 7,906,342 provides methods using mass spectral data analysis and a classification algorithm provide an ability to determine whether a non-small-cell lung cancer patient, head and neck squamous cell carcinoma or colorectal cancer patient has likely developed a non-responsiveness to treatment with a drug targeting an epidermal growth factor receptor pathway. However, these prior art references use mass spectrum obtained from a blood sample as the tool for identification and the effects are not satisfactory.

Since the molecular basis of the response heterogeneity is still unknown and no biomarker is available for response prediction, there remains a need for a technique for predicting responsiveness of a lung adenocarcinoma subject receiving EGFR treatment.

SUMMARY OF THE INVENTION

The invention relates to a method for predicting the response of an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), comprising a) providing a sample comprising genomic DNA from said EGFR-activating mutant subject; and b) analyzing said genomic DNA to determine copy number alterations (CNAs) of genes in chromosome 5p, 7p, 8q or 14q of the sample, wherein changes of CNAs in the sample of a) relative to a sample comprising genomic DNA of a EGER wild-type indicate that the EGFR-activating mutant subject has less favorable response to treatment with the EGFR-TKI.

The invention also relates to a method of predicting prognosis in an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with EGFR-TKI, comprising a) providing a sample comprising genomic DNA from said EGFR-activating mutant subject; and b) analyzing said genomic DNA to determine copy number alterations (CNAs) of genes in chromosome 5p, 8q or 14q of the sample, wherein the subject is determined to have poorer prognosis when the CNAs in the sample of a) is changed relative to the CNAs of genes in a sample comprising genomic DNA of an EGFR wild-type.

The invention further relates to a diagnostic kit for determining the response of a EGFR-activating mutant subject suffering from lung adenocarcinoma and receiving treatment with EGFR-TKI, or determining prognosis in an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with EGFR-TKI, comprising one or more probes to the genes in chromosome 5p, 8q or 14q of the sample comprising genomic DNA from said EGFR-activating mutant subject.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Sites of differential CNA found in EGFR-activating mutation status comparisons. The sites of probe-blocks displaying the differential CNA in three comparisons, the EGFR-activating mutant group versus the wild-type group, the L858R mutant group versus the EGFR wild-type group and the exon-19 in-frame deletion group versus EGFR wild-type group are shown on the right side of each chromosome ideogram. A zoom-in version of chromosome 7p is given on the right, along with the locations of some notable genes.

FIG. 2. Representative CNA profiles on chromosome 7p for the EGFR-activating mutation group and the EGFR wild-type group of lung adenocarcinoma.

FIG. 3. The Kaplan-Meier curves for both overall survival and progression-free survival analysis are provided. The clinical variables considered are EGFR mutation status, stage, age, gender and smoking status.

FIG. 4. Survival prediction by DNA copy numbers of six genes from chromosome 7p. (A) Patients are listed in an ascending order from left to right based on the CNA-risk scores. The survival time of each patient is plotted in the top panel. The bottom panel shows the copy numbers of six genes in a heat map. Pale blue dotted line represents the median of CNA-risk score dividing patients into low risk and high risk signature groups. (B) The Kaplan-Meier curves for both overall survival and progression-free survival analyses on EGFR-activating mutation patients are shown. The high and low risk groups are divided evenly based on the CNA-risk scores. (C) Same analysis as (B), applied to the EGFR wild-type group of patients.

FIG. 5. (A) Box plot for CNA-risk score distribution. Significant difference between favorable responders (partial response, 11 cases) and less favorable responders (progressive disease or stable disease, 12 cases) is shown. Two-sided t-test p value is given. (B) EGFR-TKI treatment responsiveness is associated with copy number increase in multiple genes on chromosome 7p. The Fisher exact test p value is given.

DETAILED DESCRIPTION OF THE INVENTION

The invention identifies chromosome regions with differential copy number alterations (CNAs) between the EGFR-activating mutant and EGFR wild-type tumors and found the aberration sites to cluster highly on chromosome 5p, 7p, 8q or 14q. A cluster of chromosome genes predicts the overall and the progression-free survivals for EGFR-activating mutant patients, but not wild-type. Importantly, presence of genes with changed CNA in this cluster correlates with less favorable response to EGFR-TKIs in EGFR-activating mutant patients.

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

As used herein, a “subject” refers to a vertebrate mammal, including, but not limited to, human, mouse, rat, dog, cat, horse, cow, pig, sheep, goat, or non-human primate. In some embodiments, the subject is a human. The terms “subject,” “patient” and “individual” are used interchangeably.

As used herein, a “genome” designates or denotes the complete, single-copy set of genetic instructions for an organism as coded into the DNA of the organism. A genome may be multi-chromosomal so that the DNA is cellularly distributed among a plurality of individual chromosomes. For example, in human there are 22 pairs of chromosomes plus a gender associated XX or XY pair.

As used herein, the “EGFR mutant” or “EGFR mutations” means an amino acid or nucleic acid sequence that differs from wild-type EGFR protein or nucleic acid respectively found on one allele (heterozygous) or both alleles (homozygous) and may be somatic or germ line. In an embodiment, said mutation is an amino acid or nucleic acid substitution, deletion or insertion.

As used herein, the “chromosome” refers to the heredity-bearing gene carrier of a living cell which is derived from chromatin and which comprises DNA and protein components (especially histones). The conventional and internationally recognized individual human genome chromosome numbering system is employed herein. The size of an individual chromosome can vary from one type to another with a given multi-chromosomal genome and from one genome to another.

As used herein, the “chromosomal region” is a portion of a chromosome. The actual physical size or extent of any individual chromosomal region can vary greatly. The term “region” is not necessarily definitive of a particular one or more genes because a region need not take into specific account the particular coding segments (exons) of an individual gene.

As used herein, the “copy number” of a nucleic acid refers to the number of discrete instances of that nucleic acid in a given sample.

As used herein, the “copy number alteration” refers to a variation in the number of copies of a gene or genetic region that is present in the genome of a cell. A normal diploid cell will typically have two copies of each chromosome and the genes contained therein. Copy number alterations may increase the number of copies, or decrease the number of copies.

As used herein, “copy number profile” means a collection of data representing the number of copies of genomic DNA at a plurality of genomic loci for a given sample. For instance, for three genomic loci of interest, a copy number profile represents the number of copies of DNA for the three genomic loci. In this context, “genomic locus” means a location within the genome of a cell and usually encompasses a stretch of genomic DNA between two points in the genome of a cell. This stretch of genomic DNA consists of a nucleotide sequence.

As used herein, the “prognosis” is meant response and/or benefit and/or survival.

In one aspect, the invention provides a method for predicting the response of an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), comprising a) providing a sample comprising genomic DNA from said EGFR-activating mutant subject; and b) analyzing said genomic DNA to determine copy number alterations (CNAs) of genes in chromosome 5p, 7p, 8q or 14q of the sample, wherein changes of CNAs in the sample of a) relative to a sample comprising genomic DNA of an EGFR wild-type indicates that the EGFR-activating mutant subject has less favorable response to treatment with the EGFR-TKI.

In another aspect, the invention provides a method of predicting prognosis in a EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with EGFR-TKI, comprising a) providing a sample comprising genomic DNA from said EGFR-activating mutant subject; and b) analyzing said genomic DNA to determine copy number alterations (CNAs) of genes in chromosome 5p, 7p, 8q or 14q of the sample, wherein the subject is determined to have poorer prognosis when the CNAs in the sample of a) change relative to the CNAs of genes in a sample comprising genomic DNA of an EGFR wild-type.

In a further aspect, the invention provides a diagnostic kit for determining the response of an EGFR-activating mutant subject suffering from lung adenocarcinoma and receiving treatment with EGFR-TKI, or determining prognosis in an EGFR-activating mutant subject suffering from a lung adenocarcinoma and receiving treatment with EGFR-TKI, comprising one or more probes to the genes in chromosome 5p, 7p, 8q or 14q of the sample comprising genomic DNA from said EGFR-activating mutant subject. The kits can additionally include instructional materials describing when and how to use the kit contents. The kits can also include one or more of the following: various labels or labeling agents to facilitate the detection of the probes, reagents for the hybridization including buffers, a metaphase spread, bovine serum albumin (BSA) and other blocking agents, sampling devices including fine needles, swabs, aspirators and the like, positive and negative hybridization controls and so forth.

According to the invention, EGFR tyrosine kinase inhibitors bind the ATP binding pocket of the EGFR receptor and prevent ATP from binding. As a result, binding of the inhibitor results in the suppression of EGFR mediated intracellular signaling. EGFR tyrosine kinase inhibitors include both reversible and irreversible inhibitors. Most reversible inhibitors are based on quinazolines and include, but are not limited to, gefitinib (Iressa; N-(3-Chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazo-lin-4-amine), erlotinib (Tarceva; N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine) and lapatinib (Tykerb, GW572016; N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonyleth-ylamino)methyl]-2-furyl]quinazolin-4-amine) Irreversible inhibitors permanently modify the tyrosine kinase domain of EGFR, thereby suppressing EGFR signaling. Irreversible inhibitors include, but are not limited to, CI-1033, EKB-569 and HKI-272 (See e.g., Zhang et al., 2007, JCI 117: 2051-2058). The binding of an EGFR-TKI to EGFR leads to the induction of apoptosis of the cell expressing the EGFR, thereby providing a method for cancer treatment. It should be appreciated that the terms EGFR tyrosine kinase inhibitor and EGFR kinase inhibitor are used interchangeably herein.

According to one embodiment of the invention, the lung adenocarcinoma is NSCLC.

According to the invention, the copy number alterations (CNAs) of genes change in chromosome 5p, 7p, 8q or 14q of the sample comprising genomic DNA from an EGFR-activating mutant subject. Preferably, the CNAs change in the chromosome 7p. More preferably, the CNAs change in the chromosome 7p11.2, 7p14.1, 7p15.2, 7p15.3, 8q11.21 or 8q11.23. More preferably, the CNAs change in one or more of the following representative genes, EGFR, LANCL2, VSTM2A, VOPP1, SEC61G, SEPT14 and HPVC1 located at the chromosome 7p11.2, GLI3 and C7orf10 located at the chromosome 7p14.1, NFE2L3, MIR148A and OSBPL3 located at the chromosome 7p15.2, NPY located at the chromosome 7p15.3, SDK1 located at the chromosome 7p22.2, ANK1 located at the chromosome 8p11.21 and ADAM3A located at the chromosome 8p11.23. Most preferably, the CNAs change in one or more of the six representative genes, GLI3, NFE2L3, SDK1, EGFR, VOPP1 and LANCL2 located at the chromosome 7p14.1, 7p15.2, 7p22.2, 7p11.2, 7p11.2 and 7p11.2, respectively.

In one embodiment of the invention, the changes of CNAs are DNA gain in chromosome 5p, 7p or 14q and DNA loss in chromosome 8q.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

Nucleic acid hybridization assays for the detection of target region sequences, for quantifying copy number, for sequencing, and the like, can be performed in an array-based format (such as comparative genomic hybridization (Cgh) using nucleic acid arrays). Arrays are a multiplicity of different “probe” or “target” nucleic acids (or other compounds) hybridized with a sample nucleic acid. In an array format a large number of different hybridization reactions can be run in parallel. This provides rapid, essentially simultaneous, evaluation of a large number of loci.

The nucleic acid probes are fixed to a solid surface in an array. These probes comprise portions of the target regions of the invention, optionally in combination with probes from other portions of the genome. Probes can be obtained from any convenient source, including MACs, YACs, BACs, PACs, cosmids, plasmids, inter-Alu PCR products of genomic clones, restriction digests of genomic clones, CDNA clones, amplification products, and the like. The arrays can be hybridized with a single population of sample nucleic acid or can be used with two differentially labeled collections, for example a test sample and a reference sample.

Many methods for immobilizing nucleic acids on a variety of solid surfaces are known in the art. A wide variety of organic and inorganic polymers, as well as other materials, both natural and synthetic, can be employed as the material for the solid surface. Illustrative solid surfaces include, e.g. nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, and cellulose acetate. In addition, plastics such as polyethylene, polypropylene, polystyrene, and the like can be used. Other materials which may be employed include paper, ceramics, metals, metalloids, semiconductive materials, cermets or the like. In addition, substances that form gels can be used. Such materials include proteins, lipopolysaccharides, silicates, agarose and polyacrylamides. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, proteins such as casein or BSA or mixtures of macromolecules can be employed to avoid non-specific binding, simplify covalent conjugation, enhance signal detection or the like. If the probe is to be covalently bound, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. For example, methods for immobilizing nucleic acids by introduction of various functional groups to the molecules are known. Covalent attachment of the target nucleic acids to glass or synthetic fused silica can be accomplished according to a number of known techniques and commercially available reagents. For instance, materials for preparation of silanized glass with a number of functional groups are commercially available or can be prepared using standard techniques. Quartz cover slips, which have at least 10-fold lower autofluorescence than glass, can also be silanized.

Alternatively, probes can also be immobilized on commercially available coated beads or other surfaces. For instance, biotin end-labeled nucleic acids can be bound to commercially available avidin-coated beads. Streptavidin or anti-digoxigenin antibody can also be attached to silanized glass slides by protein-mediated coupling. Hybridization to nucleic acids attached to beads is accomplished by suspending them in the hybridization mix, and then depositing them on a substrate for analysis after washing, or analyzing by flow cytometry.

Comparative genomic hybridization (CGH) can detect and map DNA sequence copy number variation throughout the entire genome in a single experiment. In one variation of CGH, the genome is provided as a cytogenetic map through the use of metaphase chromosomes. Alternatively hybridization probes are arrays of genomic sequences containing the target region sequences of the invention, optionally also including other genomic probes. Relative copy number can also be measured by hybridization of fluorescently labeled test and reference nucleic acids in both metaphase chromosome-based and array-based CGH.

In metaphase chromosome-based CGH total genomic DNA is isolated from a sample of a subject, labeled with different fluorochromes, and hybridized to normal metaphase chromosomes. Cot-1 DNA is used to suppress hybridization of repetitive sequences. The resulting ratio of the fluorescence intensities of the two fluorochromes at a location on a chromosome is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. Thus, CGH provides genome-wide copy number analysis referenced to the cytogenetic map provided by the metaphase chromosomes. However, the use of metaphase chromosome CGH limits the resolution to 10-20 megabases (Mb), prohibits resolution of closely spaced aberrations, and only allows linkage of CGH results to genomic information and resources with cytogenetic accuracy.

Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal, for example antibody-antigen or complementary nucleic acid binding. The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or radioactive label or enzymatic molecule to the antibodies. The sensitivity of the hybridization assays can be enhanced through use of a target nucleic acid or signal amplification system that multiplies the target nucleic acid or signal being detected. Alternatively, sequences can be generally amplified using nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.

Various other technologies may also be used for determining copy number. In some embodiments, the method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number using real-time PCR. Progress in the PCR reactions is monitored using fluorigenic probes and a real-time fluorescence detection system. For each reaction, the number of cycles is measured at which a defined threshold fluorescence emission is reached. Using standard curves, the copy number of the test DNA relative to a common standard DNA is determined for each locus. From the ratio of the relative copy numbers, the genomic copy number of the test locus is determined (see Wilke et al. (2000) Hum Mutat 16:431-436).

The results provided in the invention shed light on why among patients with EGFR mutation, responses to the EGFR TKI-targeted therapy are heterogeneous. This may lead to a better patient management for EGFR-mutant patients. The invention provides data to highlight chromosome 5p, 7p, 8q or 14q as the main chromosome arm enriched in notable sites of DNA copy number alterations for lung adenocarcinoma, so it is an effective predictor for both overall survival and progression-free survival of EGFR mutant patients. In this connection, chromosome 7p is the preferred embodiment. Furthermore, the invention shows that six qPCR-validated genes from chromosome 7p yield a copy-number based risk score which is an effective predictor for both overall survival and progression-free survival of EGFR mutant patients, independent of cancer staging. Yet for the EGFR wild-type patients, the invention also shows that the same signature is uncorrelated with both the overall survival and progression-free survival. This sharp contrast strongly supports the useful notion of using EGFR-mutation status to define subtypes of adenocarcinoma.

To a clinician treating the lung cancer patients, differences in the patients' ethnic and pharmacogenomic backgrounds are important factors that may heavily influence the decision in the individualized therapy. The genetic alterations clustered in chromosome 5p, 7p, 8q or 14q region (in particular chromosome 7p) that the invention identified may play a crucial role and the risk score derived from these genetic alterations may determine whether the patient will have a favorable response to EGFR-TKI therapy. The invention provides clues to why patients with EGFR-activating mutation may still have heterogeneous response to EGFR-TKI targeted therapy. The finding may also be useful for clinician to make better prediction for the treatment response. The invention also suggests that in patients with EGFR driver gene mutation, the chromosome 5p, 7p, 8q or 14q region (in particular chromosome 7p) is more vulnerable to damage by carcinogen.

EXAMPLE

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the invention. The following experimental examples are provided in order to demonstrate and further illustrate various aspects of certain embodiments of the present invention and are not to be construed as limiting the scope thereof. In the experimental disclosure which follows, the following materials and methods are used:

Patients and Methods

The 138 cancer tissues for array CGH assay were obtained from National Taiwan University Hospital (NTUH) and Taichung Veterans General Hospital (TCVGH). The 114 cancer tissues for clinical outcome prediction by genomic real-time qPCR were obtained from TCVGH. There were no overlaps between these two groups of patients. After surgical operation, the dissected tissues from lung adenocarcinoma patients were stored in the liquid nitrogen immediately and anonymized. Following the standard protocol, the genomic DNA was extracted from cancer tissue of each sample with quality checked by agarose electrophoresis.

Tumor DNA from 25 EGFR mutant (exon-19 deletion and L858R) patients of TCVGH for EGFR-TKI was obtained for treatment response study. One squamous cell carcinoma patient and one patient with insufficient information were deleted. For the remaining 23 patients, three types of responses were evaluated by physicians according to the guideline of RECIST 1.0:1. partial response (PR), 2. progressive disease (PD), 3. stable disease (SD) (P. Therasse S G A, E. A. Eisenhauer: New Guidelines to Evaluate the Response to Treatment in Solid Tumors (RECIST Guidelines). Journal of the National Cancer Institute 92:205-216, 2000).

Array Comparative Genomic Hybridization (CGH)

The whole genome NimbleGen CGH array (NimbleGen®; NimbleGen Systems Inc, Madison, Wis.) containing 385,806 probes with probe spacing of about 6,000 bp, was used for comparative genomic hybridization of DNA from cancer tissues against normal DNA extracted from the PBMC of one male and one female in a community cohort. Digital sonifier (Branson Model#450, Branson, Danbury, Conn.) was used for the DNA fragmentation. Labeling, hybridization and washing were processed according to the manufacturer's protocol. The array scanning and image generation were performed by the GenePix™ Reader (Personal 4000B, Axon Instruments, Molecular Devices, Sunnyvale, Calif.) and GenePix® Pro 6.0 software. Generation of log intensity ratio data with normalization was performed by NimbleScan™ version 2.4, SignalMap™ version 1.9 software, followed by applying cross-chip normalization. The original CNA dataset in dot pair format can be accessed at http://kiefer.stat2.sinica.edu.tw/cghdata/.

Genomic Real-Time Quantitative PCR (q-PCR)

qPCR has been established as a rapid and sensitive technique for accurate quantification of DNA in tissues. The fluorescence emitted by the reporter dye was detected on-line in real-time using the ABI prism 7900 sequence detection system (Applied Biosystem, Foster City, Calif.). The primers and probes of qPCR were designed based on 500 franking nucleotide sequences (250 upstream and 250 downstream nucleotides) of the probe location of array CGH. The sequences of primers and probes were given in Table 1.

TABLE 1

The probes and primers used for genomic real-time qPCR

Gene

Forward primer

Reverse primer

Probe

EGFR

AGGCGGCTCTCT

CTCCTCCTCTGTTG

TTGCTGCTGCTCTTTC

TCTCTCA

AAATGGATTCT

(SEQ ID NO: 57)

(SEQ ID NO: 1)

(SEQ ID NO: 2)

DGKB

TTGTTCTAAGTAC

CCCAGGTCTCCTA

TTGTCTGCTGAATTTT

CATATATAACAGA

CTTGTTGTTACT

(SEQ ID NO: 58)

ATGTTTAAATATC

(SEQ ID NO: 4)

CC

(SEQ ID NO: 3)

MEOX2

TGACTGGTGTTT

TACTCATGCATTTT

ATGCCACGTACATTTT

ACAAAAGATATTG

GAATACTCTCATTA

(SEQ ID NO: 59)

TGACA

AGTAA

(SEQ ID NO: 5)

(SEQ ID NO: 6)

ERBB2

TGTTGGTGGCTG

GGGTCTGAATCCA

CCCACAGGGCTCACC

TGACTGT

GGTAGTCTGA

(SEQ ID NO: 60)

(SEQ ID NO: 7)

(SEQ ID NO: 8)

ACTN4

GGAATGGTTTTG

TGGCACATGTTTGT

CCCTCACTGGTTCTCC

ACTCGGACTCA

CACTGTCT

(SEQ ID NO: 61)

(SEQ ID NO: 9)

(SEQ ID NO: 10)

FAM102A

GCCCACACTCCC

GCGAAGCCCAGCT

CCCACACGCTCCTCTC

TCAGC

AGGA

(SEQ ID NO: 62)

(SEQ ID NO: 11)

(SEQ ID NO: 12)

MEOX1

CCACAAATAG

CCTGGGTGTGGCT

TCCCTCCTGATGCCCC

GCCTCTCTCC

TCGT

(SEQ ID NO: 63)

TCTT

(SEQ ID NO: 14)

(SEQ ID NO: 13)

VAV3

GGTTTAAAGC

AGGAAGCTACACT

CAGGTGTCCAAATTC

TCAGTTTCAG

GAGAGTTGTGA

(SEQ ID NO: 64)

CGTTT

(SEQ ID NO: 16)

(SEQ ID NO: 15)

C10ORF130

GCTGTAAGTACA

AATGAATAGTAGTG

CTCCTGCTAAAATTTC

AAGTTATTTGATT

CTGCACAT CCA

(SEQ ID NO: 65)

TGTAGTGT

(SEQ ID NO: 18)

(SEQ ID NO: 17)

ERBB3

ACAGTGATAGCA

GCCCACGCCAGTA

ATGCTGGGCGGCACTT

GGATTGGTAGTG A

GAGAA

(SEQ ID NO: 66)

(SEQ ID NO: 19)

(SEQ ID NO: 20)

C18ORF26

AGCAGGGCCAGA

CCATTCAATAAATA

CTGCCACGGAAGTAT

TAATGTTTATGAT

CTGAGCCAGGAGT

(SEQ ID NO: 67)

AAT

A

(SEQ ID NO: 21)

(SEQ ID NO: 22)

VAV1

TCCTGCCCTGAG

CTTATCCTCCCAGC

CCAGCCTGAGGACCAG

GTCTGA

TCTTCATCTG

(SEQ ID NO: 68)

(SEQ ID NO: 23)

(SEQ ID NO: 24)

MYO3B

CCATTTGATGGT

AGAATGTGACCATA

AAGGCCAGAAAATCAC

CATGAGACTAAT

ACTATCGACTGAGT

(SEQ ID NO: 69)

GTTATCT

(SEQ ID NO: 26)

(SEQ ID NO: 25)

ERBB4 (1)

AATGCTTATCTTT

GGATATATTTATAC

TAGGCATCCCAAGCTC

CTGGTCATGAGT

ATGATAAAAACAGT

(SEQ ID NO: 70)

CTT

AGTGGTTCCTAA

(SEQ ID NO: 27)

(SEQ ID NO: 28)

ERBB4 (2)

CCTTGTTGCTTTT

CTTGCCCAAGATCA

CAATCAGCCCAAATTT

GATACACTCTCAT

CATGTCTAGTA

(SEQ ID NO: 71)

(SEQ ID NO: 29)

(SEQ ID NO: 30)

COX7B2

AGAAGTGGACAT

TTCCCACTGCACAA

CCCAGCCTGAATTAG

AAGGCCTTTAGT G

GCATACA

(SEQ ID NO: 72)

(SEQ ID NO: 31)

(SEQ ID NO: 32)

CDH12

CTTTTTTTTTCTA

TCAGAAATAGCATA

ATGGCAGGCACTAAAC

AGGTAAAATTAGT

TGTTTTTGGAGGCT

(SEQ ID NO: 73)

AACACATTTATTT

(SEQ ID NO: 34)

GGG

(SEQ ID NO: 33)

VAV2

CAAAAGTGACAC

GCTGTTCGTGTCG

CAGCCCGTCATTCGCA

TTACCCCAATTAC

TCTCCTT

(SEQ ID NO: 74)

AG

(SEQ ID NO: 36)

(SEQ ID NO: 35)

CDH1

GACAGGGCTTTA

GCACACGCCCTGA

CCCCTCCTCCCTTCTC

TGTATTAGCCAC A

GAACA

(SEQ ID NO: 75)

(SEQ ID NO: 37)

(SEQ ID NO: 38)

TWIST1

GCGCTGCGGAAG

GCTTGAGGGTCTG

CCCTCGGACAAGCTG

ATCATC

AATCTTGCT

(SEQ ID NO: 76)

(SEQ ID NO: 39)

(SEQ ID NO: 40)

TWISTNB

ACATGGGTGATG

TGTGATATTTAGTT

ATGCTGCTGGAGTATTC

AACTAGAATTTGA

TTCCCCGAATGCA

(SEQ ID NO: 77)

AGT

(SEQ ID NO: 42)

(SEQ ID NO: 41)

RPL15

AGATTGGTAAGC

CGTCTAAGCTCACA

CTCACCAGCTTCCC

TAGCAATGAATG CT

CTTGAAAGGTA

(SEQ ID NO: 78)

(SEQ ID NO: 43)

(SEQ ID NO: 44)

TFRC1

ACTTACTACACCT

AACATTTTAAGCAC

TCTTCTTGTGTCAACTTTG

GGCCATGGA

TGCAGTAAATTTGG

(SEQ ID NO: 79)

(SEQ ID NO: 45)

T

(SEQ ID NO: 46)

SDK1

TGCTGGACACTT

GAGAGGACTTCCT

CCTCCGTATACTTTCTATCCC

TCACTTGGAA

AGGGAACTTAGG

(SEQ ID NO: 80)

(SEQ ID NO: 47)

(SEQ ID NO: 48)

GLI3

AGTTTGGGAAGC

TCACCTTCTGATGA 

CTGAGCACATTTATACAGATG

CCTCCTCTAA

ACACTTTTCTGT

(SEQ ID NO: 81)

(SEQ ID NO: 49)

(SEQ ID NO: 50)

LANCL2

GCCTCAGTGGGA

CATGCCTTTATTCC

CCTGCCCGCTCTGC

ACTTCTGT

CAGCTTCTC

(SEQ ID NO: 82)

(SEQ ID NO: 51)

(SEQ ID NO: 52)

VOPP1

AGGAAACCTTCA

CCTTGAGCAGAGA

TCACACTGGAGAGGCC

GGAGCAACTC

CGTCTTTCA

(SEQ ID NO: 83)

(SEQ ID NO: 53)

(SEQ ID NO: 54)

NFE2L3

GCCCCTGGTGCG

CCAAGTGCCTCAA

TTCTGTGGCAGCCAGCTG

ACA

AGTTGCA

(SEQ ID NO: 84)

(SEQ ID NO: 55)

(SEQ ID NO: 56)

Statistical Analyses

The aCGH data were first preprocessed by averaging 10 consecutively located probes to form 36,549 disjoint blocks. A two-step statistical procedure to determine sites of amplification or deletion with high frequency of occurrence was applied. T-test to determine the DNA gain or loss status of each probe-block for each sample separately was first used and then collectively, a block as a gain-block (or loss-block) if at least 30% of the 138 samples showed gains (or losses) was claimed. To determine gain or loss status of a block, the two-sided t-test (5% significance) was used. Statistical calculation indicated that a gain/loss block claimed at 30% threshold were very unlikely to have a true prevalence less than 25% (p-value=0.0047). For comparative CNA analysis with respect to EGFR mutation status, the t-test (two-sided, 5% significance) was applied to compare two group means. Both univariate and multivariate Cox regression models were applied for prediction of patients' survival. The software, MetaCore™, was used for functional enrichment analysis. The representative CNA profile on chromosome 7p was derived by the weighted singular value decomposition method.

Example 1

CNA Profiling Results

CNA profiling on 138 tumors of lung adenocarcinoma was conducted by the array CGH of NimbleGen system. The resulting CNA profiles were shown in FIG. 1A. The statistical analysis detected a total of 3,187 probe-blocks of DNA-gain and 6,029 probe-blocks of DNA-loss with false discovery rates of 0.054 and 0.028 respectively.

Example 2

Chromosome 7p has Highest Rate of DNA-Gain for the Gene-Harboring Regions

The chromosome sites with DNA gains were examined first. It was found that relative to the arm size, chromosome 5p, 7p, and 8q had the largest region of DNA-gain (Table 2). For the gene-harboring region, the gain rate for chromosome 7p turned out the highest. Significantly, EGFR was in the list, along with other notable genes like HDAC9, DGKB, MEOX2 and POU6F2, all of which were within the top 1% genome-wide when ranking the probe-blocks according to their average CNA values across all 138 samples (Table 3).

TABLE 2

Chromosome wide DNA gain/loss percentages

Number of

Number of

Number of

Number of

gene-haboring

Number of

gene-haboring

probe-

gain probe-

gain probe-

loss probe-

loss probe-

Chromosome

blocks

blocks (%)

blocks (%)

blocks (%)

blocks (%)

 1p

1624

144 (8.9%)

 59 (3.6%)

 316 (19.5%)

 269 (16.6%)

 1q

1398

 213 (15.2%)

100 (7.2%)

103 (7.4%)

 89 (6.4%)

 2p

1252

100 (8%) 

 26 (2.1%)

 148 (11.8%)

107 (8.5%)

 2q

2038

 241 (10.5%)

111 (5.4%) 

185 (9.1%)

152 (7.5%)

 3p

1267

51 (4%) 

 20 (1.6%)

 213 (16.8%)

 179 (14.1%)

 3q

1458

 158 (10.8%)

 52 (3.6%)

101 (6.9%)

 80 (5.5%)

 4p

684

  84 (12.3%)

 19 (2.8%)

  95 (13.9%)

  79 (11.5%)

 4q

1910

 253 (13.2%)

 88 (4.6%)

 36 (1.9%)

 28 (1.5%)

 5p

639

 193 (30.2%)

 56 (8.8%)

13 (2%) 

 10 (1.6%)

 5q

1783

 182 (10.2%)

 50 (2.8%)

148 (8.3%)

116 (6.5%)

 6p

812

 48 (5.9%)

 25 (3.1%)

 115 (14.2%)

89 (11%)

 6q

1501

119 (7.9%)

 47 (3.1%)

 87 (5.8%)

 66 (4.4%)

 7p

783

 213 (27.2%)

  89 (11.4%)

 52 (6.6%)

 44 (5.6%)

 7q

1271

 187 (14.7%)

 87 (6.8%)

 161 (12.7%)

 129 (10.1%)

 8p

594

  7 (1.2%)

  2 (0.3%)

 168 (28.3%)

 123 (20.7%)

 8q

1393

 275 (19.7%)

102 (7.3%)

 69 (4.9%)

 51 (3.7%)

 9p

541

 25 (4.6%)

  9 (1.7%)

  70 (12.9%)

  61 (11.3%)

 9q

975

 17 (1.7%)

  3 (0.3%)

 307 (31.5%)

 252 (25.8%)

10p

537

  1 (0.2%)

0 (0%)

  96 (17.9%)

75 (14%)

10q

1245

 27 (2.2%)

  5 (0.4%)

 296 (23.8%)

237 (19%) 

11p

693

  72 (10.4%)

21 (3%) 

  86 (12.4%)

  78 (11.3%)

11q

1097

 84 (7.7%)

 38 (3.5%)

198 (18%) 

 172 (15.7%)

12p

474

 25 (5.3%)

 16 (3.4%)

57 (12%)

52 (11%)

12q

1325

 99 (7.5%)

 48 (3.6%)

 272 (20.5%)

 241 (18.2%)

13q

1354

 85 (6.3%)

 17 (1.3%)

 147 (10.9%)

 116 (8.6%) 

14q

1213

 143 (11.8%)

 33 (2.7%)

 160 (13.2%)

 136 (11.2%)

15q

1073

 18 (1.7%)

  9 (0.8%)

 309 (28.8%)

 244 (22.7%)

16p

406

  3 (0.7%)

0 (0%)

 143 (35.2%)

 131 (32.3%)

16q

614

12 (2%) 

  2 (0.3%)

 191 (31.1%)

 160 (26.1%)

17p

277

0 (0%)

0 (0%)

191 (69%) 

 163 (58.8%)

17q

725

22 (3%) 

  6 (0.8%)

 275 (37.9%)

 247 (34.1%)

18p

204

2 (1%)

  1 (0.5%)

  30 (14.7%)

  24 (11.8%)

18q

864

 45 (5.2%)

 18 (2.1%)

121 (14%) 

  89 (10.3%)

19p

287

3 (1%)

0 (0%)

 210 (73.2%)

 200 (69.7%)

19q

395

4 (1%)

0 (0%)

 246 (62.3%)

229 (58%) 

20p

372

 13 (3.5%)

  6 (1.6%)

  54 (14.5%)

  49 (13.2%)

20q

468

  1 (0.2%)

0 (0%)

 200 (42.7%)

 163 (34.8%)

21q

469

 45 (9.6%)

 13 (2.8%)

  82 (17.5%)

  71 (15.1%)

22q

444

0 (0%)

0 (0%)

 279 (62.8%)

 245 (55.2%)

TABLE 3

Top 1% probe blocks with highest DNA gain in 138 lung adenocarcinomas.

Gene

Cytoband

Gene name

CNA Mean

PAPP2B

1p32.2

phosphatidic acid phosphatase type 2B

0.2115

LRRIQ3

1p31.1

leucine-rich repeats and IQ motif containing 3

0.2119

Cforf173

1p31.1

chromosome 1 open reading frame 173

0.1921

Cforf173

1p31.1

chromosome 1 open reading frame 173

0.2329

TTLL7

1p31.1

tubulin tyrosine ligase-like family, member 7

0.2106

PKN2

1p22.2

protein kinase N2

0.2316

PKN2

1p22.2

protein kinase N2

0.2033

SNX7

1p21.3

sorting nexin 7

0.2149

OLFM3

1p21.1

olfactomedin 3

0.2239

OLFM3

1p21.1

olfactomedin 3

0.1962

AMY2A

1p21.1

amylase, alpha 2A (pancreatic)

0.1997

LOC100129138

1p21.1

THAP domain containing, apoptosis associated protein 3

0.1978

pseudogene

PRMT6

1p13.3

protein arginine methyltransferase 6

0.2609

PRMT6

1p12.3

protein arginine methyltransferase 6

0.2221

RPTN

1q21.3

repetin

0.1932

FLG

1q21.3

filaggrin

0.2912

NUF2

1q23.3

NUF2, NDC80 kinetochore complex component homolog

0.0078

(S. cerevisiae)

PBX1

1q23.3

pre-B-cell leukemia homebox 1

0.2012

DNM3

1q24.3

dynamin 3

0.2498

TNFSF18

1q25.1

tumor necrosis factor (ligand) superfamily, member 18

0.1984

TNR

1q25.1

tenascin R (restrictin, janusin)

0.1914

FAM5B

1q25.2

family with sequence similarity 5, member B

0.2206

HMCN1

1q31.1

hemicentin 1

0.2213

HMCN1

1q31.1

hemicentin 1

0.2415

TPR

1q31.1

translocated promoter region (to activaated MET oncogene)

0.1943

PLA2G4A

1q31.1

phospholipase A2, group IVA (cytosolic, calcium-dependent)

0.1958

PLA2G4A

1q31.1

phospholipase A2, group IVA (cytosolic, calcium-dependent)

0.1954

FAM5C

1q31.1

family with sequence similarity 5, memeber C

0.2118

FAM5C

1q31.1

family with sequence similarity 5, memeber C

0.2207

FAM5C

1q31.1

family with sequence similarity 5, memeber C

0.1915

LOC440704

1q31.2

hypothetical LOC440704

0.2018

RGS18

1q31.2

regulator of G-protein signaling 18

0.1955

RGS0

1q31.2

regulator of G-protein signaling 21

0.2464

CDC73

1q31.3

cell division cycle 73, Paf1/RNA polymerase II

0.235

complex component, homolog (S. cerevisiae)

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.2064

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.2111

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.1964

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.281

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.2141

KCNT2

1q31.3

potassium channel, subfamily T, member 2

0.196

CFH

1q31.3

complement factor H

0.2026

CRB1

1q31.3

crumbs homolog 1 (Drosophila)

0.2087

CRB1

1q31.3

crumbs homolog 1 (Drosophila)

0.2639

LHX9

1q31.3

LIM homebox 9

0.1966

MIR181A1

1q31.3

microRNA 181a-1

0.2004

CAMK1G

1q32.2

calcium/calmodulin-dependent protein kinase IG

0.2402

USH2A

1q41

Usher syndrome 2A (autosomal recessive, mild)

0.2201

USH2A

1q41

Usher syndrome 2A (autosomal recessive, mild)

0.2072

ESRRG

1q41

estrogen-related receptor gamma

0.246

ESRRG

1q41

estrogen-related receptor gamma

0.207

ESRRG

1q41

estrogen-related receptor gamma

0.1996

LYPLAL1

1q41

lysophospholipase-like 1

0.215

SMYD3

1q44

SET and MYND domain containing 3

0.2474

APOB

2p24.1

apolipoprotein B (including Ag(x) antigen)

0.2063

APOB

2p24.1

apolipoprotein B (including Ag(x) antigen)

0.2287

KLHL29

2p24.1

kelch-like 29 (Drosophila)

0.2171

SLC8A1

2p22.1

solute carrier family 8 (sodium/calcium exchanger), member 1

0.2023

SLC8A1

2p22.1

solute carrier family 8 (sodium/calcium exchanger), member 1

0.2092

NRXN1

2p16.3

neurexin 1

0.2886

ASB3

2p16.2

ankyrin repeat and SOCS box-containing 3

0.2016

CCDC85A

2p16.1

coiled-coil domain containing 85A

0.1919

FLJ30638

2p16.1

hypothetical LOC400955

0.2919

LRRTM4

2p12

leucine rich repeat transmembrane neuronal 4

0.1982

CTNNA2

2p12

catenin (cadherin-associated protein), alpha 2

0.2143

DPP10

2q14.1

dipeptidyl-peptidase 10 (non-functional)

0.2505

DPP10

2q14.1

dipeptidyl-peptidase 10 (non-functional)

0.1937

DPP10

2q14.1

dipeptidyl-peptidase 10 (non-functional)

0.2183

LRP1B

2q22.1

low density lipoprotein receptor-related protein 1B

0.2224

LRP1B

2q22.2

low density lipoprotein receptor-related protein 1B

0.2049

ARHGAP15

2q22.2

Rho GTPase activating protein 15

0.1992

DKFZp686O1327

2q22.3

hypothetical LOC401014

0.2066

KCNJ3

2q24.1

potassium, inwardly-rectifying channel, subfamily J, member 3

0.2355

DPP4

2q24.2

dipeptidyl-peptidase 4

0.1975

GRB14

2q24.3

growth factor receptor-bound protein 14

0.2435

SCN1A

2q24.3

sodium channel, voltage-gated, type I, alpha subunit

0.2394

XIRP2

2q24.3

xin actin-binding repeat containing 2

0.1977

TTN

2q31.2

titin

0.2498

UBE2E3

2q31.3

ubiquitin-conjugation enzyme E2E 3(UBC4/5 homolog, yeast)

0.2315

UBE2E3

2q31.3

ubiquitin-conjugation enzyme E2E 3(UBC4/5 homolog, yeast)

0.1988

ZNF804A

2q32.1

zinc finger protein 804A

0.1947

ZNF804A

2q32.1

zinc finger protein 804A

0.1966

SLC39A10

2q32.3

solute carrier family 39 (zinc transporter), memeber 10

0.1953

PLCL1

2q33.1

phospholipase C-like 1

0.2125

SATB2

2q33.1

SATB homebx 2

0.2014

ERBB4

2q34

v-erb-a erythroblastic luekemia viral oncogene homolog 4 (avian)

0.2414

ZNF385D

3p24.3

zinc finger protein 385D

0.2016

GADL1

3p23

glutamate decarboxylase-like 1

0.2252

EPHA3

3p11.1

EPH receptor A3

0.1977

ABI3BP

3q12.2

ABI, member 3 (NESH) binding protein

0.2059

ZPLD1

3q12.3

zona pellucida-like domain containing 1

0.2492

ZPLD1

3q13.11

zona pellucida-like domain containing 1

0.2067

PVRL3

3q13.13

poliovirus receptor-related 3

0.2266

C3orf58

3q24

chromosome 3 open reading frame 58

0.1935

C3orf58

3q24

chromosome 3 open reading frame 58

0.2105

PLOD2

3q24

procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2

0.2748

SI

3q26.1

sucrase-isomaltase (alpha-glucosidase)

0.2224

BCHE

3q26.1

butyrylcholinesterase

0.2615

LOC646168

3q26.1

hypothetical protein LOC646168

0.2086

MECOM

3q26.2

MDS1 and EVI1 complex locus

0.1927

FGF12

3q28

fibroblast growth factor 12

0.2441

PCDH7

4p15.1

protocadherin 7

0.2145

PCDH7

4p15.1

protocadherin 7

0.1924

ARAP2

4p15.1

ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2

0.222

ARAP2

4p14

ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2

0.2042

GNPDA2

4p13

glucosamine-6-phosphate deaminase 2

0.1935

EPHA5

4q13.1

EPH receptor A5

0.2336

ADAMTS3

4q13.3

ADAM metallopeptidase with throbospondin type 1 motif, 3

0.2106

AREG

4q13.3

amphiregulin

0.1974

GDEP

4q21.21

gene differentially expressed in prostate

0.2004

PDHA2

4q22.3

pyruvate dehydrogenase (lipoamide) alpha 2

0.2965

C4orf37

4q22.3

chromosome 4 open reading frame 37

0.2313

PITX2

4q25

paired-like homeodomain 2

0.1995

TRAM1L1

4q26

translocation associated membrane protein 1-like 1

0.1978

C4orf33

4q28.2

chromosome 4 open reading frame 33

0.1988

PCDH18

4q28.3

protocadherin 18

0.2112

GRIA2

4q32.1

glutamate receptor, ionotropic, AMPA 2

0.2003

LOC285501

4q34.3

hypothetical protein LOC285501

0.2217

LOC340094

5p15.32

hypothetical LOC340094

0.1976

LOC285692

5p15.2

hypothetical LOC285692

0.2127

CTNND2

5p15.2

catenin (cadherin-associated protein), delta 2

0.2152

(neural plakophilin-related arm-repeat protein)

CTNND2

5p15.2

catenin (cadherin-associated protein), delta 2

0.2293

(neural plakophilin-related arm-repeat protein)

DNAH5

5p15.2

dynein, axonemal, heavy chain 5

0.1924

DNAH5

5p15.2

dynein, axonemal, heavy chain 5

0.2171

DNAH5

5p15.2

dynein, axonemal, heavy chain 5

0.2347

DNAH5

5p15.2

dynein, axonemal, heavy chain 5

0.1948

FBXL7

5p15.1

F-box and leucine-rich repeat protein 7

0.2802

LOC401177

5p15.1

hypothetical LOC401177

0.1976

CDH18

5p14.3

cadherin 18, type 2

0.2171

CDH18

5p14.3

cadherin 18, type 2

0.2318

CDH18

5p14.3

cadherin 18, type 2

0.212

CDH18

5p14.3

cadherin 18, type 2

0.2099

CDH18

5p14.3

cadherin 18, type 2

0.2299

CDH12

5p14.3

cadherin 12, type 2 (N-cadherin 2)

0.2152

CDH12

5p14.3

cadherin 12, type 2 (N-cadherin 2)

0.2502

CDH12

5p14.3

cadherin 12, type 2 (N-cadherin 2)

0.2326

CDH12

5p14.3

cadherin 12, type 2 (N-cadherin 2)

0.2104

CDH9

5p14.1

cadherin 9 type 2 (T1-cadherin 2)

0.2323

CDH9

5p14.1

cadherin 9 type 2 (T1-cadherin 2)

0.1935

CDH9

5p14.1

cadherin 9 type 2 (T1-cadherin 2)

0.238

CDH9

5p14.1

cadherin 9 type 2 (T1-cadherin 2)

0.1923

LOC729862

5p14.1

straitin, calmodulin binding protein psuedogene

0.2219

LOC729862

5p14.1

straitin, calmodulin binding protein psuedogene

0.2457

LOC729862

5p13.3

straitin, calmodulin binding protein psuedogene

0.1979

CDH6

5p13.3

cadherin 6, type 2, K-cadherin (fetal kidney)

0.1991

CDH6

5p13.3

cadherin 6, type 2, K-cadherin (fetal kidney)

0.1968

CDH6

5p13.3

cadherin 6, type 2, K-cadherin (fetal kidney)

0.2332

CDH6

5p13.3

cadherin 6, type 2, K-cadherin (fetal kidney)

0.2033

PLCXD3

5p13.1

phosphatidylinosotol-specific phospholipase C,

0.2054

X domain containing 3

OXCT1

5p13.1

3-oxoacid CoA transferase 1

0.2194

NNT

5p12

nicotinamide nucleotide transhydrogenase

0.3061

FGF10

5p12

fibroblast growth factor 10

0.2095

HCN1

5p12

hyperpolarization activated cyclic nucleotide-gated

0.2184

potassium channel 1

HCN1

5p11

hyperpolarization activated cyclic nucleotide-gated

0.2354

potassium channel 1

HCN1

5p11

hyperpolarization activated cyclic nucleotide-gated

0.2756

potassium channel 1

HCN1

5p11

hyperpolarization activated cyclic nucleotide-gated

0.234

potassium channel 1

HCN1

5p11

hyperpolarization activated cyclic nucleotide-gated

0.2099

potassium channel 1

PDE4D

5q12.1

phosphodiesterase 4D, cAMP-specific

0.1952

MEF2C

5q14.3

myocyte enhancer factor 2C

0.2164

MEF2C

5q14.3

myocyte enhancer factor 2C

0.2027

CETN3

5q14.3

centrin, EF-hand protein, 3

0.2484

ST8SIA4

5q21.1

STB alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4

0.2113

EFNA5

5q21.3

ephrin-A5

0.227

FBXL17

5q21.3

F-box and leucine-rich repeat protein 17

0.2163

FAM170A

5q23.1

family with sequence similarity 170, member A

0.2047

KCTD16

5q32

potassium channel tetramerisation domain containing 16

0.2027

GABRG2

5q34

gamma-aminobutyric acid (GABA) A receptor, gamma 2

0.2008

MAT2B

5q34

methionine adenosyltransferase II, beta

0.2047

ODZ2

5q34

odz, add OZ/ten-m homolog 2 (Drosophila)

0.2264

OPN5

6p12.3

opsin 5

0.2037

C6orf138

6p12.3

chromosome 6 open reading frame 138

0.2069

DEFB112

6p12.3

defensin, beta 112

0.2432

PKHD1

6p12.2

polycystic kidney and hepatic disease 1 (autosamal recessive)

0.1926

MTRNR2L9

6q11.1

MT-RNR2-like 9

0.2125

EYS

6q12

eyes shut homolog (Drosophila)

0.2137

EPHA7

6q16.1

EPH receptor A7

0.2043

KLHL32

6q16.1

kelch-like 32 (drosophila)

0.192

SDK1

7p22.2

sidekick homolog 1, cell adhesion molecule (chicken)

0.2213

NXPH1

7p21.3

neurexophilin 1

0.2178

NXPH1

7p21.3

neurexophilin 1

0.198

NXPH1

7p21.3

neurexophilin 1

0.2029

NXPH1

7p21.3

neurexophilin 1

0.3486

NXPH1

7p21.3

neurexophilin 1

0.205

PER4

7p21.3

period homolog 3, (Drosophila) pseudogene

0.2273

PER4

7p21.3

period homolog 3, (Drosophila) pseudogene

0.2097

PER4

7p21.3

period homolog 3, (Drosophila) pseudogene

0.2

THSD7A

7p21.3

thrombospondin, type I, domain containing 7A

0.2272

THSD7A

7p21.3

thrombospondin, type I, domain containing 7A

0.2872

THSD7A

7p21.3

thrombospondin, type I, domain containing 7A

0.2625

TMEM106B

7p21.3

transmembrane protein 106B

0.209

ETV1

7p21.2

ets variant 1

0.2215

DGKB

7p21.2

diacylglycerol kinase, beta 90 kDa

0.2607

DGKB

7p21.2

diacylglycerol kinase, beta 90 kDa

0.2236

DGKB

7p21.2

diacylglycerol kinase, beta 90 kDa

0.2292

DGKB

7p21.2

diacylglycerol kinase, beta 90 kDa

0.2664

TMEM195

7p21.1

transmembrane protein 195

0.2006

TMEM195

7p21.1

transmembrane protein 195

0.2133

MEOX

7p21.1

mesenchyme homebox 2

0.2331

MEOX

7p21.1

mesenchyme homebox 2

0.2088

ISPD

7p21.1

isoprenoid synthase domain containing

0.2459

SNX13

7p21.1

sorting nexin 13

0.1962

PRPS1L1

7p21.1

phosphoribosyl pyrophosphate synthetase 1-like 1

0.21

HDAC9

7p21.1

histone deacetylase 9

0.2909

HDAC9

7p21.1

histone deacetylase 9

0.1928

HDAC9

7p21.1

histone deacetylase 9

0.2498

HDAC9

7p21.1

histone deacetylase 9

0.3529

HDAC9

7p21.1

histone deacetylase 9

0.1975

HDAC9

7p21.1

histone deacetylase 9

0.2718

HDAC9

7p21.1

histone deacetylase 9

0.2003

FERD3L

7p21.1

Fer3-like (Drosophila)

0.1966

TWISTNB

7p15.3

TWIST neighbor

0.268

RPL23P8

7p15.3

ribosomal protein L23 pseudogene 8

0.1998

NPVF

7p15.2

neuropeptide VF precursor

0.1949

MIR148A

7p15.2

microRCA 148a

0.1961

CCDC129

7p15.1

coiled-coil domain containing 129

0.2241

PDE1C

7p15.1

phosphodiesterase 1C, calmodulin-dependent 70 kDa

0.2017

BBS9

7p14.3

Bardet-Biedl syndrome 9

0.2467

POU6F2

7p14.1

POU class 6 homebox 2

0.2422

C7orf10

7p14.1

chromosome 7 open reading frame 10

0.2008

ABCA13

7p12.3

ATP-binding cassette, sub-family A (ABC1), member 3

0.2018

CDC14C

7p12.3

CDC 14 cell division cycle 14 homolog C (S. cerevisiae)

0.1977

CDC14C

7p12.3

CDC 14 cell division cycle 14 homolog C (S. cerevisiae)

0.2338

VWC2

7p12.3

von Willebrand factor C domain containing 2

0.2143

POM121L12

7p12.1

POM121 membrane glycoprotein-like 12

0.1945

HPVC1

7p11.2

human papillomavirus (tpe 18) E5 central sequence-like 1

0.1979

HPVC1

7p11.2

human papillomavirus (tpe 18) E5 central sequence-like 1

0.1944

EGFR

7p11.2

epidermal growth factor receptor

0.216

LOC642006

7p11.2

glucuronidase, beta pseudogene

0.2114

ZNF716

7p11.1

zinc finger protein 716

0.2088

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.2005

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.3332

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.215

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.2105

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.3048

LOC643955

7q11.21

zinc finger protein 479 pseudogene

0.2315

SEMA3D

7q21.11

sema domain, immunoglobulin domain (Ig), short basic

0.1987

domain, secreted, (semaphorin) 3D

SEMA3D

7q21.11

sema domain, immunoglobulin domain (Ig), short basic

0.2551

domain, secreted, (semaphorin) 3D

SEMA3D

7q21.11

sema domain, immunoglobulin domain (Ig), short basic

0.2206

domain, secreted, (semaphorin) 3D

GRM3

7q21.11

glutamate receptor. metabotropic 3

0.2226

GRM3

7q21.11

glutamate receptor. metabotropic 3

0.229

DMTF1

7q21.12

cyclin D binding ,yb-like transcription factor 1

0.2539

ZNF804B

7q21.13

zinc finger protein 804B

0.2178

CCDC132

7q21.3

coiled-coil domain containing 132

0.2172

CALCR

7q21.3

calcitonin receptor

0.2362

CALCR

7q21.3

calcitonin receptor

0.1957

PPP1R3A

7q31.1

protein phosphatase 1, regulatory (inhibitor) subunit 3A

0.1918

FOXP2

7q31.1

forkhead box P2

0.1946

TFEC

7q31.2

ttranscription factor EC

0.2079

TES

7q31.2

testis derived transcript (3 LIM domains)

0.2985

KCND2

7q31.31

potassium voltage-gated channel, Shal-related subfamily,

0.2694

member 2

C7orf58

7q31.31

chromosome 7 open reading frame 58

0.235

GRM8

7q31.33

glutamate receptor, metabotropic 8

0.1918

POTEA

8p11.1

POTE ankyrin domain family, member A

0.2482

POTEA

8p11.1

POTE ankyrin domain family, member A

0.2804

POTEA

8p11.1

POTE ankyrin domain family, member A

0.1915

YTHDF3

8q12.3

YTH doamin family, member 3

0.2641

LOC100130155

8q12.3

hypothetical LOC100130155

0.2114

CC8orf34

8q13.2

chromosome 8 open reading frame 34

0.1994

ZFHX4

8q.2111

zinc finger homebox 4

0.256

PEX2

8q21.12

peroxisomal biogenesis factor 2

0.2401

PKIA

8q21.12

protein kinase (cAMP-dependent, catalytic) inhibitor alpha

0.2117

SNX16

8q21.13

sorting nexin 16

0.1929

CALB1

8q21.3

calbindin 1, 28 kDa

0.2122

C8orf83

8q22.1

chromosome 8 open reading frame 83

0.1918

PGCP

8q22.1

plasma glutamate carboxypeptidase

0.2947

ZFPM2

8q22.3

zinc finger protein, multitype 2

0.2272

ZFPM2

8q23.1

zinc finger protein, multitype 2

0.3113

SYBU

8q23.2

syntabulin (syntaxin-interacting)

0.2134

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.1922

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2235

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.1952

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2199

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.3086

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2099

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2081

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2069

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.1958

CSMD3

8q23.3

CUB and Sushi multiple domains 3

0.2039

TRPS1

8q23.3

trichorhinophalangeal syndrome I

0.1925

SLC30A8

8q24.11

solute carrier family 30 (zinc transporter), member 8

0.258

SAMD12

8q24.12

sterile alpha motif domain containing 12

0.2635

MRPL13

8q24.12

mitochondrial ribosomal protein L13

0.2103

POU5F1B

8q24.21

POU class 5 homebox 1B

0.2059

MIR1208

8q24.21

mircoRNA 1208

0.2022

MIR1208

8q24.21

mircoRNA 1208

0.3121

LOC728724

8q24.21

hCG1814486

0.1974

ASAP1

8q24.21

ArfGAP with SH3 domain, ankyrin repeat and PH domain 1

0.2033

ADCY8

8q24.22

adenylate cyclase 8 (brain)

0.2013

KHDRBS3

8q24.23

KH doamin containing, RNA binding, signal transduction

0.1966

associated 3

FAM135B

8q24.23

family with sequence similarity 135, member 8

0.1976

JAK2

9p24.1

Janus kinase 2

0.3072

LINGO2

9p21.2

leucine rich repeat and Ig domain containing 2

0.1927

NELL1

11p15.1

NEL-like 1 (chicken)

0.2098

NELL1

11p15.1

NEL-like 1 (chicken)

0.2512

KCNA4

11p14.1

potassium voltage-gated channel. shaker-related subfamily,

0.2045

member 4

OR4A47

11p11.2

olfactory receptor, family 4, subfamily A, member 47

0.2163

LOC646813

11p11.12

DEAH (Asp-Glu-Ala-His) box polypeptide 9 pseudogene

0.2223

LOC646813

11p11.12

DEAH (Asp-Glu-Ala-His) box polypeptide 9 pseudogene

0.2723

OR4A5

11p11.12

olfactory receptor, family 4, subfamily A, member 8

0.2231

OR8U8

11q11

olfactory receptor, family 8, subfamily U, member 5

0.2135

MIR4300

11q14.1

microRNA 4300

0.2378

RAB38

11q14.2

RAB38, member RAS oncogene family

0.2297

GRIA4

11q22.3

glutamate receptor, ionotrophis, AMPA 4

0.2141

MGST1

12p12.3

microsomal glutathione S-transferase 1

0.1928

PLCZ1

12p12.3

phospholipase C, zeta 1

0.1966

ABCC9

12p12.1

ATP-binding cassette, sub-family C (CFTR/MRP), member 9

0.3254

SOX5

12p12.1

SRY (sex determining region Y)-box 5

0.232

SOX5

12p12.1

SRY (sex determining region Y)-box 5

0.2355

ALG10B

12q11

asparagine-linked glycosylation 10, alpha-1,2-glucosyltransferase

0.1953

homolog B (yeast)

ALG10B

12q12

asparagine-linked glycosylation 10, alpha-1,2-glucosyltransferase

0.2311

homolog B (yeast)

LRRK2

12q12

leucine-rich repeat kinase 2

0.192

FAM19A2

12q14.1

family with sequence similarity 19 (chemokine (C-C motif)-like),

0.2478

member A2

LOC283392

12q21.1

hypothetical LOC283392

0.1985

TRHDE

12q21.1

thyrotropin-releasing hormone degrading enzyme

0.1973

PPFIA2

12q21.31

protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF),

0.204

interacting protein (lipirin), alpha 2

EPYC

12q21.33

epiphycan

0.2103

PRR20A

13q21.1

proline rich 20A

0.3125

PCDH9

13q21.32

protocadherin 9

0.1972

GPC6

13q31.3

glypican 6

0.193

OR4E2

14q11.2

olfactory receptor, family 4, subfamily E, member 2

0.1944

OR4E2

14q11.2

olfactory receptor, family 4, subfamily E, member 2

0.2203

NOVA1

14q12

neuro-oncological ventral antigen 1

0.2722

NOVA1

14q12

neuro-oncological ventral antigen 1

0.1931

MIR4307

14q12

microRNA 4307

0.2268

PRKD1

14q12

protein kinase D1

0.239

PRKD1

14q12

protein kinase D1

0.2057

PRKD1

14q12

protein kinase D1

0.1934

AKAP6

14q13.1

A kinase (PRKA) anchor protein 6

0.22

NPAS3

14q13.1

neuronal PAS domain protein 3

0.2346

NPAS3

14q13.1

neuronal PAS domain protein 3

0.1919

NPAS3

14q13.1

neuronal PAS domain protein 3

0.1913

NPAS3

14q13.1

neuronal PAS domain protein 3

0.2236

MBIP

14q13.3

MAP3K12 binding inhibitory protein 1

0.2078

MBIP

14q13.3

MAP3K12 binding inhibitory protein 1

0.2042

SLC25A21

14q13.3

solute carrier family 25 (mitochondrial oxodicarboxylate carrier),

0.2344

member 21

FOXA1

14q21.1

forkhead box A1

0.2183

SEC23A

14q21.1

Sec23 homolog A (S. cerevisiae)

0.2121

FBXO33

14q21.1

F-box protein 33

0.2059

FBXO33

14q21.1

F-box protein 33

0.1988

LRFN5

14q21.2

leucine rich repeat and fibronectin type III domain containing 5

0.2004

LRFN5

14q21.2

leucine rich repeat and fibronectin type III domain containing 5

0.2475

C14orf106

14q21.3

chromosome 14 open reading frame 106

0.2477

MDGA2

14q21.3

MAM domain containing glycosylphosphatidylinosotol

0.2836

anchor 2

RPS29

14q22.1

ribosomal protein S29

0.1915

FLRT2

14q31.3

fibronectin leucine rich transmembrane protein 2

0.1917

GALC

14q31.3

galactosylceramidase

0.2127

LOC727924

15q11.2

hypothetical LOC727924

0.2004

LOC390705

16p11.2

protein phosphoatase 2, regulatory subunit B″, beta pseudogene

0.2208

CDH8

16q21

cadherin 8, type 2

0.2022

CDH8

16q21

cadherin 8, type 2

0.2014

CA10

17q21.33

carbonic anhydrase X

0.2633

KIF2B

17q22

kinesin family member 2B

0.2584

KIF2B

17q22

kinesin family member 2B

0.2473

CDH2

18q12.1

cadherin 2, type 1, N-cadherin (neuronal)

0.2013

DSC3

18q12.1

desmocollin 3

0.2018

ASXL3

18q12.1

additional sex combs like 3 (Drosophila)

0.2644

LOC100101266

19p12

hepatitis A virus cellular receptor 1 pseudogene

0.1988

LOC10101266

19p12

hepatitis A virus cellular receptor 1 pseudogene

0.2313

LOC148189

19q12

hypothetical LOC148189

0.1955

LOC148189

19q12

hypothetical LOC148189

0.2084

LOC148189

19q12

hypothetical LOC148189

0.2196

JAG1

20p12.2

jagged 1

0.2034

JAG1

20p12.2

jagged 1

0.1977

TPTE

21p11.2

transmembrane phosphatase with tensin homology

0.2796

TMPRSS15

21q21.1

transmembrane protease, serine 15

0.203

Interestingly, among the four members of the ERBB family, while EGFR and ERBB4 (2q34) have DNA-gain, ERBB2 (17q12) and ERBB3 (12q13.2) have DNA-loss. Such gain-loss disparity was also observed in several other families such as the mesenchyme homeobox genes, MEOX2 (7p21.1, gain) and MEOX1 (17p21, loss); VAV family, VAV1 (19p13.2, loss), VAV2 (9q34.2, loss) and VAV3 (1p13.3, gain) (Table 4). To confirm the high density array CGH results, genomic real time qPCR was conducted and the DNA gain/loss pattern for several genes was validated (EGFR, ERBB4, MEOX2, TWIST1, TWISTNB, DGKB, VAV3, CDH12 from the DNA-gain list and ERBB2, ERBB3, MEOX1, CDH1, VAV1, VAV2, ACTN4, FAM102A from the DNA-loss list).

TABLE 4

Summary of DNA Gain/Loss on selected gene families

Number

Number

Number

of gain

of loss

Protein family

of genes

genes

genes

ATP-binding cassette

41

4

21

aconitase

2

1

1

acyl-CoA thioesterase

5

0

3

acyl-Conenzyme A oxidase(acyl-CoA oxidase)

2

0

2

adenylate cyclase

8

2

3

aldehyde dehydrogenase

14

0

6

ankyrin(ANK)

3

2

1

adaptor-related protein complex

18

0

10

apolipoprotein

10

1

7

ATPase

48

1

24

calcium channel, voltage-dependent(CACNA)

21

2

14

calcium/calmodulin-dependent protein kinase

9

2

5

calpaio

10

0

6

cadherin

21

11

8

chloride channel(CCLA, CLCN)

9

0

4

contactin

6

4

1

collagen

41

7

15

EPH receptor

13

4

5

ERBB Family

4

2

2

fibroblast growth factor

12

3

5

fibroblast growth factor receptor

4

0

2

glutamate receptor, metabotropic

7

5

1

integrin

24

4

11

laminin

12

1

4

mesenchyme homeobox

2

1

1

procadherin

13

6

3

phosphoinositide-3-kinase

14

1

5

ribosomal protein

22

3

10

testis expressed gene

7

0

5

tropomodulin

3

0

1

transmembrane protease

12

1

4

tumor necrosis factor receptor superfamily

11

1

9

tetraspanin

15

2

9

tubulin tyrosine ligase-like family

13

1

8

tubulin

12

0

7

UDP glucuronosyltransferase

9

6

0

guanine nucleotide exchange factory (VAV)

3

1

2

wingless-type MMTV integration site family

13

0

9

tyrosine 3-monooxygenase/tryptophan 5-monooxygenase

5

0

4

activation protein

Example 3

Chromosome 7p Contains the Highest Proportion of the Most Notable Amplification for the EGFR-Mutation Group

EGFR-mutation testing for the presence of the exon-21 L858R point mutation or the exon-19 in-frame deletion was conducted. It was found a total of 81 patients had EGFR mutation of either type and 57 patients were the wild-type. The sites with most notable amplification or deletion in the EGFR-mutant patients was studied and regions with highest DNA gain and loss was located by identifying the top 1% of probe-blocks with the largest and the smallest mean values of CNA respectively. Interestingly, 81 out of 364 (22.3%) probe-blocks with highest CNA fall on chromosome 7p, outnumbering all other chromosome arms despite the smallness in the arm size (carrying only 2.25% of probe blocks in the array). The same pattern still occurred for the two EGFR mutation types studied separately. On the other hand, for the EGFR wild-type group, the pattern was completely different and chromosome 7p became insignificant.

Example 4

Chromosome 7p is Most Enriched in Containing Sites of Differential CNA Between the EGFR-Activating Mutation Group and the EGFR Wild-Type Group

The sites of significant differences between the mutation group and the wild-type group by t-test were located (FIG. 1). It was found that among all chromosomes, the greatest CNA difference is located on the chromosome 7p. Indeed, 47.13% of the probes located in this chromosome arm have CNA differences, far exceeding the percentage (20.20%) of chromosome 14q which is at the second place. For chromosome arms with higher rate of loss-blocks (17p, 19p and 19q), only very few probe sites show significant differences, indicating that the pattern of DNA loss between the mutation group and the wild-type group is more consistent.

The size of the difference (absolute value of the mean of the mutation group minus the mean of the wild type group) was also examined. It was found the largest differences to occur on a segment (869K bps of length) at 7p112 harboring EGFR, LANCL2, VOPP1, and others. The CNA values in this region are higher for the mutation group (Table 5). Co-amplification of LANCL2 with EGFR and VOPP1 with EGFR was previously reported only in some tumors (Lu Z, et al: Glioblastoma proto-oncogene SEC61gamma is required for tumor cell survival and response to endoplasmic reticulum stress. Cancer Res 69:9105-11, 2009).

TABLE 5

The genome-wide top 20 sites with the largest effect size*.

EGFR-mutant

EGFR wild-type

patients

patients

Group mean

Gene

Location

(mean ± SD)

(mean ± SD)

difference

p value**

EGFR

7p12

0.278 ± 0.348

0.128 ± 0.152

0.149

0.001

EGFR

7p12

0.239 ± 0.341

0.098 ± 0.151

0.142

0.001

LANCL2

7p11.2

0.072 ± 0.276

−0.065 ± 0.087  

0.138

5.6E−05

EGFR

7p12

0.182 ± 0.329

0.055 ± 0.106

0.127

0.002

VSTM2A

7p11.2

0.200 ± 0.232

0.084 ± 0.095

0.116

1.0E−04

VOPP1

7p11.2

0.037 ± 0.247

−0.072 ± 0.114  

0.109

0.001

LANCL2

7p11.2

−0.003 ± 0.263  

−0.111 ± 0.107  

0.108

0.001

SEPT14

7p11.2

0.034 ± 0.180

−0.066 ± 0.096  

0.100

7.8E−05

VOPP1

7p11.2

0.041 ± 0.306

−0.057 ± 0.129  

0.098

0.011

SEC61G

7p11.2

0.112 ± 0.273

0.014 ± 0.089

0.098

0.003

MIR148A

7p15.2

0.093 ± 0.159

−0.003 ± 0.093  

0.096

1.9E−05

EGFR

7p12

0.168 ± 0.254

0.074 ± 0.096

0.094

0.003

EGFR

7p12

0.119 ± 0.358

0.029 ± 0.155

0.091

0.045

C7orf10

7p14.1

0.094 ± 0.124

0.005 ± 0.097

0.090

5.0E−06

ANK1

8p11.1

−0.157 ± 0.134  

−0.068 ± 0.233  

−0.088

0.012

ADAM3A

8p11.23

0.025 ± 0.126

0.113 ± 0.134

−0.088

1.7E−04

OSBPL3

7p15

0.200 ± 0.162

0.115 ± 0.100

0.085

2.2E−04

IKZF1

7p13-p11.1

0.119 ± 0.164

0.034 ± 0.139

0.085

0.001

KIAA0146

8q11.21

−0.047 ± 0.108  

0.038 ± 0.117

−0.085

3.2E−05

SEMA5A

5p15.2

0.117 ± 0.151

0.035 ± 0.126

0.082

0.001

*Effect size is the absolute value of the mean difference between EGFR-mutant group and EGFR wild-type group

** p value is calculated by t-test.

The difference between L858R mutation and the wild-type was further compared. It was found out that chromosome 7p still was the richest arm in containing sites of differences in CNA values (Table 6).

TABLE 6

Distribution of the sites showing significantly different CAN in EGFR mutation status

comparison by t-test at 5% level

Number of

Number of

Number of

Number of

differential

differential

differential

differential

probes

probes

probes

probes

between

between

between

between

L858R

Total

EGFR-

L858R

exon-19

mutant and

number

mutant and

mutant and

deletion and

exon-19

Chromosome

of probes

wild-type (%)

wild-type (%)

wild- type (%)

deletion (%)