EP2135073A1 - Methods of determining acute myeloid leukemia response to treatment with farnesyltransferase - Google Patents
Methods of determining acute myeloid leukemia response to treatment with farnesyltransferaseInfo
- Publication number
- EP2135073A1 EP2135073A1 EP08731984A EP08731984A EP2135073A1 EP 2135073 A1 EP2135073 A1 EP 2135073A1 EP 08731984 A EP08731984 A EP 08731984A EP 08731984 A EP08731984 A EP 08731984A EP 2135073 A1 EP2135073 A1 EP 2135073A1
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- European Patent Office
- Prior art keywords
- yes
- gene
- ras
- genes
- aml
- Prior art date
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Classifications
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- 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
- G01N33/57426—Specifically defined cancers leukemia
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B26—HAND CUTTING TOOLS; CUTTING; SEVERING
- B26F—PERFORATING; PUNCHING; CUTTING-OUT; STAMPING-OUT; SEVERING BY MEANS OTHER THAN CUTTING
- B26F1/00—Perforating; Punching; Cutting-out; Stamping-out; Apparatus therefor
- B26F1/18—Perforating by slitting, i.e. forming cuts closed at their ends without removal of material
- B26F1/20—Perforating by slitting, i.e. forming cuts closed at their ends without removal of material with tools carried by a rotating drum or similar support
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B21—MECHANICAL METAL-WORKING WITHOUT ESSENTIALLY REMOVING MATERIAL; PUNCHING METAL
- B21D—WORKING OR PROCESSING OF SHEET METAL OR METAL TUBES, RODS OR PROFILES WITHOUT ESSENTIALLY REMOVING MATERIAL; PUNCHING METAL
- B21D28/00—Shaping by press-cutting; Perforating
- B21D28/02—Punching blanks or articles with or without obtaining scrap; Notching
- B21D28/14—Dies
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B21—MECHANICAL METAL-WORKING WITHOUT ESSENTIALLY REMOVING MATERIAL; PUNCHING METAL
- B21D—WORKING OR PROCESSING OF SHEET METAL OR METAL TUBES, RODS OR PROFILES WITHOUT ESSENTIALLY REMOVING MATERIAL; PUNCHING METAL
- B21D37/00—Tools as parts of machines covered by this subclass
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- 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
- FTI farnesyltransferase inhibitor
- Ras Ras
- FTIs Farnesyl transferase inhibitors
- AML Acute myelogenous leukemia
- the mutation status of the ras gene was considered to be a candidate biomarker for patient response to FTIs. This rationale was based on pre-clinical evidence that FTIs could block Ras-transformed cells, and that specific point mutations within ras genes cause constitutive activation of the Ras pathway in many cancers. End et al. (2001) Reuter et al. (2000); and Bos et al. (1989). Since it is generally accepted that tumors are heavily reliant on the activation of one or two pathways ("oncogene addiction" hypothesis), it follows that patients whose tumors are promoted by a particular pathway should respond to drugs that inhibit that pathway. Weinstein et al. (2006).
- this classifier could predict response to tipifarnib in an independent set of 54 samples from relapsed or refractory AML, with a NPV and PPV of 92% and 28%, respectively (odds ratio of 4.4). Therefore, in both newly diagnosed and relapsed or refractory AML, this classifier improves the overall response rate by approximately 50% while maintaining a high NPV, and significantly improves patient overall survival.
- the two-gene classifier was also validated by qPCR in thirty AML samples from the same clinical study demonstrating a negative predictive value (NPV) and positive predictive value (PPV) of 81% and 50%, respectively (odds ratio of 4.3). These data indicate that a simple two-gene expression assay may have utility in diagnosing a population of AML patients who are more likely to respond to tipifarnib.
- Microarray technology has been utilized to identify gene expression profiles that are predictive of response or resistance to a number of different therapeutic modalities in a variety of cancers, including chemotherapies or endocrine therapies in breast cancer, diffuse large b-cell lymphoma and leukemia.
- Figure 1 depicts the performance of the RASGRPl gene as a predictor of response to tipifarnib in AML.
- Figure 2 depicts the performance of the RASGRPl :APTX gene pair as a predictor of response to tipifarnib in AML.
- the overall survival of newly diagnosed AML patients (A) and relapsed/refractory AML patients (C) stratified with the 2-gene classifier are plotted using Kaplan-Meier analysis.
- the accuracy rates of the two-gene classifier in newly diagnosed AML (B) and relapsed/refractory AML (D) are shown.
- Figure 3 depicts the performance of RASGRPl :APTX gene classifier using qPCR.
- A The normalized RASGRPl :APTX Ct values for 20 responders and 10 patients with progressive disease. The 20 independent samples and 10 training samples that were run on microarray are shown separately. Horizontal bars indicate group means.
- B The accuracy rates of the RASGRPl gene classifier in newly diagnosed AML for all 30 patients are shown using a cutoff of 0 was used to stratify patients.
- C The associated overall survival of the stratified patients are plotted using Kaplan-Meier analysis.
- Figure 4 depicts the performance of the RASGRPl gene as a predictor of response to tipifarnib in relapsed and refractory AML.
- Figure 5 depicts the overall survival of non-FTI treated AML patients stratified with the RASGRPl :APTX gene expression ratio.
- Three cDNA probes for both RASGRPl and APTX were present in the available data set.
- Figure 6 depicts the correlation of Affymetrix and qPCR data.
- the therapeutic agents referred to in this specification are FTIs. They take on a multitude of forms but share the essential inhibitory function of interfering with or lessening the farnesylation of proteins implicated in cancer and proliferative diseases.
- the FTIs are those indicated for the treatment of leukemias such as AML.
- a patient who responds to an FTI is one in whom a reduction of more than 50% of blast cells is seen in bone marrow following treatment with the FTI.
- FTIs are within the scope of the invention and include those described in 5,976,851 ; 5,972,984; 5,972,966; 5,968,965; 5,968,952; 6,187,786; 6,169,096; 6,037,350; 6,177,432; 5,965,578; 5,965,539; 5,958,939; 5,939,557; 5,936,097; 5,891,889; 5,889,053; 5,880,140; 5,872,135; 5,869,682; 5,861,529; 5,859,015; 5,856,439; 5,856,326; 5,852,010; 5,843,941 ; 5,807,852; 5,780,492; 5,773,455; 5,767,274; 5,756,528; 5,750,567; 5,721,236; 5,700,806; 5,661,161; 5,602,098; 5,585,359; 5,578,629; 5,534,537; 5,53
- the therapeutic agents referred to in this specification are those that have an effect on the biological pathway explicated through the gene expression analysis of leukemic cells subjected to treatment with quinilone-based FTIs.
- nucleic acid sequences having the potential to express proteins or peptides within the genome is not determinative of whether a protein or peptide is expressed in a given cell. Whether or not a given gene capable of expressing proteins or peptides does so and to what extent such expression occurs, if at all, is determined by a variety of complex factors. Irrespective of difficulties in understanding and assessing these factors, assaying gene expression can provide useful information about the cellular response to a given stimulus such as the introduction of a drug or other therapeutic agent. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles.
- the gene expression profiles of this invention are used to identify and treat patients who will likely benefit from a given therapy or exclude patients from a.given therapy where the patient likely would experience little or no beneficial response to the drug or therapy.
- RNA that is produced by a gene that can code for a protein or peptide includes determining the. amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcription PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify copy DNA (cDNA) or copy RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in U. S. Patents such as: U. S. Pat. Nos.
- Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying the effect of FTIs on cell biology and the likely effect of treatment based on analysis of such effects.
- Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same.
- the product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray.
- the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells.
- mRNA mRNA
- Analysis of the expression levels is conducted by comparing such intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a tissue that has been treated with a drug can be compared with the expression intensities generated from the same tissue that has not been treated with the drug. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.
- Gene expression profiles can also be displayed in a number of ways. The most common method is to arrange a ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data is arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum.
- Commercially available computer software programs are available to display such data including "OMNIVIZ PRO" software from Batelle and "TREE VIEW" software from Stanford
- the genes that are differentially expressed are either up regulated or down regulated in diseased cells following treatment with an FTI.
- Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline.
- the baseline is the measured gene expression of the untreated diseased cell.
- the genes of interest in the treated diseased cells are then either up regulated or down regulated relative to the baseline level using the same measurement method.
- levels of up and down regulation are distinguished based on fold changes of the intensity measurements of hybridized microarray probes. A 1.5 fold difference is preferred for making such distinctions.
- the treated cell is found to yield at least 1.5 times more, or 1.5 times less intensity than the untreated cells.
- a 1.7 fold difference is more preferred and a 2 or more fold difference in gene expression measurement is most preferred.
- a portfolio of genes is a set of genes grouped so that information obtained about them provides the basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice.
- the judgments supported by the portfolios involve the treatment of leukemias with FTl's.
- Portfolios of gene expression profiles can be comprised of combinations of genes.
- One method of the invention involves comparing gene expression profiles for various genes to determine whether a person is likely to respond to the use of a therapeutic agent. Having established the gene expression profiles that distinguish responder from non-responder, the gene expression profiles of each are fixed in a medium such as a computer readable medium as described below.
- a patient sample is obtained that contains diseased cells (such as hematopoietic blast cells in the case of AML) is then obtained.
- Sample RNA is then obtained and amplified from the diseased patient cell and a gene expression profile is obtained, preferably via micro-array, for genes in the appropriate portfolios. The expression profiles of the samples are then compared to those previously determined as responder and non-responder.
- sample expression patterns are consistent with an FTI responder expression pattern then treatment with an FTI could be indicated (in the absence of countervailing medical considerations). If the sample expression patterns are consistent with an FTI non-responder expression pattern then treatment with an FTI would not be indicated.
- consistency of expression patterns is determined based on intensity measurements of micro-array reading as described above.
- gene expression profile analysis can be conducted to monitor treatment response.
- gene expression analysis as described above is conducted on a patient treated with an FTI at various periods throughout the course of treatment. If the gene expression patterns are consistent with a responder then the patient's therapy is continued. If it is not, then the patient's therapy is altered as with additional therapeutics such as tyrosine kinase inhibitor, changes to the dosage, or elimination of FTI treatment.
- additional therapeutics such as tyrosine kinase inhibitor, changes to the dosage, or elimination of FTI treatment.
- the profiles may show that three genes are up-regulated consistent with a responder but that another gene is not up-regulated as would ordinarily be the case for a responder.
- statistical algorithms can be applied to determine the probability that the patient will respond or not respond to the drug. Statistical algorithms suitable for this purpose are well known and are available.
- Articles of this invention are representations of the gene expression profiles useful for treating, diagnosing, prognosticating, staging, and otherwise assessing diseases that are reduced to a medium that can be automatically read such as computer readable media (magnetic, optical, and the like).
- the articles can also include instructions for assessing the gene expression profiles in such media.
- the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above.
- the articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format.
- a graphical recordation is one such format.
- FIG. 1 shows an example of the graphical display of such a recordation. Clustering algorithms such as those incorporated in "OMNIVIZ” and "TREE VIEW" computer programs mentioned above can best assist in the visualization of such data.
- nucleic acid arrays e.g. cDNA or oligonucleotide arrays
- cDNA or oligonucleotide arrays as described above, configured to discern the gene expression profiles of the invention.
- clustering analysis including the algorithms mentioned above
- a dynamic map was constructed based upon such expression data.
- Such a genetic network map is useful for drug discovery. For example, once basic genes of interest were identified, a list of potential up-stream regulatory genes was found using such a genetic network map. The genes so identified or their expression products were then analyzed for their use as drug targets. In some embodiments, the regulator)' function of the particular genes identified was used to identify therapeutics for use in treating leukemia.
- RNA processing and RNA editing are all accomplished by proteins which are coded by their own genes.
- DNA sequences can exert long-range control over the expression of other genes by positional effects. Therefore, the expression of genes is often regulated by the expression of other genes.
- Those regulatory genes are called upstream genes, relative to the regulated or down-stream genes.
- A++>B ⁇ >C++>D where: A, B, C, D are genes ++ up-regulates ⁇ down-regulates Gene A is an up-stream gene of gene B and B is an up-stream gene of C.
- A is an up-stream gene of gene B and B is an up-stream gene of C.
- the network is frequently looped and inter-connected.
- the expression of a gene is regulated by its own product as either a positive or negative feedback.
- Cluster analysis methods were used to group genes whose expression level is correlated. Methods for cluster analysis are described in detail in Harfigan (1975) Clustering Algorithms, NY, John Wile and Sons, Inc, and Everritt, (1980) Cluster Analysis 2nd. Ed. London Heineman Educational books, Ltd. Path analysis was used to decompose relations among variables and for testing causal models for the genetic networks. Multiple primary targets of a drug in leukemic cells were identified as were drugs/drug classes useful in treating such cells. According to the current invention, drugs are any compounds of any degree of complexity that perturb a biological system.
- the biological effect of a drug may be a consequence of drug-mediated changes in the rate of transcription or degradation of one or more species of RNA, the rate or extent of translation or post-translational processing of one or more polypeptides, the rate or extent of the degradation of one or more proteins, the inhibition or stimulation of the action or activity of one or more proteins, and so forth.
- the preferred drugs of this invention are those that modulate the MAPK/ERK signaling pathways, TGF- ⁇ , WNT or apoptotic pathways.
- tyrosine kinase inhibitors include, without limitation, tyrosine kinase inhibitors, MEK kinase inhibitors, Pl 3K kinase inhibitors, MAP kinase inhibitors, apoptosis modulators and combinations thereof.
- exemplary drugs that are most preferred among these are the "GLEEVEC" tyrosine kinase inhibitor of Novartis, U-0126 MAP kinase inhibitor, PD-098059 MAP kinase inhibitor, SB-203580 MAP kinase inhibitor, and antisense, ribozyme, and DNAzyme BcI-XL anti-apoptotics.
- Examples of other useful drugs include, without limitation, the calanolides of 6,306,897; the substituted bicyclics of 6,284,764; the indolines of 6,133,305; and the antisense oligonucleotides of 6,271,210.
- the drugs of the instant invention can be therapeutics directed to gene therapy or antisense therapy.
- Oligonucleotides with sequences complementary to an mRNA sequence can be introduced into cells to block the translation of the mRNA, thus blocking the function of the gene encoding the mRNA.
- the use of oligonucleotides to block gene expression is described, for example, in, Strachan and Read, Human Molecular Genetics, 1996.
- antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O- alkylRNA, or other antisense oligonucleotide mimetics.
- Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.
- the gene of interest can be ligated into viral vectors that mediate transfer of the therapeutic DNA by infection of recipient host cells.
- viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like.
- therapeutic DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Protocols for molecular methodology of gene therapy suitable for use with the gene is described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totowa NJ, 1996.
- compositions comprising the drugs of this invention may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences.
- a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the drug.
- the effective amount of the drug may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
- the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
- the drugs of this invention include chemical derivatives of the base molecules of the drug. That is, they may contain additional chemical moieties that are not normally a part of the base molecule.
- Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
- the drugs of this invention can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
- the drugs can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
- they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
- An effective but non-toxic amount of the compound desired can be employed as a modulating agent.
- the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day.
- the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
- An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day.
- the dosages are adjusted when combined to achieve desired effects.
- dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
- compounds or modulators used in the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
- compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
- the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
- the active agents can be administered concurrently, or they each can be administered at separately staggered times.
- the dosage regimen utilizing the compounds or modulators in the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular drug employed.
- a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
- Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
- the drugs of this invention can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
- carrier suitable pharmaceutical diluents, excipients or carriers
- the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
- suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
- suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
- Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
- Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
- the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
- suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
- Other dispersing agents include glycerin and the like.
- sterile suspensions and solutions are desired.
- Isotonic preparations which generally contain suitable preservatives, are employed when intravenous administration is desired.
- the drugs in the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
- Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
- Drugs in the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
- the drugs in the present invention may also be coupled with soluble polymers as targetable drug carriers.
- Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
- the drugs in the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
- a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
- the drugs may be administered in capsule, tablet, or bolus form or alternatively they can be mixed with feed.
- the capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate.
- suitable carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate.
- These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained.
- An inert ingredient is one that will not react with the drugs and which is non-toxic to the animal being treated.
- Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection.
- the active ingredient may also be administered by simply mixing the compound with the feedstuff or by applying the compound to the surface of the foodstuff.
- the compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraluminal, intratracheal, or subcutaneous.
- the injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier.
- Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like.
- aqueous parenteral formulations may also be used.
- the vegetable oils are the preferred liquid carriers.
- the formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.
- Bone marrow samples were collected from consenting patients before treatment with tipifarnib and mononuclear cells were processed on site. Bone marrow aspirates were diluted with PBS and centrifuged with ficoll-diatrizoate (1.077g/ml). Enriched leukemic blood cells were washed twice with PBS, resuspended in FBS with 10% DMSO and immediately frozen at - 70 0 C to -80°C. Total RNA was extracted from cell samples using the Trizol Kit (Qiagen, Santa Clarita, CA). RNA quality was determined by assessing the presence of ribosomal bands on an Agilent Bioanalyzer. Good quality samples were further processed for microarray analysis.
- cDNA and cRNA were performed according to Affymetrix (Santa Clara, CA) protocols. Since the yield of many samples was low two rounds of linear amplification was performed as previously described. 20070048782.
- 1 1 ⁇ g ofcRNA were fragmented randomly by incubation at 94°C for 35 min in 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Fragmented cRNA was hybridized to U133A arrays at 45°C for 16 h in a rotisserie oven set at 60 rpm.
- arrays were washed (with 6x SSPE and 0.5x SSPE containing Triton X-100 (0.005%)), and stained with streptavidin-phycoerythrin (SAPE; Molecular Probes, Eugene, OR). Quantification of bound labeled probe was conducted using the Agilent G2500A GeneArray scanner (Agilent Technologies, Palo Alto, CA).
- Chip performance was quantified by calculating a signal to noise ratio (raw average signal/noise). Chips were removed from further analysis if their signal-to-noise ratio was less than 20 or if the present calls on the chip was less than 30%. Genes were only included in further analysis if they were called "present” in at least 10% of the chips. Approximately 12,000 Affymetrix probe sets remained following this cut-off. The quality of the gene expression data were further controlled by identifying outliers based on principal components analysis and by analyzing the normal distributions of the gene intensities (Partek Pro V5.1).
- microarray data have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSl- XXXX. Response definitions
- HI hematological improvement
- Receiver Operator Characteristic (ROC) analysis was utilized to test the overall predictive value of individual genes and/or multigene classifiers.
- the genes that passed these criteria were ranked by AUC (Area under the ROC curve).
- the response score was used to calculate each patient's chance to response to tipifarnib therapy.
- the score was defined as the linear combination of weighted expression signals with the t statistic as the weight.
- the threshold was determined from the ROC curve of the training set to ensure 100% sensitivity and the highest specificity.
- leave-one-out cross validation (LOOCV) was carried out.
- the response scores for the 'left-out' samples based on different numbers of genes were recorded.
- the performances of the predictors with different numbers of genes were assessed based on misclassification error rate, sensitivity, specificity, p values measuring the separation of Kaplan-Meier curves of the two predicted groups. And the best predictor was selected accordingly.
- the Top Scoring Pair (TSP) algorithm was first introduced by Geman et al. (2004). In essence, the algorithm ranks all the gene pairs (genes i and j) based on the absolute difference (Dij) in the frequency of event where gene i has higher expression value than gene j in samples among class Cl to C2. In the cases of there are multiple top scoring pairs (all sharing the same Dij), we select the top pair by a secondary rank score that measures the magnitude to which inversions of gene expression levels occur from one class to the other within a pair of genes. The top pair with highest frequency of absolute Dij > 2 fold in all samples will be selected as candidate pair. The candidate pair was then assessed in an independent testing data set.
- Leave-one-out cross validation was carried out in the training data set to evaluate how the algorithm perform. The performances of the predictors were assessed based on maximum misclassification error rate. All the statistical analyses were done using R (R Development Core Team, 2006). Real-Time Quantitative RT-PCR
- RNA was reversed transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturers instructions. Samples were then incubated at 25 0 C for 10 minutes and then 37 0 C for 30 minutes for optimum RNA conversion. QPCR was performed using the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) with all samples run in triplicate.
- Each reaction contained 5 ⁇ l TaqMan® Universal PCR Master Mix containing UNG (Applied Biosystems, Foster City, CA), 4.5 ⁇ l of cDNA template and 0.5 ⁇ l of 20 x Assay on Demand Gene Expression Assay Mix or 9 pmol of both forward and reverse primer and 2.5 pmol of probe (Applied Biosystems, Foster City, CA), in a total reaction volume of 10 ⁇ l. All primer, probe sets were chosen due to the small amplicon size (less than 100 nucleotides) and FAM fluorogenic probes were used. Primers and probes used were APTX (product number 4331 182 Applied Biosystems) and RASGRPl (product number 4351372 Applied Biosystems). The RASGRPl.
- APTX expression ratio was calculated by normalizing the raw Ct values by subtracting the mean Ct from the sample set, dividing by the standard deviation, and then calculating the difference of the normalized Ct values of each gene (APTX- RASGRPl). Ma et al. (2004). Results
- N-Ras mutation N-Ras mutation, n (%) 11 (34) median age, y (range) 74 (34-85) 73 (63-85) sex, n male (%) 95 (60) 41 (61)
- CR complete response
- PR partial response
- Hl hematological improvement
- SD stable disease
- PD progressive disease
- NE not evaluable
- PGx pharmacogenomics Ras mutational status and patient outcome
- N-Ras activating mutations (codons 12, 13, 61). Thirty-four percent (11/32) of patients exhibited N-Ras mutations with one patient having mutations at multiple codons (Table 2). There was no statistically significant correlation between N-Ras mutational status and response to tipifarnib or overall survival. Table 2.
- the genes were ranked based on the area under the curve (AUC) defined from a receiver operator characteristic (ROC) analysis of the training set. This value represents the overall predictive value of the gene with an AUC of 1.0 indicating perfect classification.
- AUC area under the curve
- ROC receiver operator characteristic
- RASGRPl The predictive value of RASGRPl was not improved if additional genes were added to the classifier using a linear approach.
- TSP Top Scoring Pair
- the TSP from the training set was RASGRPl and aprataxin (APTX). RASGRPl and APTX were over- and under-expressed in responders, respectively.
- APTX aprataxin
- APTX classifier in an independent set of relapsed or refractory AML.
- the RASGRPl APTX gene classifier stratified responders with 92% NPV and 27.6% PPV (compared to 18.5% prevalence) (Fig 3C).
- the associated odds ratio for being a responder was 4.38. While this was similar to the predictive accuracy of RASGRPl alone, the application of the TSP classifier demonstrated a better NPV and an improved difference in overall survival of 98 days between predicted responders and progressors (Fig 3D), compared to only 56 days for RASGRPl (Fig 4).
- a two-gene expression ratio allows the use of a more clinically relevant qPCR detection system.
- Stratification of patient populations to predict therapeutic response is becoming increasingly valuable in the clinical management of cancer patients.
- companion diagnostics are required for the stratification of patients being treated with targeted therapies such as trastuzumab (Herceptin, Genentech) in metastatic breast cancer, and cetuximab (Erbitux, Merck) in colorectal cancer. Seidman et al. (2001); and Moroni et al. (2005).
- Predictive biomarkers are also being utilized for imatinib (Gleevec, Novartis) in gastrointestinal stromal tumors, and for erlotinib (Tarceva, OSI Pharmaceuticals) and gefitinib (Iressa, Astra-Zeneca) in lung cancer.
- imatinib Gavec, Novartis
- erlotinib Tarceva, OSI Pharmaceuticals
- gefitinib Iressa, Astra-Zeneca
- RASGRPl was the most robust single predictive gene expression marker with an overall predictive accuracy of 77% in the cross-validated training set.
- RASGRPl is a guanine nucleotide exchange factor (GEF) that specifically activates Ras. Stone (2006).
- GEF guanine nucleotide exchange factor
- Expression of RASGRPl has been found in brain, T-cells, cells of monocytic lineage, and primitive hematopoietic precursors.
- Kawasaki et al. 1998 Ebinu et al. (1998); and Tognon et al. (1998).
- RASGRP4 was previously identified as a potential oncogene in AML (Reuther et al.
- cross-validation only provides a model of performance and thus testing of an independent data set was performed to provide bona fide accuracy of response prediction.
- a simple qPCR-based diagnostic assay has wider utility in the clinic than gene expression microarrays due to the ability to assay poor quality clinical samples that may not be profiled by current microarray technologies.
- RASGRPl APTX expression ratio
- the classifier demonstrated a NPV and PPV of 81% and 50%, respectively and provided a clear overall survival advantage for those patients predicted to be responders.
- RASGRPl has been shown to activate H-RAS and N-Ras, but not K-Ras, exclusively on the golgi apparatus. Bivona et al. (2003); and Perez de Castro et al. (2004). Further, K- Ras and N-Ras can be alternatively geranylgeranylated following farnesy transferase inhibition. Whyte et al. (1997). H-Ras on the other hand is only farnesylated and this may explain the observation that tumors transformed with H-Ras are more sensitive than those transformed with N-, or K-Ras. End et al. (2001); and Lubet et al. (2006).
- H-Ras activating mutations have not been identified in AML, the specific activation of H-Ras pathways by other means (such as Ras-specific GEFs) may still be a target of FTIs in certain tumors.
- AKAP 13 is predictive of resistance to tipifarnib in relapsed/refractory AML. 200700448782.
- AKAPl 3 is also a GEF, but activates the Rho pathway. Sterpetti et al. (1999).
- expression of AKAP13 did not demonstrate predictive utility in newly diagnosed AML. This may be because the population of leukemic cells that over-express AKAPl 3 is absent in newly diagnosed disease and only proliferates in late stage AML.
- the other question that arises is why over-expression of the RASGRPl GEF increases sensitivity while over-expression of the AKAPl 3 GEF increases resistance to tipifarnib?
- Rho GEFs have been found to drive cellular transformation in a Ras- independent fashion. Reuther et al. (2001); and Sahai et al. (2002). Thus, one hypothesis is that AKAP13 activates a downstream compensatory pathway in RhoA while RAGRPl activates Ras, a clear target of FTIs. More biochemical analyses will need to be done to investigate this model. Nevertheless, the identification of two GEFs playing opposing roles in responsiveness to an FTI does highlight the importance of this class of small GTPase activators in FTI-mediated therapy. It also highlights the need for multiple markers in predicting response to targeted therapies across a wide range of diseases and disease subtypes. As GEFs are increasingly becoming attractive drug targets it may also be of interest to investigate combination therapies of FTIs and inhibitors of specific GEFs.
- the classifier has predictive utility in both newly diagnosed and relapsed or refractory AML, and improves the overall response rate by approximately 50% while maintaining a high NPV. In addition, stratification with this classifier significantly improves patient overall survival.
- Our data compare favorably to the use of FDA-approved companion diagnostics for targeted cancer therapies such as Herceptin. For instance, it has been demonstrated that stratification of metastatic breast cancer patients with over-expression of Her2/Neu improves the overall response to Herceptin and paclitaxel combination therapy from approximately 59% to 69% or 75% when using the HercepTest or PathVysion tests, respectively. Seidman et al. (2001). Our data therefore indicates that a simple two-gene expression assay may have utility in diagnosing a population of AML patients who are more likely to respond to tipifarnib.
- RasGRP Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs Science 280:1082-1086 Ehmann et al. (2006) Detection of N-RAS and K-RAS in their active GTP-bound form in acute myeloid leukemia without activating RAS mutations Leuk Lymphoma 47: 1387-
- DbI family protein found mutated in leukemia, causes transformation by activation of
- RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia J Biol Chem 277:30508-30514 Rosenwald et al. (2002) The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma N Engl J Med 346:1937-1947 Rowinsky et al (1999) Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development J Clin Oncol 17:3631-3652 Sahai et al. (2002) RHO-GTPases and cancer Nat Rev Cancer 2: 133-142 Seidman et al. (2001) Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification J
- K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors J Biol Chem 272:14459-14464 Yeoh et al. (2002) Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling Cancer Cell
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