WO2014152377A1 - Méthodes de classement et de traitement des adénocarcinomes - Google Patents

Méthodes de classement et de traitement des adénocarcinomes Download PDF

Info

Publication number
WO2014152377A1
WO2014152377A1 PCT/US2014/027272 US2014027272W WO2014152377A1 WO 2014152377 A1 WO2014152377 A1 WO 2014152377A1 US 2014027272 W US2014027272 W US 2014027272W WO 2014152377 A1 WO2014152377 A1 WO 2014152377A1
Authority
WO
WIPO (PCT)
Prior art keywords
lkbl
expression
cancer
lkb
gene
Prior art date
Application number
PCT/US2014/027272
Other languages
English (en)
Inventor
Jacob Kaufman
David Carbone
Original Assignee
Vanderbilt University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vanderbilt University filed Critical Vanderbilt University
Publication of WO2014152377A1 publication Critical patent/WO2014152377A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to the fields of oncology and molecular biology. More specifically, it relates to the classification and treatment of LKB1 -negative cancers.
  • Non-small cell lung cancer is the most common and lethal cancer worldwide. At least three major histologies of NSCLC are described: squamous carcinoma (48%), large cell carcinoma (12%) and adenocarcinoma (40%) The standard treatment for these patients is systemic chemotherapy. However, systemic chemotherapy has modest efficacy and has not greatly prolonged the median survival (8-12 months) or 5-year survival rates (2%) in these patients. Although these sub-types differ markedly in histologic appearance and gene expression, each is highly lethal, and until recently, little clinical distinction has been made among these entities.
  • Serine-threonine kinase 11 is a tumor suppressor and one of the most commonly mutated genes in non-small cell lung cancers 6"8 . It has been shown to play important roles in embryologic development, cellular polarity, motility, and transcriptional regulation and represents a 'metabolic checkpoint' via its interactions with AMPK and the mTOR pathway 9 ' 10 . Although the mTOR pathway has been reported to be active in tumors lacking LKBl, interactions between mTOR and other dysregulated pathways and the resulting tumor phenotypes remain poorly characterized. Better understanding of the biology of these tumors may identify crucial pathways that can be targeted to improve patient outcome with individualized therapy.
  • a method of identifying a patient with an LKBl -deficient cancer comprising a) obtaining a tumor sample from said patient; and b) determining a gene expression profile from cells of said tumor sample for AVPI1, BAG1, CPS1, DUSP4, FGA, GLCE, HAL, IRS2, MUC5AC, PDE4D, PTP4A1, RFK, SNF1LK, TACC2, TFF1 and TSC, wherein an increased level of expression for a majority of BAG1, HAL, IRS2, PTP4A1, RFK, SNF 1LK, TACC2, TFF1 and TSC, and a decreased level of expression for a majority of AVPI1, CPS 1, DUSP4, FGA, GLCE, MUC5AC and PDE4D, as compared to that observed in a cell expressing functional LKBl, indicates that said tumor is formed from an LKBl -deficient cancer.
  • the method may result in 10, 11, 12, 13, 14, 15, or
  • the method may further comprise administering to said patient a MEK inhibitor when said patient is determined to have an LKBl -deficient cancer.
  • the method may also further comprising administering to said patient a af inhibitor.
  • the MEK inhibitor may be selumetinib or trametinib.
  • the cancer may be is a lung cancer, and/or an adenocarcinoma.
  • the patient may be a human, and/or may be a smoker.
  • the tumor may be recurrent, metastatic and/or multi-drug resistant.
  • the tumor sample may be a biopsy or a resected tumor tissue.
  • the method may further comprise conducting histopathology and/or immunohistochemistry on said tumor sample.
  • the method may further comprise assessing Lkbl expression and/or mutational status on said tumor sample.
  • Step (b) may comprise RT- PCR, including further further preparing cR A or preparing cDNA, or and/or microarray hybridization.
  • FIGS 1A-E LKBl loss produces a characteristic pattern of gene expression.
  • FIG. 1A The significance of gene overlap is shown for pairwise comparisons of the top 200 genes over-expressed in tumors with LKB l loss in 14 studies of lung adenocarcinomas. Asterisks indicate comparisons between cell lines expressing vector control and those expressing wild- type LKBl . P-values from a hypergeometric test are color coded according to the legend (see also FIG. 5). (FIG.
  • IB Unsupervised hierarchical clustering of 178 resected lung adenocarcinomas using a 129 gene signature of LKB l loss. Tumors are shown on the horizontal axis, with loss of LKB l highlighted in red; genes are shown on the vertical axis, with four clusters of gene expression highlighted in red.
  • FIG. 1C Sensitivity and specificity of the LKBl classifier for prediction of LKBl mutations across independent testing sets; p- value represents the result of the Fisher's exact test (FIGS.
  • LKBl mRNA Expression of LKBl mRNA is shown as standard deviations from the mean among resected lung adenocarcinomas classified as LKBl loss or LKBl wild-type among tumors in which LKBl has been sequenced (FIG. ID) or with unknown LKBl mutation status (FIG. IE). Each dot represents one tumor and p-values are derived from the student's t-test comparing indicated groups.
  • FIG. 2 Pharmacologic and genetic perturbations affect the expression of transcriptional nodes. The results of gene set enrichment analysis are shown for selected perturbations that affect the expression of the three transcriptional clusters upregulated in LKBl -deficient tumors. P-values represent the significance of a hypergeometric test for the indicated comparisons.
  • FIGS. 3A-D Restoring wild-type LKBl in cell lines harboring mutations slows growth and attenuates the expression of the LKBl-deficient gene signature.
  • FIG. 3A Immunoblots of whole-cell lysates from A549, H2122, and H460 stably expressing emtpy pBABE vector, LKBl or K78I LKBl after puromycin selection.
  • FIGGS. 3B-C Changes in gene expression of A549 (FIG. 3B) or H2122 (FIG. 3C) cell lines after re-expressing LKBl or K78I LKBl were compared to the gene lists for each of the four LKBl -associated clusters using a hypergeometric test.
  • FIG. 3DA Activity of CRE-luciferase is shown for A549, H2122, and H460 cell line after stable expression of LKBl or K78I LKBl. Reporter activations were determined relative to a control luciferase with mutated CRE sites, and are shown relative to the pBABE control. P-values show the significance of unpaired student's t- tests.
  • FIGS 4A-G LKB1 loss confers sensitivity to selumetinib.
  • FIGGS. 4A-B Maximum inhibitory effect of selumetinib is shown for cell lines with high expression of the L B1 classifier compared to those with low expression in both a training (FIG. 4A) and testing (FIG.
  • FIG. 4B cohort from the CCLE dataset.
  • FIG. 4C-F Association between selumetinib and LKB l-loss signature is shown for cell lines with mutations in LKB1 (FIG. 4C), BRAF (FIG. 4D), KRAS (FIG. 4E) and wild-type for BRAF, KRAS, NRAS, and HRAS (FIG. 4F).
  • FIG. 4A-F cell lines classified as LKBl-loss are marked as 'classifier positive' while those with a wild-type signature are marked as 'classifier negative'. Distributions and medians are plotted and P-values represent the significance of Welch's t-test.
  • FIG. 5 Association matrix for down-regulated genes associated with LKBl loss. The significance of gene overlap is shown for pairwise comparisons of the 200 genes with the most significantly decreased expression in tumors with LKBl loss across 14 studies of lung adenocarcinomas. Asterisks indicate comparisons between cell lines expressing vector control and those expressing wild-type LKBl. P-values from a hypergeometric test are color coded according to the legend.
  • FIGS. 6A-F Distribution of LKBl loss scores and receiver operating characteristics in NSCLC cell lines and resected lung adenocarcinomas. Distribution of LKBl loss scores and receiver operating characteristics in NSCLC cell lines and resected lung adenocarcinomas. (FIG.
  • FIGS. 6A, 6D and 6E red points correspond to the score cutoff of 0.2 used throughout the paper.
  • FIG. 8 Comparison of LKBl loss scores derived from independent training cohorts. LKB l-loss scores are plotted for two distinct classifiers: Score A is the primary 16- gene LKB l-loss classifier used throughout this study and Score B results from an independent classifier derived by applying the same statistical approach to a different training cohort. The concordance is the percentage of tumors that are given the same LKB l-loss classification by each of the two classifiers.
  • FIGS. 9A-B TGF-beta mRNA expression is decreased in tumors with LKBl loss.
  • FIG. 9B Induction of TGF-beta mRNA relative to pBABE vector control is shown for A549 and H2122 cell lines after stable expression of wild-type LKBl . The range is plotted and p-values represent the result of student's t-test of the indicated comparisons.
  • FIG. 10 TGF-beta and c-src pathways antagonize the expression of genes in the CREB cluster.
  • the significance of gene overlap is shown for comparisons of the CREB signature to the genes perturbed by TGF-beta or dasatinib treatment of the LKBl-mutant cell line A549 at the various time points or concentrations shown.
  • P-values from a hypergeometric test are shown on the ordinate axis with positive values indicating an induction of CREB-associated genes and negative values indicating repression.
  • FIGS. 11A-B Stable expression of LKBl in cell lines slows proliferation.
  • FIGS. 12A-C Expression of wild-type LKB1 in A549, H2122, or HeLa cell lines decreases the expression of the genes in the CREB transcriptional node.
  • FIGS. 13A-B Cell lines with expression of the LKB1 classifier score show increased susceptibility to MEK inhibition.
  • FIG. 13 A The associations between L B1 classifier score and IC50 values for four MEK inhibitors are shown for the GDSC training cohorts, and for two MEK inhibitors in the CCLE training and testing cohorts.
  • FIG. 13B Associations between LKBl-loss score and the maximum inhibitory effect of selumetinib or PD-0325901 are shown in the CCLE training and testing cohorts. The values plotted are the linear regression coefficients relating the LKBl-loss score to the indicated inhibitor IC 50 , with bars representing their 95% confidence interval. P-values represent the significance associated with the linear regression coefficient.
  • FIGS. 14A-C Decreased LKB1 mRNA in association with LKB1 loss signature in resected breast cancer, lung squamous, and cervical cancer. Expression of LKB 1 mRNA is shown for tumors classified as LKBl loss or LKB 1 WT in a pooled cohort of lung squamous cell carcinomas (GSE4573; TCGA) (FIG. 14A), among the TCGA breast cancer specimens (TCGA) (FIG. 14B), or in a pooled cohort of cervical squamous cell carcinoma (GSE38964; GSE20167; TCGA) (FIG. 14C).
  • FIGS. 15A-B Comparison of protein and gene expression differences associated with known LKBl mutations or associated with predicted LKBl loss among LKBl WT tumors.
  • FIG. 15 A Differences in protein expression determined by TCGA RPPA are shown for proteins with significant association with LKBl mutations (having p ⁇ 0.01).
  • FIG. 15B Differences in mRNA expression determined by TCGA RNAseq analysis are shown for genes with significant association with LKBl mutations (having ⁇ 6 ). Dots represent individual proteins or genes.
  • the x-axis shows the difference in average expression between LKBl mutant tumors and LKBl-WT tumors with WT classification score.
  • the y-axis shows the difference in average expression between LKB1-WT tumors with a LKBl-loss classification score vs LKB1-WT tumors with a WT classification score.
  • FIGS. 16A-E Responsiveness to MEK inhibition for three previously published MEK signatures, in contrast to LKBl loss signature. Changes in gene expression corresponding to different signatures of MEK sensitivity are shown after treatment with MEK inhibitor.
  • FIG. 16A Analysis of 15 gene 'MEK functional activation' signature reported in Dry, et al. (2010).
  • FIG. 16B Analysis of 58 genes reported to be correlated with MEK sensitivity in Garnett et al. (2012).
  • FIG. 16C Analysis of 90 gene signature reported in Loboda et al. (2010).
  • FIG. 16D Analysis of 16 gene signature of LKBl loss reported here.
  • FIG. 16E Analysis of top 200 genes associated with the 16 gene LKBl loss signature.
  • the y-axis represents the log-transformed p-values for hypergeometric test of overlap significance between each signature and the genes perturbed by MEK inhibition in 34 cell lines (22 pancreatic, six skin, three breast, two colon, one lung) from two studies. Pancreatic cell lines were treated with 2 mM CI- 1040 for 24 hours, while other cell lines were treated with 50 nM PD0325901 for eight hours.
  • the inventors report the comprehensive analysis of gene expression patterns associated with LKBl functional loss in human lung adenocarcinomas, through which they identify up-regulation of the mitochondrial respiratory chain and activation of CREB and NRF2 transcription factors as key features in these tumors.
  • a 16-gene signature is predictive of both mutational and non-mutational LKBl loss across human tumors, including resected lung adenocarcinomas and cell lines of both lung and non-lung histology.
  • cell lines expressing this signature show increased sensitivity to MEK inhibition across two large studies 4,5 , independent of the sensitivity conferred by mutations in RAS and RAF family members.
  • Non-small-cell lung carcinoma is any type of epithelial lung cancer other than small cell lung carcinoma (SCLC).
  • SCLC small cell lung carcinoma
  • NSCLCs are relatively insensitive to chemotherapy, compared to small cell carcinoma. When possible, they are primarily treated by surgical resection with curative intent, although chemotherapy is increasingly being used both pre-operatively (neoadjuvant chemotherapy) and post-operatively (adjuvant chemotherapy).
  • the most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are several other types that occur less frequently, and all types can occur in unusual histologic variants and as mixed cell-type combinations.
  • non-small-cell lung cancer (not otherwise specified,” or NOS) is used generically, usually when a more specific diagnosis cannot be made. This is most often the case when a pathologist examines a small amount of malignant cells or tissue in a cytology or biopsy specimen. Lung cancer in never-smokers is almost universally NSCLC, with a sizeable majority being adenocarcinoma. On relatively rare occasions, malignant lung tumors are found to contain components of both SCLC and NSCLC. In these cases, the tumors should be classified as combined small cell lung carcinoma (c-SCLC), and are (usually) treated like "pure" SCLC.
  • c-SCLC combined small cell lung carcinoma
  • SCC Squamous cell carcinoma
  • squamous cell carcinoma is often preceded for years by squamous cell metaplasia or dysplasia in the respiratory epithelium of the bronchi, which later transforms to carcinoma in situ.
  • atypical cells may be identified by cytologic smear test of sputum, bronchoalveolar lavage or samples from endobronchial brushings.
  • squamous-cell carcinoma in situ is asymptomatic and undetectable on X-ray radiographs. Eventually, it becomes symptomatic, usually when the tumor mass begins to obstruct the lumen of a major bronchus, often producing distal atelectasis and infection. Simultaneously, the lesion invades into the surrounding pulmonary substance. On histopathology, these tumors range from well differentiated, showing keratin pearls and cell junctions, to anaplastic, with only minimal residual squamous cell features.
  • mRNA expression subtypes primary, basal, secretory, and classical
  • the primitive subtype correlates with worse patient survival.
  • These subtypes defined by intrinsic expression differences, provide a possible foundation for improved patient prognosis and research into individualized therapies.
  • LCLC Large cell lung carcinoma
  • LCLC is differentiated from small cell lung carcinoma (SCLC) primarily by the larger size of the anaplastic cells, a higher cytoplasmic -to-nuclear size ratio, and a lack of "salt-and-pepper" chromatin.
  • SCLC small cell lung carcinoma
  • Adenocarcinoma of the lung is currently the most common type of lung cancer in "never smokers" (lifelong non-smokers). Adenocarcinomas account for approximately 40% of lung cancers. Historically, adenocarcinoma was more often seen peripherally in the lungs than small cell lung cancer and squamous cell lung cancer, both of which tended to be more often centrally located. Interestingly, however, recent studies suggest that the "ratio of centrally-to-peripherally occurring" lesions may be converging toward unity for both adenocarcinoma and squamous cell carcinoma.
  • Serine/threonine kinase 11 also known as liver kinase Bl (L B1) or renal carcinoma antigen NY-REN- 19 is a protein kinase that in humans is encoded by the STK11 gene.
  • ⁇ -estradiol treatment increased L B 1 mRNA, an effect mediated by estrogen receptor a.
  • the STK11/LKB1 gene which encodes a member of the serine/threonine kinase, regulates cell polarity and functions as a tumor suppressor.
  • estradiol caused a dose-dependent decrease in LKBl transcript and protein expression leading to a significant decrease in the phosphorylation of the LKBl target AMPK.
  • ERa binds to the STK1 1 promoter in a ligand-independent manner and this interaction is decreased in the presence of estradiol.
  • STKl 1 promoter activity is significantly decreased in the presence of estradiol.
  • LKBl is a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), a necessary element in cell metabolism that is required for maintaining energy homeostasis.
  • AMPK adenine monophosphate-activated protein kinase
  • LKB l exerts its growth suppressing effects by activating a group of other -14 kinases, comprising AMPK and AMPK-related kinases.
  • Activation of AMPK by LKB l suppresses growth and proliferation when energy and nutrient levels are scarce.
  • Activation of AMPK-related kinases by LKBl plays vital roles maintaining cell polarity thereby inhibiting inappropriate expansion of tumor cells.
  • loss of LKBl leads to disorganization of cell polarity and facilitates tumor growth under energetically unfavorable conditions.
  • Germline mutations in this gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by the growth of polyps in the gastrointestinal tract, pigmented macules on the skin and mouth, and other neoplasms. Recent studies have uncovered a large number of somatic mutations of the LKBl gene that are present in lung, cervical, breast, intestinal, testicular, pancreatic and skin cancer.
  • LKBl is activated allosterically by binding to the pseudokinase STRAD and the adaptor protein M025.
  • the LKB 1-STRAD-M025 heterotrimeric complex represents the biologically active unit, that is capable of phosphorylating and activating AMPK and at least 12 other kinases that belong to the AMPK-related kinase family.
  • the crystal structure of the LKB 1-STRAD-M025 complex was elucidated using X-ray crystallography, and revealed the mechanism by which LKBl is allosterically activated.
  • LKB l has a structure typical of other protein kinases, with two (small and large) lobes on either side of the ligand ATP-binding pocket.
  • MEK inhibitors are defined herein as a chemical or drug that inhibits MEKl and/or MEK2, mitogen-activated protein kinase kinase enzymes. They can be used to affect the MAPK/ERK pathway which is often overactive in some cancers. Hence, MEK inhibitors have potential for treatment of some cancers, especially BRAF-mutated melanoma,http://en.wikipedia.org/wiki/MEK Inhibitor - cite note-ASCO2012-2 and KRAS/BRAF mutated colorectal cancer. In the context of the present disclosure, they will find particular use in treating LKB 1 -deficient cancers. Some specific MEK inhibitors include trametinib (GSK1 120212), selumetinib, MEK162, PD-325901, XL518, CI- 1040, and PD035901.
  • Trametinib (GSK1120212) is experimental cancer drug. It is a MEK inhibitor drug with anti-cancer activity. It inhibits both MEKl and MEK2. Trametinib had good results for V600E mutated metastatic melanoma in a hase III clinical trial. Its structure is shown below:
  • Selumetinib (AZD6244) is a drug being investigated for the treatment of various types of cancer, for example non-small cell lung cancer (NSCLC).
  • the gene BRAF is part of the MAPK/ERK pathway, a chain of proteins in cells that communicates input from growth factors. Activating mutations in the BRAF gene, primarily V600E, are associated with lower survival rates in patients with papillary thyroid cancer. Another type of mutation that leads to undue activation of this pathway occurs in the gene KRAS and is found in NSCLC.
  • a possibility of reducing the activity of the MAPK/ERK pathway is to block the enzyme MAPK kinase (MEK), immediately downstream of BRAF, with the drug selumetinib.
  • MAPK kinase MAPK kinase
  • selumetinib blocks the subtypes MEKl and MEK2 of this enzyme.
  • BRAF-&c ⁇ vtdX g mutations are prevalent in melanoma (up to 59%), colorectal cancer (5-22%), serous ovarian cancer (up to 30%), and several other tumor types.
  • KRAS mutations appear in 20 to 30% of NSCLC cases and about 40% of colorectal cancer. Its structure is shown below:
  • compositions containing MEK inhibitors will be necessary to prepare pharmaceutical compositions containing MEK inhibitors.
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically acceptable carrier refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or admininstration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed. The active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • a paste dentifrice may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the invention provides methods of detecting and evaluating the presence or absence of LKB1 function in a subject having cancer, including identifying those cells/cancers in which L B1 is entirely absent or expressed in a non-functional form.
  • the differentially expressed genes identified herein are used for predicting the success of certain therapeutic approaches to treating adenocarcinomas.
  • the subject is generally a mammal, and is typically human female or human male.
  • the subject typically will have been diagnosed as having cancer, and possibly has already undergone treatment for the cancer. Diagnosis of cancer is typically made through the identification of a mass on an examination, though it may also be through other means such as a radiological diagnosis, or ultrasound.
  • the signature includes the genes AVPI1 (arginine vasopressin-induced 1; GenelD: 60370), BAG1 (BCL2-associated athanogene; GenelD: 573), CPS1 (carbamoyl-phosphate synthase 1, mitochondrial; GenelD: 1373), DUSP4 (dual specificity phosphatase 4; GenelD: 1846), FGA (fibrinogen alpha chain; GenelD: 2243), GLCE (glucuronic acid epimerase; GenelD: 26035), HAL (histidine ammonia-lyase; GenelD: 3034), IRS2 (insulin receptor substrate 2; GenelD: 8660), MUC5AC (mucin 5AC, oligomeric mucus/gel-forming; GenelD: 4586), PDE4D (phosphodiesterase 4D, cAMP-specific; GenelD: 5144), PTP4A1 (protein tyrosine phosphatase type IVA, member
  • AVPI1, CPS1, DUSP4, FGA, GLCE, MUC5AC and PDE4D were found to be down-regulated in LKB1 -negative cells
  • BAG1, HAL, IRS2, PTP4A1, RFK, SNF1LK, TACC2, TFF1 and TSC were found to be up-regulated in LKB1 -negative cells.
  • PTP4A1 protein tyrosine phosphatase type IVA member 1 i 7803 NMJTO3463 BF576710
  • Immunoassays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody, a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes. More typically, expression levels of the markers described here will be determined at the nucleic acid level using any method known in the art. For example, Northern hybridization analysis using probes which specifically recognize one or more of these sequences can be used to determine gene expression. Alternatively, expression is measured using reverse-transcription-based PCR assays, e.g., using primers specific for the differentially expressed sequence of genes. RNA is isolated from cancer cells according to standard methodologies (Sambrook et al, 1989). The RNA may be converted to a complementary DNA. In one embodiment, the RNA is whole cell RNA; in another, it is poly- A RNA. Normally, the nucleic acid is amplified. The following provides a general discussion of nucleic acid assays for expression.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process. In particular
  • the probes or primers are labeled with radioactive species ( P, C, S, H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemiluminescent (luciferase).
  • radioactive species P, C, S, H, or other label
  • fluorophore rhodamine, fluorescein
  • luciferase chemiluminescent
  • PCR polymerase chain reaction
  • two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al. (1989).
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed Dec. 21, 1990.
  • Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha- thio] -triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al. (1992).
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • primers are used in a PCR.TM.-like, template- and enzyme- dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double-stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single-stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • EP 0 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCR”.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook gi a/. (1989).
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • a DNA chip is a device that is convenient to compare expression levels of a number of genes at the same time.
  • a DNA chip comprises immobilized high-density probes to detect a number of genes.
  • expression levels of many genes can be estimated at the same time by a single-round analysis. Namely, the expression profile of a specimen can be determined with a DNA chip.
  • Commercially available reagents and kits are available to perform each of the steps required for analysis.
  • the DNA chip-based method of the present invention comprises the steps of:
  • the cRNA refers to RNA transcribed from a template cDNA with RNA polymerase.
  • a cRNA transcription kit for DNA chip-based expression profiling is commercially available. With such a kit, cRNA can be synthesized from T7 promoter-attached cDNA as a template by using T7 RNA polymerase. On the other hand, by PCR using random primer, cDNA can be amplified using as a template a cDNA synthesized from mRNA.
  • the DNA chip comprises probes, which have been spotted thereon, to detect the marker genes of the present invention.
  • the chip There is no limitation on the number of marker genes spotted on the DNA chip, and for the purposes of the present invention, it is only required that the chip contain nucleic acids for the 16 gene signature, and may optionally include control nucleic acids, as well as any other genes of interest.
  • a probe for a gene whose expression level is rarely altered may be spotted on the DNA chip.
  • Such a gene can be used to normalize assay results when assay results are intended to be compared between multiple chips or between different assays.
  • the probes are designed for each marker gene selected.
  • a probe may be, for example, an oligonucleotide comprising 5-50 nucleotide residues.
  • a method for synthesizing such oligonucleotides on a DNA chip is known to those skilled in the art. Longer DNAs can be synthesized by PCR or chemically. A method for spotting long DNA, which may be synthesized by PCR or the like, onto a glass slide is also known to those skilled in the art.
  • the prepared DNA chip is contacted with cRNA, followed by the detection of hybridization between the probe and cRNA.
  • the cRNA can be previously labeled with a fluorescent dye or any other label.
  • a fluorescent dye such as Cy3(red) and Cy5 (blue) can be used to label a cRNA.
  • cRNAs from a subject and a control are labeled with different fluorescent dyes, respectively.
  • the difference in the expression level between the two can be estimated based on a difference in the signal intensity.
  • the signal of fluorescent dye on the DNA chip can be detected by a scanner and analyzed by using a special program.
  • the Suite from Affymetrix is a software package for DNA chip analysis.
  • U.S. Patent Publication 2013/0017971 (Nanostring Technologies, Seattle WA) describes methods for detecting the relative expressions of a plurality of target nucleic acid molecules in one assay.
  • the methods use a plurality of probe molecules which specifically bind to one target nucleic acid molecule of a plurality of target nucleic acids in a sample, and a plurality of reference molecules that represent each of the plurality of target nucleic acid molecules, where the probe molecules specifically bind to the plurality of reference molecules, and each of the plurality of reference molecules is present in known amounts in the composition.
  • the methods permit multiplexed detection of a plurality of target nucleic acid molecules from a biological sample including a plurality of probe molecules.
  • the probe molecules are capable of enzymatic or non-enzymatic direct detection of the target nucleic acid molecules, and preferably the detection of the target nucleic acid molecules occurs without target nucleic acid amplification.
  • the methods can be adapted to quantifying the expression of the plurality of target nucleic acid molecules. See also Geiss et al. (2008).
  • the plurality of probe molecules can include about 8 to about 50 probe molecules, about 15 to about 50 probe molecules, about 25 to about 50 probe molecules, about 50 to about 100 probe molecules or more than 100 probe molecules.
  • the probe molecules can be nucleic acid probes.
  • Each nucleic acid probe can include: (i) a target-specific region that specifically binds to a target nucleic acid molecule; and (ii) a region including a plurality of label-attachment regions linked together, wherein each label attachment region is attached to a plurality of label monomers that create a unique code for each target-specific probe, the code having a detectable signal that distinguishes one nucleic acid probe which binds to a first target nucleic acid from another nucleic acid probe that binds to a different second target nucleic acid molecule.
  • the plurality of label-attachment regions can include at least four, at least five, at least six, at least seven label attachment regions.
  • the plurality of label monomers includes at least four, at least five, at least six, at least seven label monomers.
  • the number of label monomers used can vary depending on the complexity of the plurality of target nucleic acid molecules.
  • Each of the label monomers can be selected from the group consisting of a fluorochrome moiety, a fluorescent moiety, a dye moiety and a chemiluminescent moiety.
  • the nucleic acid probe can further include an affinity tag.
  • kits including a composition for the multiplexed detection of a plurality of target nucleic acid molecules from a biological sample including a plurality of probe molecules, where each probe molecule in the plurality specifically binds to one target nucleic acid molecule in the sample, and instructions for the multiplexed detection of a plurality of target nucleic acid molecules.
  • the composition included within the kit can further include a plurality of reference molecules that represent each of the plurality of target nucleic acid molecules, wherein the probe molecules specifically bind to the plurality of reference molecules, and wherein each of the plurality of reference molecules is present in known amounts.
  • the probe molecules are capable of enzymatic or non-enzymatic direct detection of the target nucleic acid molecules.
  • the probe molecules are capable of non-enzymatic direct detection of the target nucleic acid molecules.
  • the kit can further include an apparatus which includes a surface suitable for binding, and optionally detecting, the probe molecules included with the kit.
  • the probe molecules are hybridized to the target nucleic acids or the reference molecules when bound to the surface.
  • the probe molecules may be bound to the surface by any means known in the art.
  • the kit can further include a composition for the extraction of the target nucleic acids from a biological sample.
  • the kit can further include a reagent selected from the group consisting of a hybridization reagent, a purification reagent, an immobilization reagent and an imaging reagent.
  • the present invention may utilized the NanoString nCounter® Analysis System to determine the expression levels of any or all of the genes described herein.
  • the NanoString nCounter® Analysis System (also referred to as a nanoreporter code system) delivers direct, multiplexed measurements of gene expression through digital readouts of the relative abundance of hundreds of mRNA transcripts.
  • the nCounter® Analysis System uses gene-specific probe pairs that hybridize directly to the mRNA sample in solution, eliminating any enzymatic reactions that might introduce bias in the results. After hybridization, all of the sample processing steps are automated on the nCounter® Prep Station.
  • the nCounter® Analysis System is comprised of two instruments, the nCounter® Prep Station used for post-hybridization processing, and the Digital Analyzer used for data collection and analysis.
  • the assay also requires a heat block and microcentrifuge for R A extraction and a low- volume spectrophotometer for measuring the concentration and purity of the RNA output.
  • a heat block with a heated lid is required to run the hybridization at a constant elevated temperature, and a swinging bucket centrifuge is required for spinning the Prep Plates prior to insertion into the Prep Station.
  • the nCounter® Prep Station is an automated fluid handling robot that processes samples post-hybridization to prepare them for data collection on the nCounter® Digital Analyzer.
  • total RNA or alternatively other RNA molecules extracted from FFPE (Formalin-Fixed, Paraffin-Embedded) tissue samples, or other sample types are hybridized with the Reporter Probes and Capture Probes according to the nCounter® protocol.
  • Hybridization to the target RNA is driven by excess probes. To accurately analyze these hybridized molecules they are first purified from the remaining excess probes in the hybridization reaction.
  • the Prep Station isolates the hybridized mRNA molecules from the excess Reporter and Capture Probes using two sequential magnetic bead purification steps.
  • an electric field is applied along the length of each sample cartridge flow cell to facilitate the optical identification and order of the fluorescent spots that make up each Reporter Probe. Because the Reporter Probes are charged nucleic acids, the applied voltage imparts a force on them that uniformly stretches and orients them along the electric field. While the voltage is applied, the Prep Station adds an immobilization reagent that locks the reporters in the elongated configuration after the field is removed. Once the reporters are immobilized the cartridge can be transferred to the nCounter® Digital Analyzer for data collection. All consumable components and reagents required for sample processing on the Prep Station are provided in the nCounter® Master Kit. These reagents are ready to load on the deck of the nCounter® Prep Station which can process a sample cartridge containing 12 flow cells per run in approximately 2 hours. The 12 flow cells can comprise a mixture of test samples and reference samples as required for the particular test.
  • the nCounter® Digital Analyzer collects data by taking images of the immobilized fluorescent reporters in the sample cartridge with a CCD camera through a microscope objective lens. Because the fluorescent Reporter Probes are small, single molecule barcodes with features smaller than the wavelength of visible light, the Digital Analyzer uses high magnification, diffraction-limited imaging to resolve the sequence of the spots in the fluorescent barcodes. The Digital Analyzer captures hundreds of consecutive fields of view (FOV) that can each contain hundreds or thousands of discrete Reporter Probes. Each FOV is a combination of four monochrome images captured at different wavelengths. The resulting overlay can be thought of as a four-color image in blue, green, yellow, and red.
  • FOV fields of view
  • Each 4-color FOV is captured in just a few seconds and processed in real time to provide a "count" for each fluorescent barcode in the sample. Because each barcode specifically identifies a single mRNA molecule or other nucleic acid molecule tested, the resultant data from the Digital Analyzer is an accurate inventory of the abundance of each mRNA or nucleic acid of interest in a biological sample.
  • the resulting test sample data from the Digital Analyzer are normalized to the reference sample data to generate a test result.
  • Other transformations may be included as part of the algorithm in order to generate a test result, but in the described method, at least one of the steps includes a normalization of the test sample data to the reference sample.
  • kits This generally will comprise preselected primers and/or probes, primer and/or probe sets, or arrays such as those on a DNA chip. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, Sequenase®, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Kits may also contain substances serving as control reagents for detection and/or quantification steps. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent.
  • Gene expression Gene expression datasets for resected lung adenocarcinomas, cell lines, and murine tumors were obtained from publicly available sources or derived as part of this work and made publicly available. Sources and statistical comparisons performed are given in Table 1.
  • LKBl classifier Genes associated with LKB1 loss with p-value less than 0.01 in each of two training cohorts comprised a 129-gene LKBl-loss signature. A smaller 16-gene set having the strongest association with LKB l loss in the same training cohort and was used as the LKB 1 classifier throughout the study. Clinical and mutational data were taken from the various publicly available datasets and differences between tumors classified as LKBl-loss and LKBl -wild-type were compared using the Fisher's exact test.
  • Drug sensitivity Publicly available datasets of in vitro drug sensitivity in cell lines were obtained from the GDSC 5 and CCLE 4 studies, which were used as training and testing sets. General linear models were used to determine drug sensitivity significantly associated with LKBl-loss. Multivariate linear regression was used to test the association between the LKB l classifier score and MEK sensitivity with incorporation of known mutations in the RAS/RAF pathway and previously published signatures of MEK sensitivity 5 ' 15,16 . Cell line experiments. Stable expression of LKB l and LKB l K78I was achieved by retroviral transduction with puromycin selection for at least two weeks. Protein ly sates and mRNA were collected from cells in logarithmic growth phase.
  • Drug sensitivity was determined in 96-well plates with inhibitors added 24 hours after seeding cells and relative cells numbers quantitated after 72 hours of drug exposure using the Alamar Blue colorimetric assay.
  • Activation of the CREB transcription factor was determined using a luciferase reporter driven by the CRE consensus sequence, the activity of which was measured as the ratio to an identical reporter with mutated CRE sequence.
  • LKBl mutation status was annotated using the Catalog of Somatic Mutations in Cancer (COSMIC) database, the Cancer Cell Line Encyclopedia (CCLE) resource, and individual publications.
  • COSMIC Catalog of Somatic Mutations in Cancer
  • CCLE Cancer Cell Line Encyclopedia
  • Overlap significance for all pairwise comparisons of 'up' lists was color coded and represented graphically using Cluster 3.0 and Java TreeView software, and is shown in FIG. 1A. Similarly, overlap significance for all pair-wise comparisons of 'down' lists is shown in FIG. 5.
  • LKBl-deficient gene signature Development of LKBl-deficient gene signature.
  • the inventors used a training and testing approach to develop and test a gene signature capable of classifying LKBl -deficient tumors. They generated three gene lists using statistical comparisons from two training sets: the Wash U set with comparisons to documented LKB l mutations and the Michigan samples from the Director's Challenge Consortium with comparisons to LKB l expression. The LKBl classifier was taken as the intersection of these three lists:
  • Lists B and C show a high degree of overlap, sharing more than half their genes, as they are derived from the same source and represent association with the two disticnt LKBl probesets.
  • the lm() function in the limma package of R bioconductor platform was used to determine the best fitting parameters to relate the expression of each probeset to the scores of the four LKBl-asociated gene clusters; interaction terms were not included in the model: expr(x) ⁇ a * expr(CREB) + b * expr(Mito)+
  • the accuracy of predicting LKB l mutations was assessed in resected LUAD using the pooled MSKCC2, UNC, and USC datasets, while predictions of LKBl mutations in cell lines were assessed in the pooled Sanger and CCLE datasets (FIG. 1C; FIGS. 6A-F; Tables 2-3).
  • Association with LKB l mRNA expression was assessed in tumors with unknown LKBl mutation status among the samples from the directors' challenge consortium that had not been used in the training cohort.
  • tumors with known mutation status three groups of tumors were considered: tumors with identified mutations in LKBl, tumors without observed mutations predicted to have loss of LKB l and tumors without mutations predicted to be LKBl wild-type. Expression of LKBl mRNA was compared between these groups using a student's t-test (FIG. ID).
  • LKBl mutation data was also available for the MSKCC1 dataset and these samples represented another potential test set. However, for unknown reasons univariate analysis comparing reported LKBl mutant and wild-type tumors in this dataset yielded fewer significant gene associations than would be expected by chance. In this dataset only five probesets out of 22000 passed a p-value cutoff of 0.001 ; in contrast for the Wash U and MSKCC2 cohorts, 118 probesets and 162 probesets passed this cutoff, respectively. Furthermore, the top ranked genes associated with LKB l mutations in this dataset showed no significant overlap with the consistent pattern of gene expression observed in each of the other clinical and cell line datasets (FIG. 1A; FIG. 5). Based on these findings, the inventors considered this dataset an outlier and excluded these data from the validation.
  • the inventors generated an association matrix using searches of GEO and ArrayExpress to obtain perturbations of interest to this study. Because the connectivity map did not employ a lung cancer derived cell line, they searched for all perturbations made to A549, a commonly studied lung adenocarcinoma cell line with a mutation in LKB1. The inventors next performed targeted queries for perturbations related to the hypotheses suggested by the GSEA and connectivity map analyses; specifically, they searched for perturbations involving pharmacologic or genetic modulations of the CREB pathway, the NRF2 transcription factor, mitochondria, and protein translation. Also, for the connectivity map associations highlighted in FIG. 2, the inventors downloaded Affymetrix .CEL files for the corresponding perturbations and controls and performed the analysis of gene expression changes.
  • the inventors eliminated redundant probesets to reduce the association matrix to a single probeset per gene, and then determined the top 200 over-expressed and underexpressed genes associated with each perturbation (roughly the top and bottom 2% of changes). Numeric overlap was then determined with each of the eight cluster scores and statistical significance calculated using a hypergeometric distribution by the phyper() function in the Bioconductor limma package.
  • the inventors performed univariate linear regression analysis to determine the association between the LKB1 classifier score and the IC50 values for 131 different compounds included in the GDSC study.
  • the CCLE study was split into two cell line groups. The set of cell lines that overlapped those included in the GDSC study was used as a training set confirmation, while samples that were not included among GDSC cell lines were used as an independent validation set. Linear regression was used to determine associations between cluster scores and the IC50 values and maximum inhibitory effects seen for each of the 24 inhibitors included in the CCLE study. Distributions were also compared for groups of cell lines given a binary classification as high or low LKB 1 loss score and student's t-tests were used to compare drug sensitivity between the two groups.
  • the inventors used a multivariable general linear model relating maximum inhibition to selumetinib to the LKB 1- loss score and each of three previously published MEK sensitivity signatures (Barretina et al, 2012; Dry et al, 2010; Loboda et al, 2012), as well as additional variables representing mutations in KRAS, NRAS, HRAS, BRAF, and LKB l.
  • the published gene signatures were used to calculate sensitivity scores for each cell line by averaging standardized expression for each of the published probesets.
  • A549, H2122, and H460 cell lines were generously shared with us by John Minna and Luc Girard (University of Texas, Southwestern). They were tested to ensure that they were mycoplasma negative, and were cultured in RPMI1640 containing 5% FBS, without antibiotics.
  • Empty pBABE viral plasmids, pBABE-LKBl and pBABE-LKBl-K78I were obtained from AddGene.
  • Phoenix cells were transfected with viral plasmids and retroviral particles were harvested from media supernatant 48 hours after transfection. Viruses were added to target cells with polybrene, and selection with 1 ⁇ g/ml puromycin was begun 48-72 hours after infection. Cells were then selected under puromycin for one to two weeks before performing subsequent experiments, with experiments being completed within two months.
  • Proliferation and drug sensitivity assays were performed in 96-well plates after seeding 1000 cells in each well. Quantitation of relative cell growth was made using the Alamar Blue colorimetric assay. Similarly, for drug sensitivity assays, 1000 cells per well were added to 96-well plates. Inhibitors were added at the specified concentrations 24 hours after seeding, and relative cell viability was quantified 72 hours after adding inhibitors using Alamar Blue. Selumetinib was purchased from Chemitek.
  • a 129-gene signature of LKB 1 loss was derived using two studies as a training cohort. Unsupervised clustering of these genes identified a subset of roughly 30% of lung adenocarcinomas that express an LKB 1 -deficient signature (FIG. IB).
  • a numeric LKB 1- classifier score derived from 16 of these genes identifies tumors with LKB1 mutations with high sensitivity and specificity in independent clinical testing datasets as well as in non-small cell lung cancer (NSCLC) cell lines and in non-lung cell lines of various tissue origins (FIG. 1C; FIGS. 6A-F).
  • LKB 1 mRNA is significantly lower among tumors predicted to have L B 1 loss, including the tumors that were sequenced as LKB 1 wild-type (FIGS. 1D-E). These tumors likely represent unrecognized cases of LKB 1 loss that could occur by undetected mutation, intragenic deletion, chromosomal loss, or by an epigenetic mechanism. This finding suggests that the specificity of this classifier exceeds the observed 76%, and that the LKBl-loss classifier is more sensitive than DNA sequencing for the detection of functional L B 1 loss.
  • the inventors also confirmed the findings using a second classifier derived from an independent training cohort (FIG. 8; Table 3).
  • the inventors also found the signature to be associated with LKB 1 loss in other cancer types including breast adenocarcinoma and cervical squamous carcinoma (FIGS. 14A-C).
  • FIGS. 14A-C breast adenocarcinoma and cervical squamous carcinoma
  • other genes and proteins differentially expressed by tumors with known LKBl mutations are concordantly dysregulated among the tumors with predicted loss (FIGS. 15A-B).
  • 'Mitochondrial' cluster had high expression of oxidative phosphorylation and mitochondria-associated genes as well as genes involved in protein translation; a second, referred to as the 'NRF2' cluster, expresses oxidative stress response genes driven by the NRF2 transcription factor; this phenotype was expressed by approximately half of tumors with LKBl loss.
  • the final up-regulated cluster was found to be enriched in genes with CREB consensus sequences in their promoter regions, and was also found to be up-regulated by the cAMP inducer forskolin; hence it was referred to as the 'CREB' cluster.
  • TGF-beta and stroma-related genes comprised a component of the down-regulated genes, but multiple phenotypes likely contributed to this transcriptional node (FIGS. 9A-B).
  • TGF-beta induced a significant subset of these down-regulated genes, while simultaneously attenuating both the NRF2 and CREB transcriptional components; conversely, c-SRC inhibition induced CREB activation, suggesting antagonism between these two pathways and CREB (FIG. 10).
  • LKBl a well recognized downstream target of AMPK and caused significant decrease in cell growth (GIG. 3A; FIGS. 1 1A-B).
  • Microarray analysis of gene expression changes in A549 and H2122 demonstrated restoring LKB l significantly (P ⁇ 1.0e-30 by hypergeometric test) down-regulates the CREB-driven gene cluster and increases expression of a subset of the down-regulated genes, while mitochondrial and NRF2 associated clusters are unaffected (FIGS. 3B-C; FIGS. 12A-C).
  • the attenuation of CREB activation was confirmed in three cell lines using a luciferase reporter driven by the CRE- consensus sequence, which showed a reduction in reporter activity of 30-40% (FIG. 3D; p- value less than 0.05 for each cell line).
  • LKBl loss was an independent determinant of MEK sensitivity
  • the inventors used a multivariable general linear model to account for previously reported associations with MEK sensitivity: mutations in KRAS, NRAS, HRAS, and BRAF, as well as previously reported gene signatures from three studies 4 ' 5 ' 15 ' 16 .
  • This analysis demonstrated that the signature of LKBl loss was still significantly associated with sensitivity after controlling for other factors and thus represents a novel independent predictor of response to this class of drugs (FIGS. 4C-F).
  • the inventors next wanted to determine whether this association represented a causal link between LKBl mutations and MEK sensitivity.
  • NRF2 is activated by somatic mutations in NRF2 or KEAP1 in NSCLC 23-27 , and the high frequency of NRF2 activation among LKBl -deficient tumors suggests that selective pressure exists for these mutations as a secondary protective mechanism.
  • LKBl loss is an important platform for studying the behavior of these tumors in vivo.
  • the analysis shows that LKBl loss produces distinctly different phenotypes in humans.
  • murine tumors did not express genes characteristic of CREB or NRF2 activation, while TGF-beta and related signaling pathways show increased expression in the murine model.
  • FIG. 1A A first figure.
  • LY-294002 H L60 broadinstitute. org/cmap/# avg diff: HL60; 10uM LY-294002 (n 9) vs DMSO
  • LY-294002 MCF7 broadinstitute. org/cmap/# avg diff: MCF7; 10uM LY-294002 (n 18) vs DMSO
  • PGC1A C2C12 broadinstitute.org/gsea/msigdb Mootha_PGC gene set (n 412 genes)
  • H292 broadinstitute.org/cgi-bin/cancer/datasets 1 avg diff/standard deviation: H292 cell line vs NSCLC cell lines
  • DN CREB MIN6 GSE2060 avg diff: MI N6; DN CREB (n 2) vs GFP
  • keapW- mouse liver GSE1 1287 avg diff: KEAP1-/- liver (n 3) vs control
  • siNRF2 A549 GSE28230 avg diff: A549; si RF2 (n 3) vs control siR A
  • NRF2 active 19/198 60.1 5.8 (4.0, 8.3) ⁇ 1 e-16 NRF2 low 135/653 20.7
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. VI. References
  • the CRTC1-NEDD9 signaling axis mediates lung cancer progression caused by LKB l loss. Cancer Res. 72, 6502-6511 (2012). Gu, Y. et al. Altered LKBl/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion. Oncogene 31, 469 ⁇ 79 (2012). Komiya, T. et al. Enhanced activity of the CREB co-activator Crtcl in LKBl null lung cancer. Oncogene 29, 1672-80 (2010). Mitsuishi, Y. et al Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 22, 66-79 (2012).

Abstract

L'invention concerne des méthodes de classement et de traitement des cancers, en particulier des adénocarcinomes, y compris les cancers du poumon, du sein et du col de l'utérus. Ces méthodes font appel à une signature génique qui identifie les cancers comme étant déficients en LKB1 et, en outre, comme répondant à un traitement par inhibiteur de MEK.
PCT/US2014/027272 2013-03-14 2014-03-14 Méthodes de classement et de traitement des adénocarcinomes WO2014152377A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361784757P 2013-03-14 2013-03-14
US61/784,757 2013-03-14
US201361830087P 2013-06-01 2013-06-01
US61/830,087 2013-06-01

Publications (1)

Publication Number Publication Date
WO2014152377A1 true WO2014152377A1 (fr) 2014-09-25

Family

ID=51581202

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/027272 WO2014152377A1 (fr) 2013-03-14 2014-03-14 Méthodes de classement et de traitement des adénocarcinomes

Country Status (1)

Country Link
WO (1) WO2014152377A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017180581A1 (fr) * 2016-04-15 2017-10-19 Genentech, Inc. Procédés diagnostiques et thérapeutiques relatifs au cancer

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100130527A1 (en) * 2008-11-18 2010-05-27 Lehrer Raphael Individualized cancer treatment
US20110119776A1 (en) * 2007-02-05 2011-05-19 Wong Kwok-Kin Methods of diagnosing and prognosing lung cancer
US20110236903A1 (en) * 2008-12-04 2011-09-29 Mcclelland Michael Materials and methods for determining diagnosis and prognosis of prostate cancer
WO2013019927A1 (fr) * 2011-08-03 2013-02-07 Signal Pharmaceuticals, Llc Identification d'un profil d'expression génique à titre de biomarqueur prédictif de l'état lkb1

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110119776A1 (en) * 2007-02-05 2011-05-19 Wong Kwok-Kin Methods of diagnosing and prognosing lung cancer
US20100130527A1 (en) * 2008-11-18 2010-05-27 Lehrer Raphael Individualized cancer treatment
US20110236903A1 (en) * 2008-12-04 2011-09-29 Mcclelland Michael Materials and methods for determining diagnosis and prognosis of prostate cancer
WO2013019927A1 (fr) * 2011-08-03 2013-02-07 Signal Pharmaceuticals, Llc Identification d'un profil d'expression génique à titre de biomarqueur prédictif de l'état lkb1

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GRIGORIEVA ET AL.: "D-Glucuronyl C5-epimerase suppresses small- cell lung cancer cell proliferation in vitro and tumour growth in vivo", BRITISH JOURNAL OF CANCER, vol. 105, 7 June 2011 (2011-06-07), pages 74 - 82 *
ZHANG ET AL.: "Unraveling the mystery of cancer metabolism in the genesis of tumor-initiating cells and development of cancer", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1836, 21 March 2013 (2013-03-21), pages 49 - 59 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017180581A1 (fr) * 2016-04-15 2017-10-19 Genentech, Inc. Procédés diagnostiques et thérapeutiques relatifs au cancer
CN109072311A (zh) * 2016-04-15 2018-12-21 豪夫迈·罗氏有限公司 用于癌症的诊断和治疗方法

Similar Documents

Publication Publication Date Title
Tsang et al. KRAS (but not BRAF) mutations in ovarian serous borderline tumour are associated with recurrent low‐grade serous carcinoma
Sholl et al. Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: the lung cancer mutation consortium experience
JP6144695B2 (ja) タキサン療法を用いて乳癌を処置する方法
US9670549B2 (en) Gene expression signatures of neoplasm responsiveness to therapy
Bruun et al. Prognostic, predictive, and pharmacogenomic assessments of CDX 2 refine stratification of colorectal cancer
Zhang et al. Total DNA input is a crucial determinant of the sensitivity of plasma cell-free DNA EGFR mutation detection using droplet digital PCR
MX2013013746A (es) Biomarcadores para cancer de pulmon.
De Melo et al. Mutational profile and new IASLC/ATS/ERS classification provide additional prognostic information about lung adenocarcinoma: a study of 125 patients from Brazil
Rosenbaum et al. Clinical, genomic, and transcriptomic correlates of response to immune checkpoint blockade-based therapy in a cohort of patients with angiosarcoma treated at a single center
JP2017521058A (ja) 癌処置のための個別化三剤治療を選択するための方法
WO2022053065A1 (fr) Biomarqueur utilisé pour prédire ou évaluer des patients atteints d'un cancer du poumon, procédé de détection et application
KR20160086326A (ko) 핵산 바이오마커 및 이의 용도
Yu et al. Somatic DNA mutation analysis in targeted therapy of solid tumours
Huang et al. Molecular gene signature and prognosis of non-small cell lung cancer
Song et al. Transcriptional signatures for coupled predictions of stage II and III colorectal cancer metastasis and fluorouracil‐based adjuvant chemotherapy benefit
KR20230080281A (ko) 전이성 신장암 예후 예측용 신규 바이오마커 및 이의 용도
Yokouchi et al. Detection of somatic TP53 mutation in surgically resected small-cell lung cancer by targeted exome sequencing: association with longer relapse-free survival
WO2014089055A1 (fr) Prévision de réponse au tivozanib
US20230002831A1 (en) Methods and compositions for analyses of cancer
KR102403807B1 (ko) 항암제 반응성 예측용 바이오마커 및 이의 용도
Lin et al. Oxaliplatin-based chemotherapy might provide longer progression-free survival in KRAS mutant metastatic colorectal cancer
WO2014152377A1 (fr) Méthodes de classement et de traitement des adénocarcinomes
US10100366B2 (en) Methods of detecting cancers sensitive to cabozantinib
Paquet et al. A 12‐gene signature to distinguish colon cancer patients with better clinical outcome following treatment with 5‐fluorouracil or FOLFIRI
WO2023125788A1 (fr) Biomarqueurs pour le traitement du cancer colorectal

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14768827

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14768827

Country of ref document: EP

Kind code of ref document: A1