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Patent Application Titled "Anaplastic Thyroid Cancers Harbor Novel Oncogenic Mutations of the Alk Gene" Published Online

May 26, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Xing, Michael Mingzhao (Clarksville, MD); Kannan, Murugan Avaniyapuram (Baltimore, MD), filed on May 7, 2012, was made available online on May 15, 2014 (see also Patents).

No assignee for this patent application has been made.

Reporters obtained the following quote from the background information supplied by the inventors: "Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor subfamily of receptor tyrosine kinases (RTKs), with its encoding gene located on the short arm of chromosome 2 (1, 2), ALK was initially identified as part of an oncogenic fusion gene, NPM1-ALK (also known as NPMALK), in anaplastic large-cell non-Hodgkin's lymphomas (ALCL; 3). It is also part of the fusion gene EML4-ALK in non-small-cell lung cancer (NSCLC; 4). There are a few other ALK fusion genes, such as TMP3/4-ALK and RANBP2-ALK, in inflammatory myofibroblastic tumors (IMT; 5). The tyrosine kinase activities of these fusion ALK proteins are aberrantly activated and promote cell proliferation and survival (6, 7). ALK fusion proteins have also been shown to activate various signaling pathways, among which are the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the Ras.fwdarw.Raf.fwdarw.MEK.fwdarw.extracellular signal regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway with multiple interaction points to mediate the ALK signaling (8, 9).

"Recently, ALK mutations were found in 6% to 14% of sporadic neuroblastomas (10-14). ALK mutations were also reported in familial neuroblastomas (13, 14). Moreover, genetic amplification of the ALK gene could also occur in neuroblastomas or cell lines derived from this tumor (10, 11, 13, 15). Except for occasional mutations in the juxtamembrane domain, most ALK mutations identified so far are within the tyrosine kinase domain of ALK. ALK mutations and/or copy gain were found particularly in advanced and metastatic neuroblastomas, and patients with ALK mutations had a worse prognosis (11, 12, 14). Several common ALK mutations were shown to be functional. For example, siRNA-mediated knockdown of the ALK expression in cell lines harboring ALK mutants F1174L or R1275Q caused cell apoptosis and suppression of cell proliferation (12-14). The F1174L and another mutant ALK, K1062M, were shown to display increased tyrosine kinase activity and promote cell focus formation, cell transformation, and xenograft tumorigenecity in nude mice (10). The oncogenicity of ALK F1174L and R1275Q was also shown in another study (12). Genetic copy gain of the ALK is also functionally important, as suggested by the demonstration that inhibition of ALK in neuroblastoma cell lines harboring ALK copy gain induced cell apoptosis through reduced signaling of the PI3K/Akt and MAP kinase pathways (15).

"Mutations of the ALK gene have not been reported in human cancers other than neuroblastomas. As described herein, the present inventors investigated the mutation status of the ALK gene in various thyroid cancers, including well-differentiated papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) and undifferentiated anaplastic thyroid cancer (ATC). Prompted by the finding of ALK mutations in ATC, a rapidly aggressive and deadly human cancer (16), the present inventors also examined melanoma and colon carcinoma for ALK mutation. Indeed, identifying mutations in human cancers is highly desirable because it can lead to the development of new therapeutics that target such fusion or mutant proteins, and to new diagnostics for identifying patients that have such gene mutations."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The present invention is based, at least in part, on the discovery of ALK gene mutations in thyroid cancer that may rationalize clinical evaluation of ALK inhibitors in this setting. In undifferentiated anaplastic thyroid cancer (ATC), the present inventors identified two novel point mutations in exon 23 of the ALK gene, C3592T and G3602A, in exon 23 of the ALK gene with a prevalence of 11.11%, but found no mutations in the matched normal tissues or in well-differentiated thyroid cancers. These two mutations, resulting in the L1198F and G1201E amino acid changes, respectively, both reside within the ALK tyrosine kinase domain where they dramatically increased tyrosine kinase activities. Similarly, these mutations heightened the ability of ALK to activate the PI3K/Akt and MAP kinase pathways in established mouse cells. Further investigation demonstrated that these two ALK mutants strongly promoted cell focus formation, anchorage-independent growth, and cell invasion. Similar oncogenic properties were observed in the neuroblastoma-associated ALK mutants K1062M and F1174L, but not in wild-type ALK. Overall, the results reveal two novel gain-of-function mutations of ALK in certain ATCs and they suggest efforts to clinically evaluate the use of ALK kinase inhibitors to treat patients who harbor ATCs with these mutations.

"Furthermore, although these two novel mutations are found in thyroid cancer, they likely are also present in other human cancers and can therefore make ALK an effective therapeutic target also in those non-thyroid cancers harboring them. Indeed, the molecular testing of these two novel ALK mutations in thyroid cancer as well as in other human cancers will be helpful in guiding their targeted treatments. These cancers may particularly include brain tumor, lymphoma, lung cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, melanoma, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicle cancer, bone cancer, head and neck cancer, laryngeal cancer, leukemia, myofibroblastic tumors, and other cancers.

"Accordingly, in one aspect, the present invention provides methods for treating thyroid cancer. In one embodiment, a method for treating an anaplastic thyroid cancer (ATC) patient comprises the step of administering to the patient an effective amount of an anaplastic lymphoma kinase (ALK) inhibitor. In a specific embodiment, the ALK inhibitor is crizotinib. In another embodiment, the patient has a C3592T mutation in exon 23 of the ALK gene. In yet another embodiment, the patient has a G3602A mutation in exon 23 of the ALK gene. The can further comprise the step of determining whether the patient has a C3592T and/or a G3602A mutation in exon 23 of the ALK gene prior to the administering step.

"In another embodiment, a method for treating anaplastic thyroid cancer (ATC) in a thyroid cancer patient comprises the steps of (a) determining whether the thyroid cancer patient has a C3592T and/or a G3602A mutation in exon 23 of the anaplastic lymphoma kinase (ALK) gene; and (b) treating the patient with an ALK inhibitor therapy if the patient has either or both of the mutations. Alternatively, a method for treating anaplastic thyroid cancer (ATC) in a thyroid cancer patient comprises the step of treating the patient with an ALK inhibitor therapy if the thyroid cancer patient has a C3592T and/or a G3602A mutation in exon 23 of the ALK gene. In such embodiments, the ALK inhibitor is crizotinib.

"In another aspect, the present invention provides methods for treating cancer patients that harbor the mutations described herein. For example, a method for treating a cancer patient comprises the step of administering to the patient an effective amount of an ALK inhibitor, wherein the cancer patient has a C3592T and/or a G3602A mutation in exon 23 of the ALK gene. In another embodiment, a method for treating a cancer patient comprises the steps of (a) determining whether the cancer patient has a C3592T mutation in exon 23 of the anaplastic lymphoma kinase (ALK) gene; and (b) treating the patient with an ALK inhibitor therapy if the patient has either or both of the mutations. In such embodiments, the ALK inhibitor is crizotinib. In other specific embodiments, the cancer is a neuroblastoma.

"In another specific embodiment, a method for treating a neuroblastoma cancer patient comprises the step of administering to the patient an effective amount of an ALK inhibitor, wherein the patient has a C3592T and/or a G3602A mutation in exon 23 of the ALK gene. In a more specific embodiment, the ALK inhibitor is crizotinib.

"In additional embodiments, the methods described herein can further comprise the step of administering an inhibitor of the PI3K/Akt pathway. In other embodiments, the methods described herein can further comprise the step of administering an inhibitor of the MAP kinase pathway. In a specific embodiment, a method for treating a cancer patient comprises the step of administering to the patient an inhibitor of a protein or pathway selected from the group consisting of ALK, the PI3K/Akt pathway and the MAP kinase pathway, wherein the patient has a C3592T and/or a G3602A mutation in exon 23 of the ALK gene. In a more specific embodiment, the ALK inhibitor is crizotinib. In another embodiment, the patient has ATC. In yet another embodiment, the patient has a neuroblastoma.

"In another aspect, the present invention provides diagnostic and prognostic methods relating to the mutations described herein. In one embodiment, a method for diagnosing ATC in patient comprises the step of performing an assay on a biological sample from the patient to identify the presence or absence of a C3592T and/or a G3602A mutation in exon 23 of the ALK gene according to SEQ ID NO:6, wherein the presence of either of both of the mutations correlates with a diagnosis of ATC in the patient. In another embodiment, a method for determining a patient's risk of developing ATC comprises the step of performing an assay on a biological sample from the patient to identify the presence or absence of a C3592T and/or a G3602A mutation in exon 23 of the ALK gene according to SEQ NO:6, wherein the presence of either of both of the mutations correlates with a prognosis that the patient has a higher risk of ATC than a patient without the mutations, and wherein the absence of the mutations correlates with a prognosis that the patient has a lower risk of ATC than a patient with either or both of the mutations.

"In a more specific embodiment, a method for detecting ATC in a patient comprises the step of determining the presence of a C3592T andlor a G3602A mutation in exon 23 of the ALK gene according to SEQ ID NO:6 in a blood sample of a patient, wherein the presence of the mutation indicates ATC in the patient.

"In yet another embodiment, a method for distinguishing ATC from non-ATC samples comprises the step of determining the presence of a C3592T and/or a G3602A mutation in exon 23 of the ALK gene according to SEQ ID NO:6 in thyroid sample of a patient, wherein the presence of either or both of the mutations indicates ATC and absence of either or both of the mutations indicates non-ATC. In a specific embodiment, the thyroid sample is a fine needle aspirate (FNA). In another embodiment, the thyroid sample is a tissue sample. In yet another embodiment, the thyroid sample is a cytological sample. The mutations can be detected using methods and kits known to those of ordinary skill in the art. For example, as described below, genomic DNA ean be isolated from a sample and then exon 23 can be PCR amplified and sequenced. The method may further comprise providing a diagnosis based on the presence or absence of either or both of the mutations.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1A is a sequencing electropherogram of the ALK gene. Left, the sequencing results of the matched normal tissues of the 2 ATC cases, showing the wild-type ALK gene. Middle and right, the sequencing results of 2 ATC tumors. Top, the sequencing results of sense and antisense strands of a region of exon 23 of the ALK gene in an ATC showing the heterozygous C>T mutation at nucleotide position 3,592 in codon 1,198, resulting in the L1198F amino acid change of ALK. Bottom, the sequencing results of sense and antisense strands of a region of exon 23 of the ALK gene in another ATC showing the heterozygous G>A mutation at nucleotide position 3,602 in codon 1,201, resulting in the G1201E amino acid change. Arrows indicate the mutated nucleotides. Nucleotide numbers refer to the position within the coding sequence of the ALK gene, where position 1 corresponds to the first position of the translation initiation codon. All samples were sequenced in 2 repeated experiments with independent PCR by sense and antisense primers. FIG. 1B is a schematic diagram of the ALK. Shown are the relative positions of the novel somatic ALK mutations L1198F and G1201E and the previously characterized mutations K1062M and F1174L from neuroblastoma. L1198F and G1201E are located in the tyrosine kinase domain of the ALK. FIG. 1C is an amino acid sequence alignment of the ALK proteins from 6 species. Shown are the L1198 and G1201 residues that are evolutionarily completely conserved among these different species. Numbers indicate amino acid or codon positions. Amino acid sequences are numbered with the initiation codon (methionine) of each protein defined as number 1.

"FIG. 2 shows the increased tyrosine kinase activities of ALK mutants L1198F and G1201E and their activation of the PI3K/Akt and MAP kinase pathways. FIG. 2A shows the results of an in vitro assay of tyrosine kinase activities of ALK mutants. NIH3T3 cells stably expressing Flag-tagged vector, wild-type ALK (wt-ALK), and each of ALK mutants as indicated were lysed. The cell lysates were assayed for tyrosine kinase activity as described in the Materials and Methods. The enzymatic activities were expressed as measured optical density value.times.20. Results represent mean.+-.SD of 3 independent experiments. FIG. 2B shows the activation of the PI3K/Akt and MAP kinase pathways. This is reflected by increased phosphorylation of Akt (p-Akt) and phosphorylation of ERK (p-ERK), respectively. NIH3T3 cells stably transfected with the indicated vector constructs, as described in FIG. 1A cell lysate proteins, were subjected to Western blot analyses for the indicated proteins by using appropriate antibodies as described in Materials and Methods. Successful protein expression of Flag-tagged wild-type ALK and each of the ALK mutants is shown in the top row of FIG. 1B. The key molecules of the 2 pathways are shown in the subsequent rows. Total Akt, ERK, and b-actin were used for quality control of loading proteins.

"FIG. 3 shows the focus-formation and anchorage-independent growth of cells promoted by ALK mutants. FIG. 3A shows the cell focus-forming activities of ALK mutants. Shown are images of adherent growth of NIH3T3 cells transfected with Flag-tagged vector, wild-type ALK (wt-ALK), and each of the ALK mutants indicated. Cells were cultured in reaular medium with 10% fetal calf serum under standard conditions. Images of cell foci were photographed with 10.times. magnification after appropriate culture of cells as described in the Materials and Methods. FIG. 3B shows the number of cell foci formed with the indicated transfections. The number of transformed foci was counted 14 days after cell transfection. Results represent mean.+-.SD of 3 independent experiments. FIG. 3C shows the anchorage-independent cell growth of ALK mutants on soft agar. NIH3T3 cells stably transfected with Flag-tagged vector, wild-type ALK, and each of the ALK mutants indicated were seeded in soft agar, and colonies formed 4 weeks later were photographed with 40.times. magnification. FIG. 3D shows the analyses of the number of colonies. The number of cell colonies corresponding to C that were greater than 0.1 mm in diameter was counted. Results represent mean.+-.SD of 3 independent experiments.

"FIG. 4 shows cell invasion promoted by ALK mutants. FIG. 4A shows the results from the in vitro invasion assay of NIH3T3 cells with various transfections. Cells transfected with Flag-tagged vector, wild-type ALK (wt-ALK), and each construct of the indicated ALK mutants. Cell invasion assay was conducted as described in Materials and Methods. Shown are the cells that invaded on the Matrigel matrix-coated polyethylene terephthalate (PET) membrane after removal of the noninvasive cells. FIG. 4B shows the number of invasive cells with the indicated transfections. Results of each column represent the mean.+-.SD of the numbers of invasive cells from 3 independent experiments."

For more information, see this patent application: Xing, Michael Mingzhao; Kannan, Murugan Avaniyapuram. Anaplastic Thyroid Cancers Harbor Novel Oncogenic Mutations of the Alk Gene. Filed May 7, 2012 and posted May 15, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1792&p=36&f=G&l=50&d=PG01&S1=20140508.PD.&OS=PD/20140508&RS=PD/20140508

Keywords for this news article include: Antisense Technology, Biotechnology, Patents, Genetics, Oncology, Peptides, Proteins, Cell Line, Hematology, Proteomics, Therapeutics, Bioengineering, Cultured Cells, Tyrosine Kinase, Lymphatic Diseases, Cancer Gene Therapy, Aromatic Amino Acids, Enzymes and Coenzymes, Lymphoma Gene Therapy, Immunoproliferative Disorders.

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Source: Cancer Gene Therapy Week


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