Patent Grant
8691506
This application is a section 371 U.S. National phase application of international application no. PCT/EP2006/003421, filed Apr. 13, 2006, which claims the benefit of priority to EP application no. 05008088, filed Apr. 13, 2005 and U.S. Provisional application No. 60/670,863, filed Apr. 13, 2005.
The present invention relates to a method for providing a gene expression profile being predictive for the specific response of an individual tumor to a pharmaceutically effective compound, the use thereof, a microarray wherein the nucleotide sequences attached to the substrate consist of nucleotide sequences corresponding to the predictive genes of said gene expression profile, and a diagnostic kit containing said microarray.
Gene expression profiles reflect the relative expression level of transcription units by determining the amount of mRNA expressed. Changes in the expression levels of genes up- or down-regulated in cancerous cells can be determined when comparing the gene expression profile of highly and moderately responsive tumor tissue with the gene expression profile of resistant tumor tissue.
The so-called microarray analysis is a useful method for a highly efficient analysis of gene expression profiles. A microarray is an ordered array of genes immobilized on a planar substrate that allows the specific binding of labeled nucleic acids. Microarray technologies which can be used for analyzing gene expression profiles involve depositing nucleic acids on a solid platform in a set pattern, and hybridizing a solution of complementary nucleic acids to the nucleic acid targets. Microarray technology has been applied increasingly in this field due to its capability to illustrate the cancerous changes in the cellular behavior on a genomic level.
One of the main problems when identifying genes which predict the treatment-response of an individual tumor is the lack of a sufficient large number of different tumors being characterized for both their chemosensitivity and mRNA expression. Cell lines and cell line derived xenografts do not have enough predictive capabilities for a clinical use for evaluating genes in question. In investigating tumors only a few pharmaceutically effective compounds can be evaluated in a single tumor model and today most tumor types are treated with combination chemotherapy.
In the prior art methods for obtaining gene expression profiles of tumor material are established for one individual tumor type and are not transferable to other tumor types. Further, most of the methods known in the prior art use a two sided t-test to choose the genes differentially expressed in only two classification groups for the responsiveness of a tumor to the treatment with a pharmaceutically effective compound, i.e. sensitive and resistant. The main drawback of this method is the fact, that this procedure has a high risk of getting a large number of “false positive” genes which are differentially expressed in the different groups only by chance.
Thus, the technical problem underlying the present invention is to provide a new method for predicting the responsiveness of an individual tumor to a pharmaceutically effective compound.
The solution to the above technical problem is achieved by the embodiments characterized in the claims.
In particular, the present invention relates to a method for providing a gene expression profile being predictive for the specific response of an individual tumor to a pharmaceutically effective compound, comprising the steps of
According to the present invention, the human tumor material being xenotransplanted into at least one suitable test animal, preferably a nude mouse, can be any human tumor material, e.g. derived from bladder, breast, colon, CNS, head and neck, liver, gallbladder, lung (small cell and non small cell), kidney, ovarian, pancreas, pleura, prostate, stomach, testicle, and uterus tumors, sarcomas, melanomas, and lymphomas. In a preferred embodiment of the present invention tumor material derived from different tumor types is transplanted into at least one nude mouse in step (a) of the above method, so that the predictive genes determined in step (f) of the above method are found in gene expression profiles of different tumor types.
In a preferred embodiment of the present invention each different tumor is xenotransplanted into one suitable test animal. For example, human tumor material of at least 15, more preferably at least 30, most preferably from 30 to 50 different tumors are each xenotransplanted into a different test animal, thereby resulting in at least 15, more preferably at least 30, most preferably from 30 to 50 different xenotransplanted test animals, preferably nude mice.
In a preferred embodiment of the present invention the human tumor material being xenotransplanted into the at least one test animal is derived from the tumor types shown in Table 2.
The term “xenografts” as used herein means patient-derived human tumor xenografts which have never been transformed into a cell line and have always been passaged directly into and through test animals, preferably nude mice. They have proven to have a high capability of predicting chemosensitivity to anti-cancer agents regarding the clinical situation of patients. For this reason they are a preferred choice for acquiring chemosensitivity data and gene expression profiles for the corresponding tumor samples.
The term “gene expression profile” as used herein refers to the relative expression of a plurality of mRNA transcripts or post-transcriptional level including protein amounts and post-translational modifications. A gene expression profile of a human tumor xenograft or a human tumor tissue reflects the amount of each mRNA transcript and/or post-transcriptional level in the starting sample. In a preferred embodiment of the present invention the gene expression profiles of the tumor xenografts is obtained using e.g. microarray techniques, PCR, Northern-analysis, etc.
The evaluation of the responsiveness of the tumor xenografts to the pharmaceutically effective compound has been carried out by determining the T/C (therapy/control) values of each tumor. The T/C value of each tumor can be determined by comparing the size of a tumor treated with a pharmaceutically effective compound (therapy) with the size of the untreated tumor (control). After all measurements at various time points are carried out the minimal value representing the best compound effect is taken for evaluation. If for example a tumor treated with a pharmaceutically effective compound has 20% of the size of an untreated tumor then the respective T/C value of the tumor treated with a pharmaceutically effective compound is 20% which equals to a tumor growth inhibition property of said pharmaceutically effective compound of 80%.
In preferred embodiments of the present invention a tumor xenograft is considered to be responsive when its T/C (therapy/control) value is e.g. less than 25%, a tumor xenograft is considered to be no-change when its T/C value is e.g. 25% or more and e.g. 42% or less, and a tumor xenograft is considered to be progressive when its T/C (therapy/control) value is e.g. more than 42%. In contrast to the prior art the present invention uses three groups which is much closer to the clinical situation than only using two.
In order to determine the predictive genes at least two of the groups selected from the group consisting of responsive, no-change, and progressive tumors, have to comprise more than one tumor as a minimum prerequisite for carrying out the Anova-test. Thus, the minimum prerequisites for determining the predictive genes are for example two responsive tumors, two no-change tumors, and one progressive tumor, or two responsive tumors, one no-change tumor, and two progressive tumors, or one responsive tumor, two no-change tumors, and two progressive tumors.
The health conditions of the animals used for xenotransplantation in the method according to the present invention is significantly impaired due to tumor growth. Accordingly, the life span of the test animals used for xenotransplantation in the method of the present invention is significantly shortened when compared to test animals not used in the method of the present invention. Therefore, the test animals either are sacrified early due to large tumor burden of the xenotransplanted tumor material or are killed after the completion of the method according to the present invention.
In a preferred embodiment of the present invention step (f) of the above method comprises a step of filtering out the relevant genes before the determination of the predictive genes.
In a further preferred embodiment of the above method step (f) comprises the steps of
In this embodiment the step of determining the predictive genes comprises a leave-one-out cross-validation. In k-fold cross-validation, the data is divided into k subsets of (approximately) equal size. The net is trained k times, each time leaving out one of the subsets from training, but using only the omitted subset to compute whatever error criterion is of interest. If k equals the sample size, this is called “leave-one-out” cross-validation.
Determining a list of predictive genes by taking the intersection of the n-gene-lists (each containing the 300 most specific genes), the risk of false positives is minimized due to the fact that “a gene by chance” would have to be a “false positive by chance” in any of the n-gene-lists, being much more improbable than using the procedures of the prior art. The “leave-out-method” of the present invention to get the predictive genes will also lead to a list of predictive genes, not biased by the tumor later left out in the cross-validation method since each gene which is only predictive because of a single tumor in the training-set will be discarded by using the intersection of the n-gene-lists.
In addition, combining an Anova test with the Fisher's Exact Test in the process of setting up the n-gene-lists is a promising way to get the 300 most specific genes for every time another tumor is left out.
It should be noted that cross-validation is quite different from the “split-sample” or “hold-out” method that is commonly used. In the split-sample method, only a single subset (the validation set) is used to estimate the generalization error, instead of k different subsets; i.e., there is no “crossing”.
The distinction between cross-validation and split-sample validation is extremely important because cross-validation is markedly superior for small data sets; this fact is demonstrated dramatically by Goutte (1997) in a reply to Zhu and Rohwer (1996). For an insightful discussion of the limitations of cross-validation choice among several learning methods, see Stone (1977).
In a more preferred embodiment of the present invention the expression profile of 300 genes is determined for each sub-set when determining the predictive genes in each sub-set.
In another preferred embodiment of the present invention the gene expression profile is validated by a leave-one-out cross-validation.
The pharmaceutically effective compound according to the present invention can be any pharmaceutically effective compound known in the art and/or commercially available for the treatment and/or prevention of cancerous diseases. This includes pharmaceutically effective compounds containing agents effective for preventing tumor growth and/or metastasis or for reducing tumor size. Examples of such agents are angiogenesis inhibitors, including inhibitors of endothelial cell proliferation, e.g. Angiostatin K1-3, Avastin, Endostatin, Fumagillin, and Minocycline EGFR inhibitors, e.g. Cetuximab, and inhibitors of the biosynthesis of TNF-α, e.g. Thalidomide. Further examples of such agents are DNA intercalators/cross-linkers, e.g. Bleomycin, Chlorambucil, Melphalan and Oxaliplatin, DNA synthesis inhibitors, e.g. Aminopterin, 5-Fluoro-5′-deoxyuridine, 5-Fluorouracil, Gemcitabin, Ganciclovir, and Hydroxyurea, Capecitabin, DNA-RNA transcription regulators, topoisomerase II inhibitors, e.g. Actinomycin D, Daunorubicin, Doxorubicin, and Idarubicin, enzyme inhibitors, e.g. Curcumin, Etoposide, and Trichostatin A, agents causing demethylation of the DNA, e.g. 5-Aza-2′-deoxycytidine and 5-Azacytidine, agents having an antiproliferative effect, e.g. Cholecalciferol, microtubule inhibitors, e.g. Colchicine, Docetaxel, Nocodazole, Paclitaxel, Vinblastine, Vincristine, Vindesine, and Vinorelbin, and apoptosis causing agents, e.g. Troglitazone and Thapsigargin. In a preferred embodiment of the present invention the pharmaceutically effective compound is Avastin, a VEGF inhibitor.
In a more preferred embodiment of the present invention, the pharmaceutically effective compound is Avastin and the human tumor material being xenotransplanted into the test animals is derived from colon, non small cell lung, breast and/or renal tumors.
In a particularly preferred embodiment of the present invention the pharmaceutically effective compound is Avastin and the predictive genes of the gene expression profile obtained by the above method consists of genes characterized by SEQ. ID. No. 1 to SEQ. ID. No. 118.
In another preferred embodiment of the present invention the present invention the pharmaceutically effective compound is Avastin and the predictive genes of the gene expression profile obtained by the above method consists of genes characterized by the sequences shown in Table 3.
It is a further object of the present invention to provide a use of a gene expression profile obtained by the above method in the manufacture of a medicament carrying out an individual, tumor-specific diagnosis or treatment of a cancerous disease.
Another object of the present invention is the provision of a method for predicting the responsiveness of an individual tumor to a treatment with a pharmaceutically effective compound comprising the steps of
Using the above method it is possible to predict the prospect of success of a treatment of a patient suffering from a cancerous disease. Therefore, the above method can be used to avoid unnecessary treatment with chemotherapeutics which are a great physical and psychological burden on a tumor patient. Thus, the above method is useful for optimizing the individual treatment of a cancerous disease and avoiding the costs and serious side-effects of an ineffective chemotherapy.
In a preferred embodiment of the present invention the gene expression profiles of the tumor to be treated is obtained using microarray techniques.
The present invention also relates to the use of Avastin for a gene expression profile-dependent treatment of a cancerous disease. Avastin may e.g. be used in the treatment of a patient having a tumor, wherein the dosage of Avastin has been lowered or increased depending on the gene expression profile of said tumor when compared to the dosage which would have been used in the treatment of said patient without obtaining the gene expression profile of said tumor. In a preferred embodiment of the present invention, the gene expression profile-dependent treatment of a cancerous disease is a combination therapy comprising the use of at least one other pharmaceutically effective compound. In a more preferred embodiment of the present invention the decision whether Avastin is to be used in said combination therapy is dependent on the gene expression profile of the tumor to be treated. In a most preferred embodiment of the present invention the decision whether Avastin is to be used in said combination therapy is dependent on a gene expression profile consisting of the genes listed above.
The present invention also relates to a microarray, wherein the nucleotide sequences attached to the substrate consist of nucleotide sequences corresponding to the predictive genes of a gene expression profile obtained by the method for providing a gene expression profile being predictive for the specific response of an individual tumor to a pharmaceutically effective compound of the present invention.
The term “microarray” as used herein means any arrangement of biomolecules, such as nucleic acids, antibodies, preferably oligonucleotides in addressable locations on a substrate resulting in a so-called “biochip”. The substrate may be any substrate suitable for use in a microarray known in the art, like e.g. membranes, glass, plastic, silicon wafers, and metal alloys. The information obtained when using microarray techniques can also be obtained by other methods known in the art for determining gene expression profiles, e.g. PCR, Northern-analysis, etc.
It is a further object of the present invention to provide a diagnostic kit containing the above microarray.
The figure shows:
The present invention will now be further illustrated in the following examples without being limited thereto.
The activity of Avastin has been tested against a set of 46 of patient derived tumor xenografts in nude mice derived from different tumor types preferably known to have the chance of being responsive to Avastin e.g. colon, non small cell lung, renal, or breast cancers. The gene expression profiles for the same tumors have been obtained by using the Affymetrix gene-expression Arrays HG-U133A/B and HG-U 133 Plus 2.0. These gene expression arrays determine the expression of about 34,000 genes.
For identifying genes, being predictive for the specific response of an individual tumor to a specific compound, the following steps are carried out, starting with a list of all gene expressions determined by the arrays:
After these “quality filtering” steps the main determination of predictive genes is carried out:
Each gene in this list has to fulfill the following requirements:
The procedure carried out for all 46 sub-training-sets, results in 46 lists of 300 genes respectively. By setting up a gene-list containing the intersection of all these 300 gene containing lists, the result is a set of genes which were specific in all sub-training-sets and is not dependent on a single tumor sample. This step minimizes the risk of “false positive” genes, which otherwise could have been wrongly identified as being specific/predictive.
The final outcome is a list of 118 gene expression in 46 tumors (Table 1, horizontal: different tumors tested, vertical: Affymetrix genes) which is predictive for the response of these tumors to Avastin. The tumor types used determining said gene profile are shown in Table 2 and the sequences of the predictive genes are shown in Table 3. The gene signature is validated by a “leave-one-out cross-validation” using e.g. either a “k-nearest neighbours” or a “support vector machine” classification algorithm.
After validation, the gene expression profile can be used for further prospective validation or to predict tumor samples with unknown chemosensitivity. Using e.g. a “support vector machine” of order 1 for a “leave-one-out cross-validation” showed that the detected genes have a very good ability to discriminate the tumors into the three classification groups and predict tumor response to Avastin (
Methods
It should be noticed that k-nearest neighbours and support vector machines are available in the prior art.
A. Welch Anova (Parametric)
Let i index over the G groups formed by distinct levels of the comparison parameter.
Let Xik be the expression values, with k running over the replicates for each situation, interpreted according to the current interpretation (ratio, log of ratio, fold change).
Let:
Ni=the number of non-missing data values for each group,
be the group means, and
be the within-group sum of squares.
In all calculations, missing values (No Data) or (NaN) are left out of the sums, not propagated. If any of the Ni are zero, drop that parameter level from the analysis, and readjust G accordingly. If G is not at least 2, exit (p-value=1).
First, it has to be checked that each group has Ni greater than or equal to 2 and SSi greater than 0. If not, remove it from consideration and recompute G. If G is not at least 2, exit (p-value=1). (This reflects the more stringent requirements of not assuming the variances equal—if the variance estimate is pooled, replicates are only needed for at least one group, if variances are separately estimated then replicates are needed for each group.) Then compute:
the group weights
the sum of weights
the weighted mean
the denominator degrees of freedom
The (approximate) p-value is calculated by looking up W in the upper tail probability of an F distribution with d1 and d2 degrees of freedom. Note that d2 will not, in general, be an integer.
B. Fisher's Exact Test:
Fisher's Exact Test looks for an association between expression level and class membership. Each gene is tested for its ability to discriminate between the classes. Genes with the lowest p-values are kept for the subsequent calculations.
In this method, all the measurements for a given gene are ordered according to their normalized expression levels. For each class (parameter value), the predictor places a mark in the list where the relative abundance of the class on one side of the mark is the highest in comparison to the other side of the mark. The genes that are most accurately segregated by these markers are considered to be the most predictive. A list of the most predictive genes is made for each class and an equal number of genes (lowest p-value using Fisher's Exact Test) are taken from each list.
Homo sapiens
Homo sapiens cDNA,
Homo sapiens cDNA, 3'
| Number | Date | Country | Kind |
|---|---|---|---|
| 05008088 | Apr 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/003421 | 4/13/2006 | WO | 00 | 12/11/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2006/108659 | 10/19/2006 | WO | A |
| Number | Date | Country |
|---|---|---|
| 03057916 | Jul 2003 | WO |
| 2004111603 | Dec 2004 | WO |
| Entry |
|---|
| Zembutsu et al. IDS—Citation No. 2, Oct. 11, 2007. |
| Zembutsu, H., et al., “Gene-expression profiles of human tumor xenografts in nude mice treated orally with the EGFR tyrosine kinase inhibitor ZD1839,” International Journal of Oncology, 2003, 23(1):29-39. |
| Zembutsu, H., et al., “Genome-wide cDNA Microarray Screening to Correlate Gene Expression Profiles with Sensitivity of 85 Human Cancer Xenografts to Anticancer Drugs,” Cancer Research, Jan. 15, 2002, 62:518-527. |
| Bergsland, E., et al., “Maximizing the Potential of Bevacizumab in Cancer Treatment,” The Oncologist, 2004, 9(suppl 1):36-42. |
| Ghadimi, B.M., et al., “Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy,” Journal of Clinical Oncology, Mar. 20, 2005, 23(9):1826-1838. |
| Number | Date | Country | |
|---|---|---|---|
| 20110243925 A1 | Oct 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60670863 | Apr 2005 | US |
