WO2001090413A2

WO2001090413A2 - Method and markers for prognosticating efficacy of anticancer agents

Info

Publication number: WO2001090413A2
Application number: PCT/US2001/016004
Authority: WO
Inventors: Rafael Rosell; Mariano Monzo
Original assignee: Bristol-Myers Squibb Company
Priority date: 2000-05-19
Filing date: 2001-05-17
Publication date: 2001-11-29
Also published as: WO2001090413A3; AU2001261725A1

Abstract

Methods and markers for prognosticating the efficacy of anti-cancer agents in patients suffering from cancer by detection of mutant tubulin genes are provided.

Description

METHOD AND MARKERS FOR PROGNOSTICATING EFFICACY OF

ANTICANCER AGENTS

This application is a continuation-in-part of U.S. Patent Application Serial No. 09/323,971, filed June 2, 1998.

Background of the Invention

Microtubules are a major filament of the cytoskeleton and are involved in various biologic phenomena including mitosis, cell shape determination, cell locomotion and movement of intracellular organelles. Tubulin is one of the major microtubular components. Polymerization and depolymerization of tubulin regulate microtubular dynamics. Microtubules are considered one of the most important molecular targets for cancer chemotherapy.

Antimitotic agents which disrupt microtubules, also referred to herein as anti-tubulin agents, can be classified into two categories based on mechanism of action. These are the vinca alkaloids such as estramustine, rhizoxin and E7010, which inhibit microtubule polymerization, and taxanes such as paclitaxel and docetaxel which promote polymerization of microtubules and enhance microtubule stability.

The anti-mitotic anticancer agent paclitaxel is active against solid tumors. Paclitaxel is a microtubule-disrupting agent that primarily targets tubulin. In the absence of guanosine triphosphate (GTP) , paclitaxel induces polymerization and stabilizes tubulin to cold- or calcium- induced microtubule depolymerization, thereby blocking cell cycle in the M phase. Tubulin is a heterodimer that consists of the alpha- and beta-tubulin subunits that form the microtubule . Clinical trials with taxanes such as paclitaxel and docetaxel have revealed these agents to be effective against several cancers which were advanced or resistant to other anticancer drugs, especially breast cancer, ovarian cancers and non small cell lung carcinoma (NSCLC) . With respect to NSCLC, a number of randomized clinical trials have demonstrated that survival in patients with advanced stage III or IV NSCLC can be prolonged with paclitaxel . Preliminary studies with paclitaxel showed response rates of 21% and 24% and an impressive 1-year survival rate of 45% in one trial (Chang et al . J. Natl Cancer Inst. 85:388-394, 1993; and Murphy et al. J. Natl Cancer Inst. 85:384-388, 1993). Paclitaxel is now often used in combination with other cytotoxic drugs including cisplatin and carboplatin in patients with NSCLC.

However, the acquisition of drug-resistant tumor cells is still a major problem in the medical treatment of malignant disease. The hydrophobic nature of drugs such as paclitaxel is known to induce overexpression of the MDR1 gene (Horwitz et al. J. Natl Cancer Inst. 15:55-63 1993). However, paclitaxel resistant human lung cancer cells selected in the presence of low levels of paclitaxel do not express MDR1 (Kavallaris et al . J. Clin, Invest. 100:1282-1293, 1997). Further, cells expressing high levels of the multidrug resistance (MDR) -associated protein MRP display no or low resistance to paclitaxel (Zames et al . Proc. Natl Acad. Sci. USA 91:8822-8826 1994) . It has now been found that mechanisms of drug resistance to anti-cancer agents, and in particular anti-tubulin agents, in cancer are related to mutations in the beta-tubulin gene which affect microtubule dynamics. Summary of the Invention An object of the present invention is to provide prognostic markers for efficacy of anti-cancer agents in patients suffering from cancer, wherein the prognostic markers comprise mutant tubulin genes.

Another object of the present invention is to provide a method of prognosticating the efficacy of anti-cancer agents in a patient suffering from cancer which comprises obtaining a biological sample from the patient and analyzing the sample for mutant tubulin genes. In this method, the presence of a mutation in the GTP-binding site (codons 127-270) of exon 4 of a tubulin gene is indicative of resistance to anti-cancer agents and poor survival rates in patients with cancer. In this method, the presence of a mutation in the carboxy- terminal region (codons 271-445) of exon 4 of a tubulin gene is indicative of increased survival in patients with cancer and sensitivity of the cancer to anticancer agents. Detailed Description of the Invention

Point mutations in the tubulin gene have now been linked with survival in cancer patients as well as anti-tubulin agent sensitivity of the cancer cells in the clinical setting. For example, a group of patients without beta-tubulin gene mutations had a 39.4% response rate with a 10 month median survival time, and 1-year, 3-year and 5-year survival rates of 33.3%, 12.1% and 3%, respectively. In contrast, patients with beta-tubulin mutations showed no response to paclitaxel treatment and poor survival.

Further, genetic analysis was performed on forty-nine patients with non small cell lung carcinomas. Forty-eight patients underwent biopsy before starting chemotherapy to obtain tissue for histologic diagnosis. A portion of the tumor specimen was also processed for genetic analysis. One patient had a biopsy performed after receiving paclitaxel. Forty-three patients were treated with paclitaxel 210 mg/m² in a 3-hour infusion every 9 weeks. Six patients were treated with paclitaxel 200 mg/m² in a 24-hour infusion every 3 weeks. After three courses of paclitaxel, a decision was made regarding continuing treatment. All tumor responses were submitted to a peer-review process by two independent radiologists. Responders received up to a maximum of 10 courses. Responses were graded as complete if all evidence of disease disappeared on follow-up computed tomography scans. A partial response was defined as more than a 50% reduction in the sum of products at the largest perpendicular diameter of all indicator lesions. Survival was calculated from the date of first treatment to the most recent follow-up contact or to the date of death and included all patients in the study. Median follow-up from the time of treatment for the entire series of patients was 7 months (range 1 to 67 months) . The genomic DNA of exons 1 through 4 of the beta-tubulin gene was sequenced in all 49 patients. Findings from this analysis provided evidence for mutations. To distinguish somatin mutations from rare germline variants, variations in normal control DNA for each patient were also determined. Normal control DNA was obtained either: from nonepithelial normal tissue in archival paraffin-embedded biopsy samples; by isolation of DNA from distant normal nonepithelial, archival paraffin blocks other than the biopsy samples; or in six patients, by venipuncture and isolation of lymphocyte DNA. This analysis showed that patients' tumors contained true somatic mutations when matched with normal control DNA. Somatic mutations are shown in Table 1. Of the 19 somatic mutations identified in 16 patients, 13 were missense mutations, one was a single base-pair insertion, three were 1 base pair deletions and 2 were nonsense mutations. This analysis showed that 16 patients (33%; 95% CI, 20.7% to 45.3%) had beta-tubulin mutations.

There were no differences in baseline characteristics, including performance status and stage, in patients with and without beta-tubulin mutations. However, none of the patients with beta-tubulin mutations attained an objective response; one had stable disease and 15 had progressive disease. In contrast, of the remaining 33 patients without beta-tubulin mutations (including one patient who had a beta-tubulin polymorphism), 13 had partial or complete response (39.4%; 95% CI, 22.8% to 56%; p=0.01). Median survival for the 16 patients with beta-tubulin mutations was 3 months (95% CI, 2 to 3.9 months), whereas for the 33 patients with no beta- tubulin mutations, median survival was 10 months (95% CI, 7.9 to 12.1 months, p=0.0001). A set of monoclonal antibodies to detect and discriminate tubulin isotypes I, II and III showed no differences in histopathologic data, clinical data, or survival .

All patients with beta-tubulin mutations were chemotherapy naive, whereas in the group of patients with tubulin mutations, three North American patients had been unsuccessfully treated with cisplatin. Although one patient

(Case Al in Table 1) with beta-tubulin mutations survived for

23 months, the individual did not attain even a partial response. No second-line chemotherapy was foreseen for nonresponders, although 6 of the 14 patients with stage IIIB received radiotherapy after completion of the study.

Table 1: Beta-Tubulin Mutations Found in Non Small Cell Lung Carcinoma Patients

DOD=died of disease AWD=alive with disease

I I

In additional studies, one hundred and thirty one patients with non small cell lung cancer were examined for tubulin gene mutations. Fifty patients in this study had resected non small cell lung cancer and 81 patients had advanced metastatic disease. DNA was extracted and purified from 200 μl aliquots of serum. DNA was also extracted from 110 healthy individuals to be used as controls. The β-tubulin mutations were detected by direct sequencing of the amplified polymerase chain reaction (PCR) products, and the tumor mutations were confirmed by direct sequencing in both directions. Quantitative real-time PCR analysis was performed to assess the average of copy number of the β-tubulin gene in patients and control. β-tubulin mutations were detected in serum DNA from 55/131 (36%) of the patients. No mutations were detected in control subjects. Almost all the β-tubulin mutations were exon 4, clustered in two domains: the GTP- binding site (codons 127-270) and the carboxy-terminal region

(codons 271-445) . Patients with advanced non small cell lung cancer harboring mutations at codons 127-270 showed worse survival, while patients with mutations at codons 271-445 showed significantly better survival (P=0.03). Table 2 shows the base-line characteristics of patients from this study evaluated for β-tubulin mutations in serum DNA. Table 3 shows the base-line characteristics of patients from this study broken down by presence or absence of β-tubulin mutations. Table 4 shows the various β-tubulin mutations identified in these patients.

Table 2: Base-line Characteristics of 131 Non Small Cell Lung Cancer Patients Evaluated for β-tubulin Mutations in serum DNA

Table 3: Base-line Characteristics of 131 Non Small Cell Lung Cancer Patients, Broken Down by Presence or Absence of β-tubulin Mutations

Table 4: β-tubulin Mutations in Non Small Cell Lung Cancer Patients

I

I

a Type of mutation: miss, missense mutation; null, null mutation (nonsense and frameshift) ^b Type of mutation: trans, transition; transv, transversion 0 BS : Paclitaxel Binding Site d BS: GTP Binding Site

I

As demonstrated by these studies, the presence or absence of mutant tubulin genes, and in particular mutant beta-tubulin genes, in a patient suffering from cancer can be used to prognosticate the efficacy of anti-cancer agents, and in particular anti-tubulin agents such as taxanes including, but not limited to, paclitaxel and docetaxel, in the treatment of the cancer in the patient, as well as survival of the patient. Thus, mutant beta-tubulin genes serve as prognostic markers for efficacy of anti-cancer agents in patients suffering from cancer. In particular, patients with mutations in the GTP-binding site of exon 4 of the β-tubulin gene (codons 127-270) exhibit increased resistance to anticancer agents and worse survival while patients with mutations in the carboxy-terminal region of exon 4 of the β-tubulin gene (codons 271-445) exhibit significantly better survival. Accordingly, efficacy of anti-cancer agents, and in particular anti-tubulin agents, can be prognosticated in a patient suffering from cancer by obtaining a biological sample from the patient and then analyzing the sample for the presence or absence of mutant tubulin genes. In a preferred embodiment, the sample is analyzed for the presence of mutant beta-tubulin genes in either the GTP-binding region (codons 127-270) of exon 4 or the carboxy-terminal region (codons 271-445) of exon 4. The presence of a mutant tubulin genes, and in particular a beta-tubulin gene with a mutation in the GTP-binding region of exon 4, in the biological sample of the patient is indicative of a resistance of the tumor to anti-cancer agents, particularly anti-tubulin agents such as taxanes. Accordingly, other treatment regimes should be selected for these patients. The presence of a mutant tubulin gene, and in particular a beta-tubulin gene with a mutation in the carboxy-terminal region of exon 4, in the biological sample of the patient is indicative of a sensitivity of the tumor to anti-cancer agents, particularly anti-tubulin agents such as taxanes. The absence of any mutation in the tubulin genes in the biological sample of the patient is also indicative of anti-cancer agents, and in particular anti-tubulin agents such as taxanes being effective against the tumor, thereby decreasing the progression of the tumor and increasing the survival time of the patient . Biological samples which can be screened for these mutations are samples containing DNA. Examples include, but are not limited to, tumor biopsy samples and blood or serum samples obtained from the patient. Mutations in tubulin genes can be detected in these samples in accordance with well known methods including, but not limited to, those described herein.

The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1: Patients and DNA Extraction in 49 Patient Study

Tumor specimens were analyzed from 49 patients with stage IIIB or IV NSCLC submitted for paclitaxel treatment (43 Spanish patients from three Spanish Centers and six North American patients from the M.D. Anderson Cancer Center in Houston, TX) . Of the six North American patients, three had received no prior treatment . The remaining three were treated unsuccessfully with cisplatin. Only one specimen was a tumor biopsy taken after the start of paclitaxel treatment, and normal tissue from the same patient was used as its control. Genomic DNAs were extracted from paraffin-embedded specimens, incubated in 10 mmol/L Tris, pH 8.1, 2.5 mmol/L MgCl₂, 50 mmol/L KC1₂, 0.5% TWEEN 20 (monolaurate polyoxyethylenesorbitan) and 1 mg/ml proteinase K. The mixture was then phenol-chloroform extracted, ethanol precipitated, vacuum dried and resuspended in H₂0.

For this polymerase chain reaction, different pairs of oligonucleotides were designed to amplify specific regions of the beta-tubulin gene (Genbank Accession No. J00314) that code for the GTP- and paclitaxel-binding sites. The samples were denatured at 95° for 5 minutes and then subjected to 35 cycles of denaturing for 1 minute at 95°C, annealing for 1 minute at various temperatures, and extension for 2 minutes at 72°C, followed by a final period of extension at 72°C for 5 minutes. The products were separated by electrophoresis in agarose gels and visualized with ethidium bromide staining under ultraviolet light.

Example 2: DNA Sequencing of 49 Patients

All DNA samples were also examined by automatic DNA cycle sequencing. Primers and primer dimers contained in the

PCR-amplified products were removed using S300 HOUR Sephacryl microcolamine (Pharmacia Biotec, Uppsala, Sweden) . Purified

PCR products were used as a template in a cyclic sequencing reaction. Following this reaction, 4 μl of stop solution containing formamide and dextran blue was added, and the mixture was denatured at 95°C for 3 minutes before loading into a prewarmed, denaturing, 6% polyacrylamide-3 mol/L urea gel on an ALF-express DNA sequencer. Samples were run at 40W for 2 to 3 hours and the sequencing data obtained were compared with the wild-type beta-tubulin sequences.

Independent PCR products derived from each genomic DNA sample were analyzed at least twice.

Example 3 : Immunofluorescence

Paraffin sections of tumor blocks were deparaffinized with xylene, rehydrated and washed for 10 minutes in tri- buffered saline (TBS) . The slides were blocked with 2% BSA in TBS (TBS-BSA) for 30 minutes and washed three times with 0.02% TWEEN-20 in TBS (TBS-TWEEN) . Primary monoclonal antibodies, anti I-II and III beta-tubulin isotypes (Sigma, St. Louis, MO) were diluted 1:400 in TBS-BSA and incubated overnight at 4°C in a humid chamber, followed by extensive washing with TBS-TWEEN. The secondary antibody, a goat antibody to mouse immunoglobulin coupled to fluorescein (Dako, Denmark, Copenhagen), was diluted 1:200 in PBS-BSA and incubated for 30 minutes in a dark and humid chamber. The slides were then washed and mounted with cytofluoromedium (Sigma) .

Example 4: Patients and samples in 131 Patient Study

Serum from 131 non-small-cell lung cancer (NSCLC) patients was used for DNA extraction. All patients gave their signed informed consent. The serum DNA from the patients was compared with that from 110 healthy blood donors as controls. Control subjects were interviewed for demographic factors, medical history. All interviewed subjects were asked to provide a blood sample for storage of serum. Various controls were used to identify polymorphisms and rule out possible incorrect mutations. We analyzed the DNA from lymphocytes (10 samples) from patients included in the study, normal lung tissue (10 samples) and serum from healthy volunteers (10 samples) .

Example 5: DNA isolation

From Serum Two hours after extracting 10 ml of blood in a vacutainer with gel and clot activator from p atients and controls, the tubes are centrifuges at 12.000 rpm for 10 minutes at 4°C. 200 μl of serum are used for the DNA extraction, which is performed according to standard procedures with a commercial kit (QIAamp DNA Blood Mini Kit (250) , Qiagen, Germany) . From Tissue

DNA was extracted from surgical tissue from 19 patients included in the study that had undergone surgery. The tissue was immediately frozen in liquid nitrogen after surgery and convserved at -80°C until used in the laboratory.' Approximately 10 μg of tissue were used for the DNA extraction which was performed according to a commercial kit (DNeasy Tissue, Qiagen, Germany) .

Example 6: DNA quantification

Spectrop otometer After DNA extraction, the DNA was quantified with a spectrophotometer (Scan2000, Pharmacia Biotech, Sweden) at 260/280/320 nm. The DNA was also run on a 2 percent agarose gel to check the purity, and then stored at 4°C until it was used for PCR amplification.

Real - Time PCR

All DNA was quantified using PCR-RT (ABI PRISM 7700, Perkin Elmer, USA) . Special primers and probe were designed for the β-actin gene: sense primer 5 ' -CCAGTGTGACATGGTGCATCT-3 ' (SEQ ID NO:l), antisense primer 5 ' -ACAGCCTGGATAGCAACGTACA-3 ' (SEQ ID NO:2) , and 5 ' -Fluoresecent Label, 6-FAM probe 5'- CAGATCATGTTTGAGACCTTCAACACCCC-3' (SEQ ID NO : 3 ) . The conditions for the PCR were 25μl TaqMan Mix (Perkin Elmer, USA), 0.014μl forward primer, 0.01 μl reverse primer, 0.6μl probe, 5ml DNA, for a final volume of 50μl. Real-time quantification was performed using ABI PRISM 7700 Sequence Detection System computer program.

RNA isolation lOμg of normal or tumoral tissue were used for RNA extraction; which was performed according to a commercial kit (Ultraspec Tm RNA, Bioteck, USA) .

PCR amplification

All four exons of β-tubulin were amplified in three separate PCRs . Exon 1 and exon 4 were amplified separately and exon 2 and 3 were amplified together. The primers were specifically designed for each PCR. To ensure that only the functional m40 clone was being amplified the primers wre designed in intronic regions (to rule out possible pseudogenes) and subsequently checked using the BLAST system (www.ncbi.nlm.nih.gov/blast) to further guarantee the specificity to the functional clone.

Primer name TUXIA is SEQ ID NO: 4; primer name TUXIB is SEQ ID NO: 5; primer name TUBEX2U is SEQ ID NO: 6; primer name TUBEX2L is SEQ ID NO: 7; primer name TUB4U is SEQ ID NO: 8; primer name TUB4L is SEQ ID NO : 9.

The PCR components of the Master Mix were 5μl lOx Buffer (Ecogen, Spain), 2μl 50mM MgCl₂ (Ecogen, Spain), 0.6μl lOmM dNTP mix (Boehringer Manheim, Germany), 0.7μl lower primer (lOμM) , and 0.5μl Taq Polymerase (Ecogen, Spain) for a final folume of 50 μl . The PCR amplifications were carried out in a thermal cycler (GeneAmp PCR SYstem 9700, Perkin Elmer, USA) . The conditions of the PCR were as follows: 10 minutes at 95°C, 35 cycles of 30 seconds at 94°C, 60 seconds at Tm, 90 seconds at 72°C, and a final extension of 7 minutes at 74°C. The PCR amplifications were checked on a 1 percent agarose gel.

RT-PCR cDNA was synthesized from total RNA from normal tissue or tumoral tissue with murine leukemia virus reverse transcriptase and random hexamers (GeneAmp RNA PCR Kit, Perkin Elmer, USA) according to manufacturer's instructions with the following modification, the RT times were as follows: 15 min at 50°C, 5 min at 90°C and 5 min at 5°C. Amplification for the purpose of sequencing was performed with human β-tubulin- specific primers for the forth exon: sense 5'- TGGCCAGATCTTTAGACCAGACA-3 ' (SEQ ID NO: 18) and antisense 5'- CGGTGGCATCCTGGTACTGC-3 ' (SEQ ID NO: 19). Amplification was performed through 35 cycles (30 s at 94°C, 1 min at 65°C, and 1 min 30 s at 72°C) with an initial 5 min at 95°C and a final extension step of 10 min at 72°C.

PCR purification The PCR and RT-PCR products were purified before being used for sequencing. A commercial kit (Usb, USA) was used which contained exonuclease 1 (lOU/μl in 20mM Tris-HCl, pH 7.5, ImM MgC12, 0. lmM ZnC12, 50 percent glycerol) , and shrimp alkaline phosphatase (2U/μl in 25mM Tris-HCl, pH 7.6, ImM MgCl₂, 0. ImM ZnCl₂, 50 percent glycerol) . Each tube contained lOμl PCR or RT-PCR product, 4μl exonuclease 1, and 4μl shrimp alkaline phosphatase. The mixture was incubated at 37°C for 45 minutes and the enzymes were inactivated by heat at 80°C for 15 minutes in a thermal cycler (GeneAmp PCR System 9700, Perkin Elmer, USA) .

Example 7: DNA sequencing of 131 Patients

All DNA sequencing was carried out with a fluorescently labeled primer on an ALFwin Express Sequencer (Pharmacia

Biotech, Sweden) . A total of 8 sequencing reactions were carried out so as to sequence the entire 445 amino acids of the β-tubulin gene.

Primer name SE1 is SEQ ID NO: 10; primer name TS2360 is

SEQ ID NO: 11; primer name TS2491 is SEQ ID NO: 12; primer name

TS2955 is SEQ ID NO: 13; primer name TS3190 is SEQ ID NO: 14; primer name TS3430 is SEQ ID NO: 15; primer name TS3661 is SEQ ID NO: 16; and primer name TS3856 is SEQ ID NO: 17.

The PCR conditions for each primer were: 5 minutes at 95°C, 25 cycles of 30 seconds at 94°C, 30 seconds at Tm, and 45 seconds at 72°C followed by a final extension step of 72°C for 10 minutes. The sequencing reaction was carried out with Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Sweden) . The reaction was carried out in 4 separate tubes containing 1 μl (5'Cy5 labeledprimer lpmol/ l) , 5μl template DNA (0.5μg) and 2 μl of A, C, G or T reagent. Each reagent contained the following components: Tris-HCL (pH9.5), MgC12 , Tween20, Nonidet p-40, 2-mercaptoethanol, dATP, dCTP, 7-deaza-dGTP, dTTP, thermostable pyrophosphatase and Thermo Sequenase DNA polymerase. Each individual reagent also contained the specific ddNTP. The PCR reaction was carried out in a thermal cycler (GeneAmp PCR System 9700, Perkin Elmer, USA) .

Formamide loading dye with EDTA and fuchsin was added to the tubes and after a 3 minute denaturation at 90°C they were loaded in the sequencer. The samples were run for 300 minutes or less (for exons 1,2 and 3) on a 8 percent acrylamide gel (Reprogel High Resolution, Pharmacia Biotech, Sweden) at 1500V, 60mA, 25W and 55°C. Example 8: DNA analysis

The serum DNA from the non-small-cell lung cancer patients was compared to the GenBank m40 β-tubulin sequence

(accession number:LJ00314) . The sequence was also compared to the controls used (lymphocytes, serum from healthy controls, normal lung tissue) .

Example 9: Statistical analysis

The primary outcome of this study was overall survival, measured from the date of first treatment of NSCLC. The study was carried out to determine the prognostic importance of β- tubulin mutations in serum DNA in addition to known prognostic factors .

The univariate associations between the presence or absence of β-tubulin mutations and baseline prognostic factors were analyzed with a Fisher exact test for categorical variables . Survival curves were prepared according to the Kaplan-Meier method. Univariate survival distributions were compared using the log-rank test. All patients ere followed from first treatment until death or until data were censored (and the patient considered to be alive) as of Marc 30, 2000. A multivariate survival analysis was carried out according to the Cox proportional-hazards model.

All factors were treated as single categorical variables, with the exception of age at diagnosis and serum DNA concentration, which were analyzed as continuous variables. All reported P-values are 2-sided. Statistical significance was defined as P<0.05. Continuous variables with non-normal distribution were compared by means of the Mann- Whitney U test. All calculations were performed using SPSS software version 9.0.

Claims

What is Claimed is:

1. A method of prognosticating efficacy of anti-cancer agents in a patient suffering from cancer comprising:

(a) obtaining a biological sample from the patient; and (b) analyzing the sample for mutant tubulin genes, wherein the presence of a mutation in a GTP-binding site of exon 4 of the tubulin gene is indicative of increased resistance of the cancer to anticancer agents and the presence of a mutation in the carboxy-terminal region of exon 4 of the tubulin gene is indicative of sensitivity of the cancer to anticancer agents .

2. The method of claim 1 wherein the mutant tubulin gene is a mutant beta-tubulin gene.

3. The method of claim 1 wherein the anticancer agent is an anti-tubulin agent.

4. The method of claim 1 wherein the patient is suffering from lung cancer.

5. A prognostic marker for efficacy of anti-cancer agents in patients suffering from cancer comprising a tubulin gene with a mutation in a GTP-binding site or a carboxy- terminal region of exon 4 of a tubulin gene.

6. The prognostic marker of claim 5 wherein the mutant tubulin gene is a mutant beta-tubulin gene.