# Patent

## Title
Use of GTF21 mutations in the prognosis of thymic cancers

## Abstract

Disclosed are methods of determining the prognosis of thymic cancer in a subject comprising detecting a mutation in the general transcription factor IIi (GTF2I) genetic sequence or protein. The presence of a GTF2I mutation indicates that the thymic cancer is indolent.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This patent application claims the benefit of U.S. Provisional Patent Application No. 61/975,222, filed Apr. 4, 2014, which is incorporated by reference in its entirety herein.


INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
Incorporated by reference in its entirety herein is a computer-readable nucleotide sequence listing submitted concurrently herewith and identified as follows: One 260,721 Byte ASCII (Text) file named “720112ST25.TXT,” dated Mar. 11, 2015.
BACKGROUND OF THE INVENTION
While some thymic cancers may be relatively benign, there are some subtypes that may be aggressive. The currently available histological classification and staging systems may not be sufficiently informative about the prognosis of these cancers. Accordingly, there is a need for improved methods of determining the prognosis of thymic cancers.
BRIEF SUMMARY OF THE INVENTION
An embodiment of the invention provides a method of determining the prognosis of thymic cancer in a subject, the method comprising: obtaining genetic material from the subject; assaying the genetic material to detect a mutation in at least one copy of general transcription factor IIi (GTF2I) genetic sequence; and correlating the presence of a GTF2I mutation with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent.
Another embodiment of the invention provides a method of determining the prognosis of thymic cancer in a subject, the method comprising: obtaining a sample from the subject; assaying the sample to detect a mutation in GTF2I protein; and correlating the presence of a mutation in the GTF2I protein with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent.
Another embodiment of the invention provides a method for detecting a GTF2I mutation in a subject, wherein the subject has thymic cancer, the method comprising: obtaining genetic material from the subject; and assaying the genetic material to detect a mutation in at least one copy of GTF2I genetic sequence.
Still another embodiment of the invention provides a method for detecting a mutated GTF2I protein in a subject, wherein the subject has thymic cancer, the method comprising: obtaining a sample from the subject; and assaying the sample to detect a mutation in GTF2I protein.


DETAILED DESCRIPTION OF THE INVENTION
It has been discovered that a mutation in the general transcription factor IIi (GTF2I) gene is present in indolent thymic tumors and is rarely found in more aggressive thymic tumors. GTF2I is a transcription factor that regulates the transcription of genes that control cell proliferation (e.g., c-FOS), the cell cycle (e.g., cyclin-D1), and developmental processes. GTF2I binds specifically to several DNA sequences and mediates growth factor signaling. Human GTF2I is assigned Gene NCBI Gene ID No. 2969, and an Mendelian Inheritance in Man (MIM) No. 601679. The human GTF2I gene is found on chromosome 7 at 7q11.23. A wild type genomic GTF2I DNA sequence comprises SEQ ID NO: 1. Five (wild type) transcriptional variants include the GTF2I alpha (α) isoform (SEQ ID NO: 3), beta (β) isoform (SEQ ID NO: 4), gamma (γ) isoform (SEQ ID NO: 5), delta (δ) isoform (SEQ ID NO: 6), and epsilon (∈) isoform (SEQ ID NO: 7).
An embodiment of the invention provides a method of determining the prognosis of thymic cancer in a subject, the method comprising: obtaining genetic material from the subject; assaying the genetic material to detect a mutation in at least one copy of GTF2I genetic sequence; and correlating the presence of a GTF2I mutation with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent. The inventive methods may provide many advantages. For example, the inventive methods may, advantageously, help the clinician decide whether treatment is needed (for example, additional treatment after surgery) or what type of treatment is needed (such as, for example, an aggressive course of treatment for more aggressive cancers). The inventive methods are also, advantageously, more objective than currently available histological classification and staging systems.
The thymic cancer may be any thymic cancer. In an embodiment of the invention, the thymic cancer is a thymic epithelial tumor (TET). Preferably, the TET is a thymic carcinoma (TC) or a thymoma. According to the 2004 World Health Organization (WHO) classification system, thymomas are further classified into A, AB, B1, B2 and B3 types according to their histological features (Travis et al., Pathology and genetics: Tumors of the lung, pleura, thymus and heart, IARC Press, Lyon, France (2004)). Type A and AB thymomas have the best prognosis, with 10-year survival rates close to 100%, wheras TCs are the most aggressive TETs with 50% 10-year survival (Kelly et al., J Clin. Oncol., 29: 4820-7 (2011)). Preferably, the subject is a human.
In an embodiment, the method comprises obtaining genetic material from the subject. Obtaining genetic material from the subject may be carried out in any suitable manner known in the art. In an embodiment of the invention, the genetic material is obtained from tumor tissue. The genetic material may be obtained, for example, from tumor resection material or a tumor biopsy.
In an embodiment, the inventive method involves assaying genetic material obtained from a test subject to detect a mutation in at least one copy of the GTF2I genetic sequence. The genetic material can be, for example, DNA (for example, genomic DNA or complementary DNA (cDNA)) or RNA (e.g., (messenger RNA (mRNA)). In an embodiment of the invention, the genetic material is cDNA or mRNA of a GTF2I isoform selected from the group consisting of the GTF2I α isoform, GTF2I β isoform, GTF2I γ isoform, GTF2I δ isoform, and GTF2I ∈ isoform.
The genetic material may be obtained directly from a tumor of the test subject, or the genetic material can be copied or amplified from genetic material within the test subject's tumor cells (e.g., via polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), or other suitable technique). For example, epithelial cells can be harvested from the tumor tissue to obtain genetic material. To ensure that a sufficient quantity of genetic material is available for testing, genetic material may be amplified from cells obtained from the test subject, and the amplified genetic material is assayed in accordance with the inventive method. Preferably, a PCR or RT-PCR strategy is employed using primers flanking all or a portion of the GTF2I gene, so as to amplify this sequence from the test subject for the assay. While the method may comprise amplifying and assaying one copy of the GTF2I gene, preferably, the method comprises amplifying both copies of the GTF2I gene from the test subject, so that both can be assayed in accordance with the inventive method.
However obtained, the method comprises assaying the genetic material to detect a mutation in the GTF2I gene (e.g., a mutation at least one of the two GTF2I alleles). Any test able to detect mutations appropriate to the type of genetic material (e.g., genomic DNA (gDNA), cDNA, RNA) may be employed. The assaying may comprise obtaining the sequence of at least a portion of the GTF2I genetic sequence or obtaining the sequence of substantially all of the GTF2I genetic sequence. In an embodiment, the method may further comprise comparing the sequence of the genetic material of the subject to the sequence of the wild type GTF2I genetic sequence and identifying any differences between the sequence of the genetic material of the subject and the wild type GTF2I genetic sequence to detect any mutations. Examples of wild type GTF2I genetic sequences may include, for example, SEQ ID NO: 1 (wild type GTF2I genomic DNA), SEQ ID NO: 2 (wild type GTF2I exon 15), SEQ ID NO: 3 (wild type GTF2I alpha (α) isoform cDNA), SEQ ID NO: 4 (wild type beta (β) isoform cDNA), SEQ ID NO: 5 (wild type gamma (γ) isoform cDNA), SEQ ID NO: 6 (wild type delta (δ) isoform cDNA), or SEQ ID NO: 7 (wild type epsilon (∈) isoform cDNA). Other examples of wild type GTF2I genetic sequences may include Genbank Accession Nos. ABBA01063881.1, ABBA01063882.1, AC004883.3, AC005231.3, AC083884.6, AC211433.4, CH471200.2, AA641906.1, AF015553.1, AF035737.1, AF038967.1, AF038968.1, AF038969.1, AK057670.1, AK093663.1, AK096095.1, AK292832.1, AK294935.1, AK297238.1, BC004472.2, BC070484.1, BC099907.1, BT007450.1, CB118501.1, and DC346283.1. In an embodiment of the invention, the assaying comprises carrying out a PCR assay that specifically detects the mutation. Examples of PCR assays that specifically detect the mutation may include any one or more of (i) carrying out PCR using primers that amplify the mutated GTF2I genetic sequence but not the wild type GTF2I genetic sequence; (ii) carrying out PCR using primers that amplify the wild type GTF2I genetic sequence but not the mutated GTF2I genetic sequence; and (iii) carrying out PCR using primers that amplify the mutated GTF2I genetic sequence, the wild type GTF2I genetic sequence, and GTF2I pseudogenes, but the sequences of the PCR products make it possible to distinguish the mutated GTF2I genetic sequence from the wild type GTF2I genetic sequence as well as GTF2I pseudogenes. In an embodiment of the invention, the primers used in the PCR assay amplify the GTF2I genetic sequence (wild type or mutated) but not GTF2I pseudogenes.
The GTF2I mutation may be any type of gene mutation. For example, the GTF2I mutation may be any one or more of a missense mutation, a nonsense mutation, an insertion, a deletion, a duplication, and a frameshift mutation. Preferably, the GTF2I mutation is a missense mutation.
The GTF2I mutation may located anywhere in the coding sequence of the GTF2I gene. In an embodiment of the invention, the GTF2I mutation is located in exon 15 of the GTF2I gene. Preferably, the mutation is chr7:74146970 T/A. In an embodiment of the invention, the mutation is selected from the group consisting of: (a) g.75,041T>A, with reference to SEQ ID NO: 1; (b) c.1208T>A, with reference to SEQ ID NO: 3; (c) c.1211T>A, with reference to SEQ ID NO: 4; (d) c.1271T>A, with reference to SEQ ID NO: 5; (e) c.1148T>A, with reference to SEQ ID NO: 6; and (f) c.1205T>A, with reference to SEQ ID NO: 7. The GTF2I genetic mutations g.75,041T>A, c.1208T>A, c.1211T>A, c.1271T>A, c.1148T>A, and c.1205T>A are defined herein by reference to the wild type genomic DNA sequence (SEQ ID NO: 1) or one of the five wild type isoform cDNA sequences of GTF2I (SEQ ID NO: 3-7). Thus, these GTF2I genetic mutations are described herein by reference to genomic DNA (“g.”) or cDNA (“c.”), followed by the particular position in the sequence at which the mutation is taking place, followed by the native nucleotide at that position, followed by the nucleotide with which the native nucleotide is being replaced.
In an embodiment of the invention, the genetic material comprising the mutation comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 8 (mutated GTF2I cDNA); SEQ ID NO: 9 (mutated GTF2I genomic DNA); SEQ ID NO: 10 (mutated GTF2I alpha isoform cDNA); SEQ ID NO: 11 (mutated GTF2I beta isoform cDNA); SEQ ID NO: 12 (mutated GTF2I gamma isoform cDNA); SEQ ID NO: 13 (mutated GTF2I delta isoform cDNA); SEQ ID NO: 14 (mutated GTF2I epsilon isoform cDNA); and a complement of any one of SEQ ID NOs: 8-14.
The method further comprises correlating the presence of a GTF2I mutation with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent. An indolent cancer may be characterized by any one or more of slow growth, less metastasis, fewer symptoms, an earlier cancer stage (e.g., at diagnosis), a higher chance of complete resection, and a longer survival time as compared to a cancer that lacks the GTF2I mutation. In an embodiment of the invention, the absence of a GTF2I mutation indicates that the thymic cancer is aggressive. An aggressive cancer may be characterized by any one or more of fast growth, more metastasis, more symptoms, a later cancer stage, a lower chance of complete resection, and a shorter survival time as compared to a cancer that has the GTF2I mutation. In an embodiment of the invention, the prognosis indicates the likelihood of the subject's 10-year survival. In this regard, the presence of the mutation indicates that the subject will have a significantly higher chance to survive 10 years and the absence of the mutation indicates that the subject will have a significantly lower chance to survive 10 years.
In an embodiment of the invention, the presence of the mutation indicates that the thymic cancer is, or has a high likelihood of being, a thymoma histotype A or AB according to the 2004 WHO classification (Travis et al., Pathology and genetics: Tumors of the lung, pleura, thymus and heart, IARC Press, Lyon, France, (2004)). In an embodiment, the absence of the mutation indicates that the thymic cancer is, or has a high likelihood of being, thymoma histotype B1, B2, or B3 or a TC according to the 2004 WHO classification. As described in the Examples, the presence of GTF2I mutation has been seen significantly more frequently in histologically indolent tumors (WHO type A and AB) than in more aggressive histological tumors (WHO B3 and thymic carcinoma). In addition, aggressive tumors (such as B3 thymomas and thymic carcinomas) that have the GTF2I mutation have a better prognosis than those tumors without the mutation. According to the 2004 WHO classification, A type thymomas present bland spindle/oval epithelial tumor cells with few or no lymphocytes. Grossly, they are usually encapsulated and easily separable from the surrounding organs even in case of tumors of conspicuous dimension. Type B thymomas show epithelial cells with a predominantly round or polygonal appearance. Type B1 thymomas display tumor epithelial cells with very little atypia, scattered in a prominent population of immature non-neoplastic thymocytes that resemble the structure of normal thymus cortex. Type B2 thymomas are characterized by large polygonal epithelial tumor cells arranged in a loose network containing numerous immature T lymphocytes. B3 thymomas are composed of medium size round or polygonal epithelial tumor cells with slight atypia; these cells are mixed with a minor component of intraepithelial thymocytes. AB thymomas are composed of a lymphocyte-poor type A and a more lymphocyte-rich type B component.
In an embodiment of the invention, the presence of the mutation indicates that the cancer is, or is more likely to be, in an early stage (cancer stage I-II). In an embodiment of the invention, the absence of the mutation indicates that the cancer is, or is more likely to be, in an advanced stage (cancer stage III-IV) (Masaoka et al., Cancer, 48(11): 2485-92 (1981)).
Another embodiment of the invention provides a method for detecting a GTF2I mutation in a subject, the method comprising: obtaining genetic material from the subject; and assaying the genetic material to detect a mutation in at least one copy of GTF2I genetic sequence. Obtaining a sample from the subject and assaying the genetic material may be carried out as described herein with respect to other aspects of the invention. In an embodiment, the subject has thymic cancer, which may be as described herein with respect to other aspects of the invention. The GTF2I mutation may also be as described herein with respect to other aspects of the invention.
In an embodiment of the invention, the method further comprises treating thymic cancer in the subject based on the presence or absence of the mutation. The inventive methods of determining the prognosis of thymic cancer in a subject may, advantageously, make it possible for an attending clinician to determine whether a particular treatment is necessary and, if so, how much treatment is necessary. In an embodiment of the invention, the method comprises treating thymic cancer in the subject when the mutation is present by surgically removing all or part of the thymic cancer without administering one or more of, two or more of, or all three of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy in an amount effective to treat thymic cancer in the subject. In this regard, when the mutation is present, treating thymic cancer in the patient may comprise not treating the patient with one or more of, two or more of, or all three of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy. Accordingly, the inventive methods may, advantageously, make it possible for a patient with the mutation to avoid or reduce exposure to one or more of, two or more of, or all three of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy and the accompanying side effects. In addition, the inventive methods may, advantageously, make it possible for a patient with an indolent thymic cancer to avoid or reduce the harmful effects on the heart that are associated with radiotherapy.
In an embodiment of the invention, the method may comprise treating thymic cancer in the subject when the mutation is absent by surgically removing all or part of the thymic cancer and administering one or more of, two or more of, or all three of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy in an amount effective to treat thymic cancer in the subject. The chemotherapy may comprise any suitable chemotherapy. For example, the chemotherapy may comprise administering any one or more of doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE), belinostat, cisplatin, carboplatin, cyclophosphamide (CYTOXAN), ifosfamide (IFEX), vincristine (ONCOVIN), etoposide (VP-16), paclitaxel (TAXOL), pemetrexed (ALIMTA), 5-fluorouracil (5-FU), methylprednisolone, octreotide, gefitinib, imatinib, and gemcitabine (GEMZAR) to the subject in an amount effective to treat thymic cancer in the subject. Belinostat has antitumor activity in heavily pretreated thymic malignancies (Giaccone et al., J. Clin. Oncol., 29: 2052-2059 (2011)). The immunotherapy may comprise any suitable immunotherapy. For example, the immunotherapy may comprise administering one or both of bevacizumab and cixutumumab to the subject in an amount effective to treat thymic cancer in the subject. Cixutumumab may be useful for treating thymoma (Rajan et al., Lancet Oncol., 15: 191-200 (2014)). Various treatments for thymic cancer are described in Rajan et al., Curr. Treatment Options Oncol., 9: 277-287 (2008).
The term “treat,” as well as words stemming therefrom, as used herein, does not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment of cancer in a subject. Furthermore, the treatment provided by the inventive method can include treatment of one or more conditions or symptoms of the disease, e.g., cancer, being treated.
Another embodiment of the invention provides a method of determining the prognosis of thymic cancer in a subject, the method comprising: obtaining a sample from the subject; assaying the sample to detect a mutation in GTF2I protein; and correlating the presence of a mutation in the GTF2I protein with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent.
In an embodiment, the method comprises obtaining a sample from the subject. In an embodiment, the sample is a tumor tissue sample. Obtaining a sample from the subject may be carried out in any suitable manner known in the art, and the sample may be from any suitable source, for example, from tumor resection material or a tumor biopsy.
The method further comprises assaying the sample to detect a mutation in GTF2I protein. For example, the GTF2I protein can be purified from the sample (either partially or substantially and assayed via immunohistological techniques (e.g., Western blotting, ELISA, immunoprecipitation, etc.) using one or more antibodies recognizing mutant GTF2I protein but not wild type GTF2I protein. In this regard, the assaying may comprise contacting the sample with an antibody that specifically binds to mutant GTF2I protein and does not bind to wild type GTF2I protein, thereby forming a complex, and detecting the complex. Alternatively, or in conjunction, the GTF2I protein sample from the test subject can be assayed using one or more antibodies recognizing wild type GTF2I protein but not mutant GTF2I protein. In this regard, the assaying may comprise contacting the sample with an antibody that specifically binds to wild type GTF2I protein and does not bind to mutant GTF2I protein, thereby forming a complex, and detecting the complex. In an embodiment, the wild type GTF2I protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31 (wild type alpha isoform GTF2I protein), SEQ ID NO: 32 (wild type beta isoform GTF2I protein), SEQ ID NO: 33 (wild type gamma isoform GTF2I protein), SEQ ID NO: 34 (wild type delta isoform GTF2I protein), and SEQ ID NO: 35 (wild type epsilon isoform GTF2I protein). In an embodiment, the GTF2I protein with the mutation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 (mutated alpha isoform GTF2I protein), SEQ ID NO: 16 (mutated beta isoform GTF2I protein), SEQ ID NO: 17 (mutated gamma isoform GTF2I protein), SEQ ID NO: 18 (mutated delta isoform GTF2I protein), and SEQ ID NO: 19 (mutated epsilon isoform GTF2I protein).
In an embodiment, the mutation is selected from the group consisting of: (a) p.Leu403His, with reference to SEQ ID NO: 31; (b) p.Leu404His, with reference to SEQ ID NO: 32; (c) p.Leu424His, with reference to SEQ ID NO: 33; (d) p.Leu383His, with reference to SEQ ID NO: 34; and (e) p.Leu402His, with reference to SEQ ID NO: 35. The GTF2I protein mutations pieu403His, p.Leu404His, p.Leu424His, p.Leu383His, and p.Leu402His are defined herein by reference to one of the five wild type isoform amino acid sequences of GTF2I (SEQ ID NO: 31-35). Thus, these GTF2I protein mutations are described herein by reference to protein (“p.”), followed by the native amino acid residue being replaced, followed by the particular position in the sequence at which the mutation is taking place, followed by the amino acid residue with which the native amino acid residue is being replaced.
The method further comprises correlating the presence of a mutation in the GTF2I protein with the prognosis of thymic cancer in the subject, wherein the presence of the mutation indicates that the thymic cancer is indolent. Correlating the presence of a mutation in the GTF2I protein with the prognosis of thymic cancer in the subject may be carried out as described herein with respect to other aspects of the invention.
Another embodiment of the invention provides a method for detecting a mutated GTF2I protein in a subject, the method comprising: obtaining a sample from the subject; and assaying the sample to detect a mutation in GTF2I protein. Obtaining a sample and assaying the sample may be carried out as described herein with respect to other aspects of the invention. The mutated GTF2I protein may be as described herein with respect to other aspects of the invention. In an embodiment, the subject has thymic cancer, which may be as described herein with respect to other aspects of the invention.
In an embodiment of the invention, the method further comprises treating thymic cancer in the subject based on the presence or absence of the mutation in the GTF2I protein. Treating thymic cancer in the subject based on the presence or absence of the mutation may be carried out as described herein with respect to other aspects of the invention.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Examples
Materials and Methods
Tumor samples of 286 patients were collected from four different institutions: National Cancer Institute (NCI) (Bethesda Md.), Pisa University Hospital (Pisa, Italy), Padua University Hospital (Padua, Italy) and IRCCS Istituto Clinico Humanitas (Rozzano, Italy). All patients selected for this study were enrolled in protocols approved at the participating institutions and a written informed consent for genome profiling (including array comparative genomic hybridization (CGH) and sequencing) was obtained from all study participants.
Nucleic Acid Extraction:
Samples of thymic epithelial tumors were collected during surgical procedures or through an image guided tumor biopsy. The collected specimens were immediately frozen in liquid nitrogen. Samples were embedded in optimal cutting temperature compound (OCT) and 8 μm slices were cut using a cryostat. A pathologist evaluated the slices after haematoxylin and eosin (H&E) staining in order to select regions rich of tumor cells for macro-dissection. Samples were annotated with pathologist's estimation of tumor cellularity. DNA and RNA were extracted at the same time from the selected tumor portion using ALL PREP RNA/DNA kit (Qiagen, Valencia, Calif.).
From paraffin embedded (FFPE) blocks, 4 and 10 μm slices were cut using a microtome. 4 μm slides were stained with haematoxylin and eosin and a pathologist confirmed the tumor diagnosis and selected the tumor material. 10 μm slides were deparaffinized using HISTOCHOICE Clearing Agent (Sigma-Aldrich, St. Louis, Mo.) and rehydrated through alcohol series. Thereafter, the stained and the 10 μm slides, from the same block, were matched and the selected regions rich in tumor cells were scraped from the rehydrated slides. DNA was extracted using DNEASY Blood and Tissue kit (Qiagen) according to vendor's protocol but with an extended proteinase K digestion of at least 16 hours (h) at 70° C.
Patient's blood (5 mL) was collected in ethylenediaminetetraacetic acid (EDTA) tubes and frozen at −80° C. DNA was extracted from the whole blood using QIAAMP DNA Blood Maxi Kit (Qiagen).
Array Comparative Genomic Hybridization:
Tumors were chosen for array Comparative Genomic Hybridization (aCGH) depending on the availability of frozen material and on their tumor cell content: only samples rich in cancer cells (>80%) were selected. Array CGH was performed in 65 cases. Array CGH was performed as previously described (Petrini et al., Cell Death Dis., 3: e351 (2012)). The reference human genome was the NCBI version 37.1. Data were analyzed using Nexus 7 (Biodiscovery Inc., El Segundo, Calif., USA) according to the following pipeline. A systematic correction was applied to the data in order to limit the wave-like artifacts due to the genomic regions rich in GC nucleotides. The bias estimations were determined using a linear model that took into account the percentage of CG content and the length of the fragments. Bias estimations were then subtracted from the Log2Ratio of the probes. Thereafter, probes were re-centered through normalization to the median Log2Ratio of the diploid regions that were determined sample by sample. Segmentation was performed using Rank Segmentation algorithm according to the following settings: a significant threshold of 5.0E-6, maximum contiguous probe spacing of 1000 kb and a minimum of 10 probes per segment. Sex chromosomes were removed from the analysis. The presence of CN aberrations, candidate drivers of the tumor growth, was assessed using GISTIC algorithm (Beroukhim et al., PNAS, 104: 20007-12 (2007)). Regions with a Q-bound lower than 0.25 and a G-score higher than one were considered significant. GISTIC peaks related to germline CN variations were filtered out. The Toronto database of genomic variants (Iafrate et al., Nat. Genet., 36: 949-51 (2004)) was adopted to define regions of germline CN variations. If a GISTIC peak was fully mapped in one of these regions, the peak was removed from the list of significant results. Chromosome arm level CN aberrations were defined when more than 80% of a chromosome arm (p or q) was covered by CN gains or losses. For example, chromosome 1q is 100,313,968 base pairs (bp) long. A single CN gain of a portion of chromosome 1q longer than 80,251,174 bp (80% of 100′313′968) will be classified as an arm level CN gain of chromosome 1q. Also, an arm level CN gain of chromosome 1q will be described if the sum of the lengths of three regions of CN gains mapped on 1q exceeds 80,251,174 bp. As previously described (Petrini et al., Cell Death Dis., 3: e351 (2012)), the 80% cutoff for the definition of chromosome arm level CN aberration was chosen based on the frequency distribution of the length of CN aberrations. A hierarchical cluster of tumors was built using complete linkage of their chromosome arm level CN aberrations.
Transcriptome Sequencing:
Samples were selected for transcriptome sequencing if their RNA integrity number was >8, the H&E staining demonstrated a proportion of cancer cells >80%, and copy number aberrations larger than 5 Mb were detected using aCGH, in order to safely exclude germline copy number variations that usually (99%) are shorter than 2.3 Mb (Ostrovnaya et al., BMC Bioinformatics, 11: 297 (2010)). Type A thymomas were exceptions because typically they do not present copy number aberrations. Type A thymomas were safely included upon pathology review since they always present a scant thymocyte component (non-neoplastic precursors of lymphocytes). Transcriptome sequencing was conducted at the NCI sequencing facility according to the ILLUMINA mRNA sample preparation kit (Illumina, San Diego, Calif.). Briefly, using poly-T-bound magnetic beads, poly-A mRNAs were captured from total RNA. First and second strand cDNA were serially synthetized. Overhanging fragments' ends were repaired using T4 DNA and Klenow DNA polymerases and adaptors linked using T4 DNA Ligase. Ligation products were run on an agarose gel, the 200 bp band was excised and used for DNA extraction. cDNA libraries were generated from the purified products and subsequently validated with the 2100 Agilent bioanalyzer (Agilent). According to the instructions, cDNA libraries were hybridized to a flow cell, amplified, linearized and denatured using Illumina Cluster Station, in order to generate pair-end clusters ready for sequencing. Genome Analyzer II or HiSeq2000 were used for sequencing.
Transcriptome Sequencing Data Analysis Workflow:
Human genome hgl9 was chosen as reference and only sequences mapped to human chromosomes chrl-22, X, Y, and M were retained. FASTQ files were obtained directly from the sequencing machine programmed to automatically trim the adaptors and barcode sequences. The reads were mapped to the reference genome using TopHat (Trapnell et al., Bioinformatics, 25: 1105-11 (2009)). Quality of the RNA sequencing results was assessed using CollectRnaSeqMetrics available in Picard tools and with FASTQC software.
Estimation of Gene Expression:
Gene expression was estimated from the mapped reads using Cufflinks algorithm. The log10 transformation of the FPKM+1 values was used to cluster thymic epithelial tumors. Samples were clustered using a hierarchical cluster and uncentered Pearson Correlation. Two samples with high duplication rate and the cell lines were removed from the cluster analysis.
Prediction and Validation of Fusion Transcripts:
Candidate fusion transcripts were identified from the FASTQ files using two independent algorithms: FusionMap with MONO Version 2.10.8 (Ge et al., Bioinformatics, 27: 1922-28 (2011)) and DeFuse (McPherson et al., PLoS Comput. Biol., 7: el001138 (2011)). The following parameters were used according to previous reports (Ge et al., Bioinformatics, 27: 1922-28 (2011)): MinimalFusionAlignmentLength=25, FusionReportCutoff=1 and NonCanonicalSpliceJunctionPenalty=4. Moreover, at least 20 seed reads were required to support the predicted candidates.
Fusion candidates were identified and filtered as previously described (McPherson et al., PLoS Comput. Biol., 7: el001138 (2011)). The predicted fusion transcripts identified by both methods were evaluated using the BLAT tool from the UCSC website. Predicted fusions were excluded from the candidates' list if one fusion arm had multiple possible alignments with an identity >95% or if they overlapped a region of human chained self alignment (Chiaromonte et al., Pac. Symp. Biocomput., 115-26 (2002)) or a region annotated with: segmental duplications (Bailey et al., Science, 297: 1003-7 (2002)), repeat maskers (Jurka et al., Trends Genet., 16: 418-20 (2000)), interrupted repeats (Jurka et al., Trends Genet., 16: 418-20 (2000)) and simple repeat (Benson et al., Nucleic Acids Res., 27: 573-80 (1999)). The fusion transcripts included in this filtered list of candidates were validated using reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. In brief, reverse transcription was performed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif.). PCR primers were designed on opposite sides of the fusion junction and were tagged with M13 forward and reverse primer sequences. The reactions were carried out using Taq DNA polymerase (Invitrogen) and two negative controls: cDNA from normal thymus of two unrelated subjects were included for each fusion candidate in order to exclude nonspecific amplification. Amplicons were run on an agar gel and if the predicted size band was detected, PCR products underwent EXOSAP-IT (USB, Cleveland, Ohio) purification and Sanger sequencing. Fusions were considered validated if the forward and reverse sequences were uniquely mapped to the predicted fusion transcript and if at least one of them spanned the junction sequence. The confirmed fusion transcripts were visualized using Circus-0.64.
Exome Sequencing:
Samples were included in the exome sequencing analysis if they fulfilled the following criteria: 1) tumor and normal DNA from the same patient were available, 2) the tumor sample selected for DNA extraction presented at least 80% of tumor cells in H&E stained slides from the same specimen and 3) array CGH analysis revealed the presence of CN aberrations larger than 5 Mb. Type A thymomas were included based on pathology review only, since they usually do not present CN aberrations, but are rich in epithelial cells. Exonic sequences were enriched using different capture-based platforms. Exome capture procedures were performed according to the respective vendors' instructions. Exon-enriched libraries were subsequently paired-end sequenced using Illumina's Genome Analyzer-II or HiSeq2000.
Exome Sequencing Data Analysis Workflow:
Raw FASTQ sequence reads were firstly mapped to human genome 19 (USCS) using Novoalign and then local realignment was performed around insertions/deletions (INDELs) using Genome Analysis Tool Kit (GATK). The duplicated reads were removed using Picard tool (http://picard.sourceforge.net/) and base quality score recalibration was performed using Genome Analysis Tool Kit (GATK). Using VarScan2 (Kobolt et al., Bioinformatics, 25: 2283-5 (2009)), somatic mutations were identified by comparing the tumor and normal bam files of each patient. The detected somatic single nucleotide variations (SNVs) and INDELs were annotated using snpEff. Predicted SNVs and INDELs were further filtered and accepted if all of the following requirements were met: 1) at least 4 reads carrying the mutation in tumor bam file, 2) mutations present in more than 20% of the reads mapped to the mutation locus, 3) at least eight reads covering the mutation locus in the normal bam file and 4) no more than 2% of reads carrying the mutation in normal bam file. In order to further reduce the false positive calls, mutations identified as germline in at least one different patient blood control were filtered out.
The somatic mutations of the coding regions identified using exome sequencing have been further annotated using Annovar (Wang et al., Nucleic Acids Res., 38: e164 (2010)) with NCBI dbSNP Build 137data, Cosmic database, SIFT (Kumar et al., Nat. Protoc., 4: 1073-81 (2009)) and Polyphen2 (Adzhubei et al., Nat. Methods, 7: 248-9 (2010)) scores.
Confirmation and Re-Sequencing of the Selected Mutated Genes:
The exome sequencing data was validated using independently prepared libraries for high depth sequencing on MISEQ sequencers (Illumina) using a custom panel of 197 genes. DNAs were extracted and fragmented by sonication. Then, indexed DNA libraries were prepared by three successive steps of end-repair, A-tailing and adapter ligation to the DNA fragments. In subsequent PCR amplification steps, primers containing a flow cell attachment site (P5), sequencing primer sites for index read (Index SP) and application read two (Rd2 SP), unique six by indices (Index) and a second flow cell attachment site (P7), were incorporated. The indexed libraries were then pooled in groups of up to 12, target enriched (Agilent), and sequenced. Sequence data were processed with in-house variant calling pipeline, which includes BWA alignment (Li et al., Bioinformatics, 25: 1754-60 (2009)), GATK local realignment (McKenna et al., Genome Res., 20: 1297-303 (2010)), Strelka somatic variant calling (Saunders et al., Bioinformatics, 28: 1811-7 (2012)), SnpEff and SnpSift variant annotation (Cingolani et al., Fly (Austin), 6: 80-92 (2012)).
Sanger Sequencing of GTF2I Locus:
Sanger sequencing is able to detect mutations if present in a substantial percentage of cells (15-20%). This limits the possibility to detect GTF2I mutations if the gene and the pseudogene sequences were amplified at the same time with non-specific primers because the mutation would be present in only 1:6 (17%) of the amplicons if heterozygous. In order to design specific primers for GTF2I, the nucleotide difference in exon 15 (C in the gene and T in the pseudogenes) was noted. A forward primer (ATCCCGTACCCTCTTTTCC) (SEQ ID NO: 20) was designed with its last 3′ base covering the C nucleotide that is specific for the GTF2I gene. The reverse primer (AGACAAGAGTTCAACAGG) (SEQ ID NO: 21) anneals to both GTF2I and pseudogene sequences. These primers were tagged with M13 forward and reverse sequences. DNA containing exclusively GTF2I sequence or the pseudogene sequences was used for the optimization of the PCR conditions. The plasmids containing exclusively GTF2I exon-15 or the pseudogene sequences were generated during the TopoTA cloning experiments. With a melting temperature of 62.5° C. only the GTF2I sequence was amplified. PCR was performed using Taq DNA Polymerase (Invitrogen) with 1.5 mM MgCl2, and 200 nM of forward and reverse primers, according to the following amplification STEPs: STEP 1: 94° C. 1:00 min; STEP2: 94° C. 30 sec, 62.5° C. 30 sec, 72° C. 45 sec (×35 times); STEP3: 72° C. 7:00 min. The amplicons were purified using ExoSap-IT (USB) and sequenced according to Sanger method with M13 Forward and M13 Reverse primers. The chr7:74146970 locus was inspected for mutations on both strands using Mac Vector.
GTF2I Deep Sequencing:
Using PCR amplification followed by direct deep sequencing, the chr7: 74146970T/A mutation of GTF2I was sequenced in 250 samples. Forward and reverse primers were tailed with Illumina Adapter tags for downstream next generation sequencing using the BioMark HD System (Fluidigm) and Access Array Integrated Fluidic Circuit (IFC) chips and kits (Fluidigm). Additionally, PCR products were indexed using an 8-mer oligo barcode. DNA was sequenced using 500-cycle MISEQ Reagent Kits V2 (Illumina) and the MISEQ Benchtop Sequencer (Illumina).
GTF2I Deep Sequencing Data Analysis:
In order to avoid potential alignment problems arising from the presence of the two pseudogenes homologous to GTF2I in the genome, a new algorithm was developed to avoid the alignment step altogether. The sequence of the primers used in the target selection/library preparation of the DirectSeq protocol are present at the 5′ end of each (valid) sequencer read. Only reads with a perfect match to the first 10 nucleotides (nt) of any of the DirectSeq primers were retained for further analysis. Depending on the relative position (Tables 2 and 3) with respect to the recognized primer sequence, the nucleotide N1 discriminating between gene/pseudogene and the nucleotide N2 discriminating between variant/wild type were identified. Across the entire dataset, the number of all possible combinations was counted for N1, N2 for each primer. This count included nucleotide combinations not fitting the combinations CT (gene/WT), CA (gene/mutant), TT (pseudogene/WT), TA (pseudogene/mutant) expected in the gene/pseudogene, WT/variant model as controls. These non-canonical combinations typically made up less than 1% of the reads associated with the primer, in line with the typical error rate of Illumina sequencers. Subsequently, this number was used as an estimate for the read error rate R as long as this estimate exceeded R>0.5%, otherwise R=0.5% was used as the noise estimate. A variant was called if the number of reads compatible with a mutated gene exceeded the number of reads expected as a result of read errors by at least 5*R, i.e.




  
    
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P-values for the association of the mutation status with WHO classification and stage were estimated using a Chi-square test utilizing a flat distribution as the null-model.

Survival Analysis:
Kaplan-Meier method was used to generate survival curves. Disease Related Survival (DRS) was calculated from the date of the first histological diagnosis to the date of death due to tumor progression. DRS was chosen instead of the overall survival because, given the expected long survival, especially in the most indolent thymomas, and the advanced age of many patients, death was often not related to the tumor. Survival curves were compared by the Log Rank test. A Cox proportional hazard model was initially built for the univariate analysis, which included WHO groupings (A, AB, B1 vs B2, B3, TC), stage (I-II vs III-IV), completeness of resection (R0 vs R1-R2) and GTF2I mutation status. Subsequently, multivariate analysis was performed including prognostic factors found in the univariate analysis (p<0.1). All tests were performed using the SPSS version 20 (SPSS, Inc., Chicago, Ill.).
Structural Model and Molecular Dynamics of GTF2I:
A structural model of GTF2i was based on the solution structure of GTF2I (Brookhaven Protein Data Bank [PDB]: 2DN4). GTF2I was energy minimized using the consistent valence force field AMBER 10.0 simulation package. The cutoff for nonbonded interaction energies was set to 00 (no cutoff); other parameters were set to default. The dielectric constant was set at ∈=4 to account for the dielectric shielding found in proteins. The minimization was conducted in two steps: the first using steepest descent minimization for 5000 cycles and then using conjugate gradient minimization until the average gradient fell to <0.01 kcal/M.
Using the energy-minimized structure of GTF2i as the initial model, three ns molecular dynamics (MD) simulations with a distant-dependent dielectric constant were conducted by using the SANDER module of the AMBER 10.0 simulation package with the PARM98 force-field parameter. MD simulations were performed using 0.001-ps time steps with temperature set at 300° K. The SHAKE algorithm was used to keep all bonds involving hydrogen atoms rigid. Temperature and pressure coupling algorithms (Berendsen et al., J. Chem. Phys., 81: 3684 (1984)) were used to maintain constant temperature and pressure. Electrostatic interactions were calculated with the Ewald particle mesh method (Darden et al., J. Chem. Phys., 98: 10089 (1993)), and a dielectric constant at one Rij and a nonbonded cutoff of 14 Å was used to the approximate electrostatic interactions and van der Waals interactions. Structural analyses were done using the SYBYL X.1 (Tripos International, St. Louis, Mo.) molecular modeling program.
Ectopic Expression of GTF2I Mutation:
pEBB plasmids containing GTF2I beta (NM_033000.2) and delta (NM_001518.3) isoforms were purchased from Addgene. The GTF2I sequence in the plasmid was sequenced and two synonymous and one non-synonymous mutations were identified in both isoforms. The non-synonymous mutations were corrected using site directed mutagenesis. Similarly, the chr7:74146970 T/A mutation was introduced in the plasmid using QuikChange Site-Directed Mutagenesis Kit (Agilent), according to the vendor protocol. The primers to introduce the mutation were designed using primer X and included the following: sense primer: (SEQ ID NO: 22) and antisense primer: (SEQ ID NO: 23).
A beta L404H and a delta L383H mutated isoform were generated in a pEBB plasmid. The mutated and wild type GTF2I isoforms were first moved into a donor vector (pDONR221vector) through a recombinase reaction (GATEWAY BP CLONASE II Enzyme mix, Invitrogen) and subsequently moved into a lentiviral vector (pLenti6.3/V5-DEST GATEWAY Vector kit, Invitrogen) using GATEWAY LR CLONASE II Enzyme mix (Invitrogen), according to the vendor's instructions. The pLenti6.3/V5-DEST plasmids with GTF2I WT and mutated isoforms were transfected into NIH-3T3 cells (purchased from ATCC, Manassas, Va.) using Lipofectamine LTX (Invitrogen). NIH-3T3 cells were grown in DMEM (Gibco, Invitrogen) supplemented with 50 U/mL penicillin, 50 U/mL streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (Invitrogen) and grown in a 37° C. incubator with humidified 5% CO2 atmosphere. P-BABEpuro vector containing HRASV12H was purchased from Addgene and used as positive control for softagar assay (Li et al., J. Biol. Chem., 279(36): 37398-406 (2004)). Stable clones were selected using 8 μg/ml blasticidin or 1 μg/ml puromycin when appropriate (Gibco, Invitrogen). For each GTF2I isoform (both wild type (WT) and mutated), four independent stable pool transfectants were obtained. Stable ectopic GTF2I expression was confirmed by western blot using anti-V5 antibody (Invitrogen) and anti-α-Tubulin (Sigma-Aldrich) as loading control.
Cell Proliferation and Soft Agar Assay:
HRASV12H and mock-transfected NIH-3T3 cells were the positive (Li et al., J. Biol. Chem., 279(36): 37398-406 (2004)) and negative controls, respectively. 1000 cells/well were plated in 96-well plates and tested for cell proliferation using a luminescent method (CELLTITER-GLO Luminescent Cell Viability Assay, Promega, Madison, Wis.) at 24, 48, 72 and 96 hours. For WT and mutated GTF2I isoforms, four different stable pools were included in the experiments. Each experiment was replicated at least three times and the average of cell proliferation was calculated for both WT and mutated β- and δ-GTF2I isoforms. Soft agar assay was performed as previously described (Chen et al., Int. J. Oncol., 37(4): 963-71 (2010)) using 5000 cells for each well of a 6-well plate. Experiments were performed at least three times and averages were calculated from the results of four distinct pools of each GTF2I variant. Expression of endogenous GTF2I was tested by western blot using anti-GTF2I antibody (Cell Signaling, Danvers, Mass.) in frozen primary thymic epithelial tumors for which GTF2I mutation status was available.
Sequencing of RNA from GTF2I and its Pseudogenes:
Primers were designed in order to selectively amplify the transcripts of GTF2I or of its pseudogenes. Primers specific for GTF2I were located on its exon 10 (5′-SEQ ID NO: 24) and on the junction of exon 16-17 (5′-SEQ ID NO: 25). Primers specific for the pseudogenes were on their exon 1 and on exon 5-6 junction (reverse primers have the same sequence). All the primers were flanked with M13 primer sequences. Tumor RNA was converted into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif.). Pseudogenes and GTF2I fragment were amplified by PCR using Taq DNA Polymerase (Invitrogen) according to the following program: STEP1: 94° C. 1:00 min; STEP2: 94° C. 30 sec, 55° C. 30 sec, 72° C. 45 sec (×35 times); STEP3: 72° C. 7:00 min. Amplicons of 503 bp (delta) and 566 bp (beta) were verified with a run on a 1.2% agarose gel and then purified from unincorporated nucleotides and residual primer using EXOSAP-IT (USB). PCR products were sequenced using M13 primers and Sanger technology.
TopoTA Cloning for Detection of T/a Mutation in GTF2I Gene and Pseudogenes:
Primers able to amplify the genomic DNA of GTF2I and its pseudogenes at the same time were designed. Forward (CTCAAGCCATAAAAGCCA) (SEQ ID NO: 26) and reverse (AGACAAGAGTTCAACAGG) (SEQ ID NO: 27) primers were purchased from Integrated DNA Technologies (IDT Inc., Coralville, Iowa) in order to amplify a fragment of 218 bp containing C or T, a signature that distinguishes GTF2i gene from its pseudogenes, and the T/A mutation locus. PCR was performed using Taq DNA Polymerase (Invitrogen) according to the following amplification STEPs: STEP1: 94° C. 1:00 min; STEP2: 94° C. 30 sec, 55° C. 30 sec, 72° C. 45 sec (×35 times); STEP3: 72° C. 7:00 min. PCR amplicons were cloned into a pCR4-TOPO vector using TopoTA Cloning Kit for Sequencing (Invitrogen), according to vendor's instructions. E. Coli DH5α bacteria were transformed with the plasmid and plated on an LB-agar Petri dish with 100 μg/mL kanamycin selection and incubated overnight at 37° C. Colonies, selected for sequencing, were resuspended in 5 mL of LB media with 100 μg/mL kanamycin and grown overnight at 37° C. in a shaking incubator. DNA was extracted using QIAPREP Spin Miniprep Kit (Qiagen) and plasmid sequenced using M13 Forward and Reverse primers.
Example 1
This example demonstrates the identification of copy number aberrations in aggressive histotypes of thymic epithelial tumors (TETs).
Large CN aberrations, affecting an entire chromosome arm (arm level CN aberrations), were present in more aggressive histotypes, in particular in TC, B3 and B2, but uncommon in the more indolent A and AB thymomas. Two major clusters of TETs were identified according to their arm level CN aberrations, the first encompassing most of A and AB tumors, and the second including mainly the aggressive histotypes. The aberrations defined two clusters of TETs: one with few arm level CN aberrations and one rich in arm level CN aberrations. These clusters trend to correlate with WHO histotypes and with the presence GTF2I mutations. Overall, the most frequent arm level CN losses involved chromosomes 6 (6p 26%; 6q 29%), 3p (22%) and 13q (18%). The most frequent arm level CN gains affected chromosomes 1q (55%), 7 (7p 20%; 7q 15%) and 20p (17%). Of particular interest were focal CN aberrations that appeared to be within the significant peaks identified using GISTC, an analysis aimed at identifying significant regions of CN aberrations possibly driving the cancer growth. These included the focal amplification of BCL2 locus, which correlated with an increased expression of BCL2 transcript according to RNAseq data. The average fragments per kilobase of exon per million fragments mapped (FPKM) value measured with BCL2 amplification was 41.52 with a standard deviation (SD) of 12.94 (n=3), and the average FPKM value measured without BCL2 amplification was 9.13 with a SD of 7.844 (n=8) (Mann Whitney p=0.0121).
Example 2
This example demonstrates the identification of a GTF2I mutation in TETs.
Exome sequencing revealed 722 somatic single nucleotide variations (SNVs) and 68 insertions/deletions (INDELs) in the coding regions of 28 TETs. An average of 28 mutations per sample was estimated (range 3-94). Thymic carcinomas (TCs) had a significantly higher number of mutations than thymomas (Mann Whitney-U; p=0.001). Moreover, mutations in several cancer genes were observed in more than one case of TC, but they were usually single events in thymomas. A customized 197-cancer gene panel was designed to sequence 52 TETs including 26 of the 28 tumors already characterized by whole exome sequencing. The two methods were highly correlated (Chi square p<0.001).
In thymomas, recurrent mutations were frequently observed in only one gene: GTF2I. This mutation was strikingly prevalent in A and AB histotypes, and all the GTF2I mutated cases presented the same single nucleotide change T/A at the same position chr7:74146970 (Table 1). This mutation was not previously described as a polymorphism in dbSNP137 and ESP5400 databases, or as a somatic mutation in tumors (COSMIC database). The missense mutation of GTF2I led to a leucine to histidine substitution and consequently altered deleteriously the protein structure and/or function according to SIFT and Polyphen2 predictions. The mutation affects the second conserved “GTF2I repeat domain” of the protein in proximity to its DNA binding site. Sanger sequencing confirmed the presence of mutations in tumors but not in normal DNA in all the mutated cases detected by exome sequencing.
The mutation was found in GTF2I sequence but not its pseudogenes: GTF2IP1 and LOC10093631 (Tables 2 and 3; see also Examples 7 and 8). GTF2I mutation (T/A) was mapped on exon 15. This region matches exon 4 of the pseudogenes, and differs by only one nucleotide: C in GTF2I and T in pseudogene sequences. The allele frequencies theoretically present in a cell were as follows: one GTF2I mutated allele (1:6,  ̃17%), one GTF2I WT allele (1:6,  ̃17%) and four pseudogenes wild type alleles (4:6,  ̃67%). TopoTA cloning was performed in four tumors with GTF2I mutation. Sequencing of cloned amplicons identified the mutation only in GTF2I but not in the pseudogenes. Deep sequencing was performed on five tumors with GTF2I T/A mutation. The mutation was found in GTF2I only and not the pseudogenes, which equals the mutation rate of  ̃17% or 1 out of 6 alleles (2 GTF2I+4 pseudogene alleles). The mutation was not identified in the negative controls.
The data demonstrate an increasing number of mutations and CN aberrations from type A to TC with the exception of GTF2I mutations.









TABLE 1





Number of Patients




Sequenced
WHO Histotype
Frequency of GTF2I Mutation







56
A
82%


54
AB
74%


28
B1
32%


32
B2
22%


62
B3
21%


36
TC
 8%



















TABLE 2







WT reads
Mutation (MUT) reads




















Normal Samples





GTF2I
31.2%
0.0%



Pseudogenes
68.2%
0.0%



WT Tumors



GTF2I
31.2%
0.1%



Pseudogenes
67.8%
0.1%



Mutated Tumors



GTF2I
28.3%
4.1%



Pseudogenes
65.7%
0.4%




















TABLE 3







Normal Samples
homozygous N1/N2 =
GTF2I
33%



C/T



homozygous N1/N2 =
LOC100093631
67%



T/T
GTF2IP1


WT Tumors
homozygous N1/N2 =
GTF2I
33%



C/T



homozygous N1/N2 =
LOC100093631
67%



T/T
GTF2IP1


Mutated Tumors
N1/N2 = C/A
GTF2I
17%



N1/N2 = C/T
GTF2I
17%



homozygous N1/N2 =
LOC100093631
67%



T/T
GTF2IP1









Example 3
This example demonstrates the prevalence of the GTF2I mutation in TETs.
The frequency of the GTF2I mutation was assessed on a total of 274 TETs (Table 4). Tumors rich in cancer cells (>50%) were evaluated for GTF2I mutation using Sanger sequencing (199 TETs). The GTF2I mutation was further sequenced in 250 TET samples, using a deep sequencing approach, which also included 78 tumors relatively rich in non-neoplastic thymocytes (cancer cells <50%). A total of 172 cases were sequenced using both Sanger and deep sequencing that demonstrated a good concordance in mutation detection (Examples 7 and 8). GTF2I mutation (chr7:74146970T/A) was observed in 199 of the 270 TETs evaluated (44%), and most commonly in A (82%) and AB (74%) thymoma (78% overall, Table 1). The frequency of mutation progressively decreased in more aggressive histological types to only 8% in TC (3/36). More mutations were observed in early stages (I-II, 57%) than in advanced stages of the disease (III-IV, 19%, Chi-Square p<0.001). Survival data were available for 214 patients (median follow-up 39.4 months 95% CI: 30.3-48.5). Patients with tumors bearing GTF2I mutations had a better prognosis than wild type (WT) GTF2I (96% vs 70% 10-year survival, respectively; Log-Rank p<0.001), reflecting the higher mutation frequency in less aggressive tumors. All of the three TCs with GTF2I mutation were alive with a median follow-up of 27.6 months (95% CI 0-70%). Within thymomas there was a more favorable outcome in tumors with mutated than WT GTF2I (96% vs 88% 10-year survival; Log-Rank p=0.057). In models of multivariate analysis that include only two covariates, both GTF2I status and WHO classification were prognostic factors independent of disease stage. In contrast, GTF2I status and WHO classification were dependent from each other. Combining stage, WHO classification and GTF2I status in the same model, only stage was an independent prognostic factor.













TABLE 4








GTF2I
MUT
WT



Patient characteristics

Total
Sequenced
GTF2I
GTF2I
p-value





















Total in study

286
274
43%
57%



Patients

282
270
44%
56%


Age

median 56



range (20-86)


Sex
Female
139
135
41%
59%
0.327



Male
143
135
47%
53%



uk
4


WHO
A
58
56
82%
18%
p < 0.001*



AB
55
54
74%
26%



B1
28
28
32%
68%



B2
33
32
22%
78%



B3
65
62
21%
79%



TC
41
36
 8%
92%



NEC
4
4
0
100% 



Micronodular
2
2
50%
50%


Stage
I
41
40
58%
42%
p < 0.001**



IIA
55
53
64%
36%



IIB
73
71
51%
49%



III
29
29
35%
65%



IVA
21
19
16%
84%



IVB
34
32
 6%
94%



uk
33
30
37%
63%


Resection
R0
139
136
49%
51%
p = 0.0267



R1
18
18
33%
67%



R2
13
13
15%
85%



uk
116
107
41%
59%


Paraneoplastic
All
66
65
43%
57%
p = 0.636***


Syndromes
Myasthenia
63
62
45%
55%



No
145
136
39%
61%



uk
75
73
52%
48%












CGH
65
53
32%
68%



Whole Exome Sequencing
28
28
21%
79%


Transcriptome Sequencing
25
25
28%
72%


197-gene Re-sequencing
52

—
—


Sanger Sequencing
199
199
61%
39%


GTF2I Deep Sequencing
250
250
42%
58%


Samples Sequenced for GTF2I

274
43%
57%









Example 4
This example demonstrates the expression of the GTF2I mutation in thymomas.
RNAseq demonstrated the expression of GTF2I mutation in all thymomas evaluated (five type A and two type AB thymomas). Both the mutated and WT GTF2I alleles were expressed. The median number of reads covering the mutation locus was 1114 and the mutated allele was present in an average of 47% (range 44-49%) of total GTF2I reads. Sanger sequencing of the cDNA confirmed the mutant GTF2I in the transcripts of all these samples. There are five known isoforms of GTF2I (Roy et al., Gene, 492: 32-41 (2012)) that differ by alternative splicing of exons 10 and 12. Using transcriptome sequencing, it was demonstrated that exon 10 was almost not expressed; whereas exon 12 was expressed with approximately half of reads both in mutated and WT samples observed in the neighboring exons. Indeed, according to cufflinks estimates, the expression of β and δ isoforms was significantly higher than the other three. These results suggest that the β and δ-isoforms are predominantly expressed in TETs. Using RT-PCR and specific primers designed for β- or δ-isoforms, the expression of the two isoforms was confirmed and the T/A mutation was detected in both of them. The observed T/A mutation corresponded to L404H in β-isoform and L383H in δ-isoform of GTF2I.
Gene expression was estimated from RNAseq data. Tumors with GTF2I mutations tended to cluster together in a group rich in A and AB thymomas, similarly to what was observed in CGH results.
Example 5
This example demonstrates the functional characterization of the GTF2I mutation.
GTF2I mutation is included within the amino acid sequence RILLAKE (SEQ ID NO: 28) that may represent a non-canonical destruction box resembling the destruction box (RXXLXX[LIVM]) (SEQ ID NO: 29) found in cyclins, PLK1 and Securin (King et al., Mol. Biol. Cell, 7: 1343-57 (1996)). The RILLAKE (SEQ ID NO: 28)>RILHAKE (SEQ ID NO: 30) mutation may render GTF2I unrecognizable by the protein degradation machinery (Desgranges et al., Mol. Cell Biol., 25: 10940-52 (2005)). Mutant tumors showed higher GTF2I expression than WT tumors at protein (Table 5) but not mRNA level (Table 6). To understand the biological significance of the GTF2I mutation and the elevated protein expression in mutant tumors, L404H and L383H β- and δ-isoforms of GTF2I were created by site-directed mutagenesis. pLenti6.3/-V5Dest expression vectors carrying the WT and mutated isoforms were stably introduced into NIH-3T3 cells. All β- and δ-isoforms accelerated cell proliferation in comparison to mock-transfected cells. Both β- and δ-mutant isoforms increased cell proliferation more than their WT counterparts. In contrast, no significant differences were observed in soft-agar colony formation between the mutant and WT GTF2I-transfected cells. The mutant clones (both β- and δ-isoforms) exhibited higher levels of GTF2I protein than WT ones whereas such a difference in expression was not observed at mRNA level. These results indicate that L404H and L383H mutations may augment GTF2I expression post-transcriptionally which in turn may accelerate cell proliferation by upregulation of cell cycle control proteins (Ashworth et al., Cell Cycle, 8: 596-605 (2009)).










TABLE 5








Normalized quantification of



histotype
GTF2I protein expression




















WT
B2
4.06



WT
B3
1



WT
B3
0.31



WT
B3
0.29



MUT
A
4.16



MUT
A
13.03



MUT
B1
5.33



MUT
A
8.05



WT
B1
0.02



WT
AB
0.01



WT
B2
1



WT
B1
0.02



MUT
A
3.67



MUT
A
2.56



WT
B2
1



WT
B2
0.07



WT
TC
0.51



WT
B3
0.03



MUT
AB
1.07



MUT
A
2.63



MUT
AB
3.14



















TABLE 6









Average of GTF2I mRNA expression (FPKM)










Mutated Tumors
Wild Type Tumors













Delta (NM_001518)
140,000
210,000


Beta (NM_033000)
90,000
150,000









Example 6
This example demonstrates the presence of fusion genes in TETs.
The presence of fusion genes was investigated using RNAseq data and two independent algorithms: FusionMap and DeFuse. All the predicted fusions were confirmed using RT-PCR followed by Sanger sequencing. Fusion transcripts were identified in seven of the 25 tumors evaluated including the TY82 TC cell lines known to carry the BRD4-NUT fusion. In these tumors the number of fusion genes ranged between 1-16. There was an average of one fusion transcript in each case (range 0-16). A B2 thymoma presented a remarkably high number of fusion transcripts (16 fusions) compared to the other samples. Although fusion proteins of GTF2I have recently been described in angiofibromas (Arbajian et al., Genes Chromosomes Cancer, 52: 330-1 (2013)), none of the fusion transcripts observed in TETs involved GTF2I sequence.
Example 7
This example demonstrates that the T/A mutation identified in Example 2 maps to the GTF2I locus but not to its pseudogenes.
GTF2I is a gene that spans 35 exons and is mapped on the long arm of chromosome 7 (chr7:74,072,030-74,175,022). The chr7:74146970 T/A mutation identified by exome sequencing is located in exon 15 of GTF2I. The T/A mutation was aligned to the same position of GTF2I exon 15 using either BWA (data not shown) or Novoalign algorithms.
There are two known pseudogenes of GTF2I: LOC100093631 and GTF2IP1. GTF2I exon 15 sequence differs by only one nucleotide from the sequence of the two pseudogenes (According to BLAT in the UCSC website: 99.5% identity, chr7:74629125-74629308 and chr7:72593127-72593310). Both these pseudogenes map to the long arm of chromosome 7 (chr7:72569012-72621336 and chr7:74601104-74653445, respectively). GTF2IP1 maps on the negative strand of chromosome 7, whereas LOC10093631 and GTF2I reside on the positive strand.
These two pseudogenes possess exons and their transcripts are processed into mature mRNAs, but proteins are not translated from either of them (de Jong et al., Eur. J. Cancer, 44: 123-30 (2008)). The two pseudogenes have a head (exon 1) that has no homology with GTF2I sequence and a tail (exon2—3′-UTR) that is very similar (99% identical; GTF2I/GRF2IP1 31,794 bp identical on 31,929 bp of sequence and GTF2I/LOC100093631 31,782 bp identical on 31,925 bp of sequence).
The transcripts of the two pseudogenes are almost identical since their sequences differ by only three nucleotides out of 3631 bp. Part of the GTF2I RNA sequence, exon13-3′UTR, is closely related to the portion of pseudogene sequences exon2-3′UTR. GTF2I exon15-3′UTR and pseudogene exon2-3′UTR RNA sequences are 99% identical. Among 3218 bp of shared sequence, GTF2I transcript differs only by four and three nucleotides from LOC100093631 and GTF2P1, respectively. The first exon of the two pseudogenes is not related to GTF2I sequence but closely resembles the first exon of GATS (93.9% identity) and GATSL2 (98.9% identity) genes. GTF2I sequence from exon 1 to exon 12 is unique and BLAT search did not reveal close similarity to other genomic regions.
Because the chr7:74146970 T/A mutation was mapped to GTF2I exon 15, it resides in the region of high homology between the gene and the pseudogenes. Therefore, it was necessary to demonstrate that this T/A mutation really belongs to GTF2I locus rather than to the pseudogenes. At the genomic level, exon 15 of GTF2I is  ̃4500 bp away from the point where GTF2I and the pseudogene sequences start to differ. In contrast, at mRNA level the distance is only 217 bps apart in the 8-isoform. Therefore, it was possible to design specific primers able to distinguish mRNA sequences of GTF2I from those of the pseudogenes. The T/A mutation was observed only in GTF2I cDNA but not in the cDNA from the pseudogenes in the five samples tested. Mutations were not identified in GTF2I or in the pseudogenes in the negative controls (four samples without GTF2I mutation). To further demonstrate that the mutation belongs to GTF2I locus at the genomic level, it was noted that GTF2I exon 15 and pseudogenes exon 4 differ by one nucleotide. The nucleotide chr7:74146870 is a cytidine in GTF2I sequence whereas the corresponding chr7:72593177 and chr7:74629258 in LOC100093631 and GTF2IP1 were thymidines. Because polymorphisms have not been described in these three positions, according to dbSNP137, the C/T single nucleotide difference could be used as a marker of GTF2I and pseudogenes sequences. Therefore, if the sequenced DNA strand contains both the C/T marker and the T/A mutation, one can ascertain whether the sequences with the mutation come from the gene or from the pseudogenes. Thus, it was possible to design primers that indistinctly amplify GTF2I and pseudogene sequences and then to determine if the T/A mutation belongs to the gene or to the pseudogenes using the C/T marker. In order to sequence just one strand of DNA, two strategies were adopted. The first was based on TopoTA cloning and the second on deep-sequencing technologies (MiSeq, Illumina). For TopoTA cloning, primers were designed in order to amplify a 218 bp DNA fragment that includes the C/T (gene/pseudogenes) marker and the site of mutation (chr7:74146970 T/A). The amplicons, generated using PCR reactions, were cloned into a pCR4-TOPO plasmid so that the expression of the toxic ccdB gene in the vector backbone, was disrupted. E. Coli DH5α bacteria were transformed and plated in a Petri dish with Ampicillin selection. Only bacteria carrying the amplicons, but not those carrying the empty vector, were able to grow. Colonies (17-40 for each tumor) that carry a single copy of DNA amplicons were picked, expanded and their DNA sequenced using specific sequencing primers. According to exome sequencing results the chr7:74146970 T/A mutation was expected to be heterozygous. Because the two pseudogenes were expected to be homozygous wild type, the mutated GTF2I amplicons should be 1:6 ( ̃17%) of the amplicon sequenced. Four different tumors have been studied using TopoTA cloning, in all of them approximately 1:3 of the colonies were from GTF2I (average 35%; range 30-40%) and included all the T/A mutations. Colonies with a copy of mutated GTF2I were about 12% (6-18%), slightly less than the 17% expected, which is compatible with some normal cell contamination of the samples.
A customized deep sequencing assay was developed in order to discriminate mutations in GTF2I or pseudogenes. It was based on two pairs of primers (P1 and P3) able to amplify a region that includes the T/A mutation site and the Gene/Pseudogene marker (C/T). An additional pair of primers (P2) was included in the deep sequencing assay exclusively for genotyping purposes and was designed in order to enrich the amplification of GTF2I sequences. Twelve samples were multiplexed on a MISEQ flow cell in order to obtain extremely high read counts over the region of interest (average number of total reads was 2,306,186 range 1,137,605-4,122,859; average number of informative reads was 585,714 range 167,054-1,449,423). Five samples had GTF2I mutation and seven were negative controls that included three normal DNA, three tumors without GTF2I mutation and a thymic carcinoma cell line without GTF2I mutation. The deep sequencing assay demonstrated GTF2I mutation only in the five positive cases but not in the negative controls. The reads with the mutations belonged exclusively to GTF2I sequence in three cases, whereas two tumors presented 1% of the pseudogene reads with the mutation. The frequency of GTF2I reads with the mutation was close to what expected (average frequency 11.5%, range 8-18%, expected frequency 17%). The few pseudogene mutated reads (1% in two cases) did not support the presence of a pseudogene allele carrying the T/A mutations (expected 17%). These reads may be related to polymerase errors introduced in the amplification step or they can represent a real pseudogene T/A mutation present in a subclone of few tumor cells.
When all samples were evaluated, for which the GTF2I mutation was genotyped using MISEQ (n=250), the results matched the expectation (Tables 2 and 3). Results were evaluated separately for the 12 normal samples, the WT tumors and the tumors with GTF2I mutations. Normal and WT tumors have an inconspicuous proportion of mutated reads either from GTF2I or from the pseudogenes. In the tumors with GTF2I mutations, the average mutated reads were 4.01% from GTF2I and 0.4% from the pseudogenes. This low proportion of mutated GTF2I reads was expected for the presence of tumors with extensive components of non-neoplastic thymocytes. Even in the tumor with highest fraction of mutated reads belonging to the pseudogenes (2.96%), the reads from GTF2I were significantly higher (16.13%).
The results of TopoTA cloning, MISEQ and the transcript sequences demonstrated that the chr7:74146970 T/A mutation unambiguously involves the GTF2I sequence.
Example 8
This example demonstrates the frequency of the GTH2I mutation.
According to the exome sequencing and transcriptome sequencing results, GTF2I mutation was common in the A and AB subgroups of thymomas. A larger cohort of patients was then screened for GTF2I mutation using standard Sanger technology and the deep sequencing approach described above. Sanger sequencing revealed GTF2I T/A mutation in 78 (39%) out of 199 thymic epithelial tumors. The somatic nature of the mutation was confirmed by the absence of GTF2I mutation in normal DNA from patients' blood. A limitation of the standard Sanger methodology is the presence of non-neoplastic thymocytes that can outnumber the epithelial tumor cells in some histotypes (in particular some AB, B1, B2). Therefore, only tumors with at least 50% cancer cells were sequenced using Sanger technology. Alternatively, a deep sequencing approach was considered for screening for the presence of the GTF2I mutation in thymocytes-rich tumors. However, samples with an extremely high proportion of nonneoplastic thymocytes represent a challenge for the detection of GTF2I mutations even using deep sequencing. This can be the case for some B1 thymomas. According to the deep-sequencing assay, 106 tumors (42%) out of 250 had the GTF2I mutation. Sanger and deep sequencing technologies showed good concordance on the 172 samples assayed with both methods. The two methods detected GTF2I mutations in 59 cases and excluded its presence in 88 tumors. The deep sequencing approach was designed to be more sensitive than the Sanger method in lymphocyte rich tumors, where GTF2I mutation was identified in 20 additional cases. These cases were considered mutated. In five samples (3%), mutations were observed only using the Sanger method but not using the deep sequencing approach. The mutation status of these samples was considered undetermined. When discordant results were observed in cases with three different sequencing technologies, samples were considered mutated when two of these technologies detected the mutation.
Combining exome sequencing, Sanger sequencing and GTF2I deep sequencing data, GTF2I mutation was observed in 119 tumors out of the 274 evaluated (43%). The frequency of mutation was higher in thymomas (50%) than in thymic carcinomas (8%; Fisher exact test p<0.001).
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A method for detecting a GTF2I mutation in a tumor, the method comprising:
obtaining genetic material from the tumor;
assaying the genetic material; and
detecting a mutation in at least one copy of GTF2I genetic sequence, wherein the mutation is selected from the group consisting of:
(a) the T>A mutation at position 75,041 of SEQ ID NO:1;
(b) the T>A mutation at position 1208 of SEQ ID NO: 3;
(c) the T>A mutation at position 1211 of SEQ ID NO: 4;
(d) the T>A mutation at position 1271 of SEQ ID NO: 5;
(e) the T>A mutation at position 1148 of SEQ ID NO: 6; and
(f) the T>A mutation at position 1205 of SEQ ID NO: 7.

2. The method of claim 1, wherein the tumor is a thymic epithelial tumor (TET).

3. The method of claim 2, wherein the thymic epithelial tumor is thymoma or thymic carcinoma.

4. The method of claim 1, wherein assaying comprises carrying out a polymerase chain reaction (PCR) assay that specifically detects the mutation.

5. The method of claim 1, wherein the genetic material is genomic DNA.

6. The method of claim 1, wherein the genetic material is RNA or cDNA of GTF2I β isoform or GTF2I δ isoform.

7. The method of claim 1, wherein the genetic material is RNA.

8. The method of claim 1, wherein the genetic material comprising the mutation comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8-9, 11, and 13 and complements thereof.

9. The method of claim 1, wherein the genetic material comprising the mutation comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8-14 and complements thereof.

10. The method of claim 1, wherein the method comprises detecting the presence of the T>A mutation at position 75,041 of SEQ ID NO: 1.

11. A method for detecting a mutated GTF2I protein in a tumor, the method comprising:
obtaining a sample from the tumor;
assaying the sample;
and detecting a mutation in GTF2I protein selected from the group consisting of:
(a) the mutation of Leu to His at position 403 of SEQ ID NO: 31;
(b) the mutation of Leu to His at position 404 of SEQ ID NO: 32;
(c) the mutation of Leu to His at position 424 of SEQ ID NO: 33;
(d) the mutation of Leu to His at position 383 of SEQ ID NO: 34; and
(e) the mutation of Leu to His at position 402 of SEQ ID NO: 35.

12. The method of claim 11, wherein assaying comprises contacting the sample with an antibody that specifically binds to mutant GTF2I protein and does not bind to wild type GTF2I protein, thereby forming a complex, and detecting the complex.

13. The method of claim 11, wherein assaying comprises contacting the sample with an antibody that specifically binds to wild type GTF2I protein and does not bind to mutant GTF2I protein, thereby forming a complex, and detecting the complex.

14. The method of claim 11, wherein the tumor is a thymic epithelial tumor.

15. The method of claim 11, wherein the GTF2I protein with the mutation comprises the amino acid sequence of SEQ ID NO: 16 or 18.

16. The method of claim 11, wherein the GTF2I protein with the mutation comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1549.

17. A method of treating a thymic epithelial tumor in a human subject, the method comprising:
obtaining a sample from the thymic epithelial tumor;
assaying the sample; and
detecting one or both of (I) and (II):
(I) a mutation in at least one copy of GTF2I genetic sequence, wherein the mutation is selected from the group consisting of:
(a) the T>A mutation at position 75,041 of SEQ ID NO: 1;
(b) the T>A mutation at position 1211 of SEQ ID NO:4; and
(c) the T>A mutation at position 1148 of SEQ ID NO: 6;

(II) a mutation in GTF2I protein selected from the group consisting of:
(a) the mutation of Leu to His at position 404 of SEQ ID NO: 32; and
(b) the mutation of Leu to His at position 383 of SEQ ID NO: 34; and


treating the thymic epithelial tumor in the human subject when the mutation is present by surgically removing all or part of the thymic epithelial tumor without administering one or more of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy in an amount effective to treat thymic epithelial tumor in the human subject.

18. The method of claim 17, comprising treating the thymic epithelial tumor in the human subject when the mutation is absent by surgically removing all or part of the thymic epithelial tumor and administering one or more of (i) radiotherapy, (ii) chemotherapy, and (iii) immunotherapy in an amount effective to treat the thymic epithelial tumor in the human subject.

19. The method of claim 18, wherein the chemotherapy comprises administering one or more of doxorubicin, epirubicin, belinostat, cisplatin, carboplatin, cyclophosphamide, ifosfamide, vincristine, etoposide, paclitaxel, pemetrexed, 5′ fluorouracil, methyiprednisolone, octreotide, gefitinib, imatinib, and gemcitabine to the human subject in an amount effective to treat the thymic epithelial tumor in the human subject.