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© 1999 American Society for Clinical Oncology Paclitaxel Resistance in Non–Small-Cell Lung Cancer Associated With Beta-Tubulin Gene MutationsFrom the Department of Pathology and the Laboratory of Molecular Biology of Cancer, Medical Oncology Service, Hospital Germans Trias i Pujol, Badalona, Barcelona; Departamento de Estadística, Universidad Autónoma de Madrid, Madrid; Medical Oncology Service, Hospital Clínico Madrid, Madrid; Medical Oncology Service, Hospital Arnau de Vilanova, Valencia, Spain; and the Departments of Pathology and Thoracic/Head and Neck Medical Oncology, M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Rafael Rosell, MD, Medical Oncology Service, Hospital Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain; email rrosell{at}ns.hugtip.scs.es
PURPOSE: The mechanisms that cause chemoresistance in non–small-cell lung cancer (NSCLC) patients have yet to be clearly elucidated. Paclitaxel is a tubulin-disrupting agent that binds preferentially to beta-tubulin. Tubulins are guanosine triphosphate (GTP)–binding proteins. Beta-tubulin is a GTPase, whereas alpha-tubulin has no enzyme activity. We reasoned that polymerase chain reaction (PCR) and DNA sequencing of the beta-tubulin gene could reveal more information regarding the connection between beta-tubulin mutations and primary paclitaxel resistance. PATIENTS AND METHODS: Constitutional genomic DNA and paired tumor DNA were isolated from 49 biopsies from 43 Spanish and six American stage IIIB and IV NSCLC patients who had been treated with a 3-hour, 210 mg/m2 paclitaxel infusion and a 24-hour, 200 mg/m2 infusion, respectively. Oligonucleotides specific to beta-tubulin were designed for PCR amplification and sequencing of GTP- and paclitaxel-binding beta-tubulin domains. RESULTS: Of 49 patients with NSCLC, 16 (33%; 95% confidence interval [CI], 20.7% to 45.3%) had beta-tubulin mutations in exons 1 (one patient) or 4 (15 patients). None of the patients with beta-tubulin mutations had an objective response, whereas 13 of 33 (39.4%; 95% CI, 22.8% to 56%; P = 0.01) patients without beta-tubulin mutations had complete or partial responses. Median survival was 3 months for the 16 patients with beta-tubulin mutations and 10 months for the 33 patients without beta-tubulin mutations (P = .0001). CONCLUSION: We have identified beta-tubulin gene mutations as a strong predictor of response to the antitubulin drug paclitaxel; these mutations may represent a novel mechanism of resistance and should be examined prospectively in future trials of taxane-based therapy in NSCLC.
LUNG CANCER IS the most lethal cancer in the Western world; more than one half of non–small-cell lung cancer (NSCLC) patients have advanced disease at diagnosis and are beyond the limits of surgery. However, a number of randomized clinical trials and recent meta-analyses1 have demonstrated that survival in patients with advanced stage III or IV NSCLC can be prolonged with chemotherapy.2 Several new chemotherapeutic agents have recently shown encouraging activity in NSCLC, including paclitaxel (Taxol, Bristol Myers Squibb Company, Princeton, NJ). Preliminary studies reported response rates of 21% and 24%, and a particularly impressive 1-year survival rate of 45% in one trial.3,4 Since then, paclitaxel has been used in combination with other cytotoxic drugs, chiefly either cisplatin or carboplatin. Paclitaxel is a microtubule-disrupting agent that targets primarily tubulin; in the absence of guanosine triphosphate (GTP), it can induce polymerization and can stabilize tubulin to cold- or calcium-induced microtubule depolymerization,5,6 thus blocking cell cycle in the M phase.7-11 Tubulin is a heterodimer that consists of the alpha- and beta-tubulin subunits that form the microtubule.12 A network of microtrabecular filaments forms the cytoplasmic matrix, giving rise to the concept of the cytoskeleton, which comprises microtubules, actin, and intermediate filaments. Microtubules display a remarkable versatility of functions and are involved in multiple biologic phenomena, including mitosis, cell shape determination, cell locomotion, and movement of intracellular organelles.13 A well-established property of tubulin is that each subunit can bind GTP.14-17 Although a taxoid photoaffinity probe has revealed binding not only with the beta-tubulin subunit but also, to a lesser degree, with the alpha-tubulin subunit, GTP–alpha-tubulin binding seems to have primarily a structural role. In contrast, GTP–beta-tubulin binding stimulates microtubule assembly (polymerization), whereas hydrolysis to guanosine diphosphate induces depolymerization.16,17 The beta-tubulin gene is localized to chromosome region 6p21.3. The protein has a sequence of 445 amino acids, encoded by four exons of the gene.17 Ultraviolet photoaffinity labeling and proteolysis methods have identified several exchangeable GTP-binding sites of beta-tubulin. Among these sites, phosphate binds with amino acids 140 to 146, guanine binds with either amino acids 60 to 77 or 241 to 245, and ribose binds with amino acids 178 to 181.14,15 In addition, Shivanna et al16 found another potentially crucial GTP-binding site between amino acids 3 to 19. Moreover, a direct photo–cross-linking of radiolabeled paclitaxel to beta-tubulin has been observed in the N-terminal 31 amino acids (Fig 1).18
Drug resistance is often a multifactorial process, and the hydrophobic nature of paclitaxel can induce overexpression of the MDR1 gene.6 However, paclitaxel-resistant human lung cancer cells selected in the presence of low levels of paclitaxel did not express MDR1.19 Cells expressing high levels of the multidrug resistance (MDR)–associated protein MRP displayed no or low resistance to paclitaxel.20 Alternative mechanisms of drug resistance could be related to point mutations that affect microtubule dynamics. Indeed, the discovery of tubulin deletions in Chinese hamster ovary (CHO) cells21 selected for resistance to and/or dependence on paclitaxel led us to search for point mutations in the beta-tubulin gene in paclitaxel-treated NSCLC patients. Moreover, resistant cells were found to be paclitaxel-dependent for growth in paclitaxel selected CHO cell lines and in a human small-cell lung cancer cell line.22-24 Recently, beta-tubulin point mutations have also been identified in paclitaxel-resistant human ovarian cancer cells in the class I isotype M40 gene, which produces the majority of beta-tubulin mRNA.25 Beta-tubulin point mutations could thus represent a process indicative of intrinsic drug resistance. In the present study, we addressed two important questions: Are beta-tubulin gene mutations present in the genomic DNA of human NSCLC tumors? Do beta-tubulin mutations correlate with survival in paclitaxel-treated NSCLC patients?
Patients and DNA Extraction 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 American patients from the M.D. Anderson Cancer Center in Houston, TX). Of the six American patients, three had received no prior treatment, and the remaining three had been treated unsuccessfully with cisplatin.26 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 KCl, 0.5% Tween-20, and 1 mg/mL proteinase K. The mixture was then phenol-chloroform extracted, ethanol precipitated, vacuum dried, and resuspended in H2O. For this polymerase chain reaction (PCR) assay, different sets of oligonucleotides were designed to amplify specific regions of the beta-tubulin gene that code for the GTP- and paclitaxel-binding sites (Fig 1 and Table 1). The samples were denatured at 95°C for 5 minutes and then subjected to 35 cycles of denaturing for 1 minute at 95°C, annealing for 1 minute at different temperatures (as shown in Table 1), 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.
DNA Sequencing
Immunofluorescence
Statistical Analysis
The aim of the present study was to examine whether gene mutations in the GTP- and paclitaxel-binding regions of beta-tubulin influenced paclitaxel monotherapy response in patients with stage IIIB and IV NSCLC (46 who were previously untreated and three who were unsuccessfully treated with cisplatin) by using PCR and automatic DNA sequencing. The protocol was approved by the ethical committees of the three participating centers in Spain and by the M.D. Anderson Cancer Center's Surveillance Committee in the United States, and all patients gave their written informed consent. Forty-eight patients underwent biopsy before starting chemotherapy to obtain tissue for histologic diagnosis, and part of the tumor specimen was processed for genetic analysis; one patient had a biopsy performed after receiving paclitaxel. The 43 Spanish patients were treated with paclitaxel 210 mg/m2 in a 3-hour infusion every 3 weeks, and the six American 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). We sequenced the genomic DNA of exons 1 through 4 of the beta-tubulin gene in all 49 patients. Our findings provide evidence for mutations; however, to distinguish somatic mutations from rare germline variants, we determined which of the variations were present in normal lung epithelium for the same patient. This analysis showed that patients' tumors contained true somatic mutations when matched with normal control DNA obtained in one of three ways: (1) from nonepithelial normal tissue in the previously mentioned archival, paraffin-embedded biopsy specimens; (2) if necessary, by isolation of DNA from distant (normal) nonepithelial, paraffin-embedded tissue, from archival paraffin blocks others than the biopsy specimen; or (3) in six patients, by venipuncture and isolation of lymphocyte DNA. Of the 19 somatic mutations identified in 16 patients, 13 were missense mutations, one was a single base-pair (bp) insertion, three were 1-bp deletions, and two were nonsense mutations (Table 2). This analysis showed that 16 patients (33%; 95% CI, 20.7% to 45.3%) had beta-tubulin mutations, as shown in Tables 2 and 3 and Figs 1 and 2. The characteristics of all patients are listed in Table 3. There were no differences for all the baseline characteristics, including performance status and stage according to the presence or absence of beta-tubulin mutations. 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 = .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 = .0001) (Fig 3). 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 (data not shown).
All patients with beta-tubulin mutations were chemotherapy-naive, whereas in the group of patients without tubulin mutations, three American patients had been unsuccessfully treated with cisplatin. Although one patient (case A1 in Table 2) with beta-tubulin mutations survived for 23 months, that individual did not attain even a partial response. No second-line chemotherapy was foreseen for nonresponders, although six of the 14 patients with stage IIIB received radiotherapy after the completion of the study.
Our results provide the first demonstration of tubulin point mutations being linked to paclitaxel resistance in human tumors in the clinical setting. The 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. These results are in stark contrast to the lack of response and poor survival of patients with beta-tubulin mutations. Paclitaxel has a binding site on the N-terminal 31 amino acids of beta-tubulin,6,8,18 encoded by exons 1 to 2, and several GTP-binding sites are located primarily on amino acids encoded by exon 4.15-17 Whereas GTP promotes normal microtubule assembly, paclitaxel-induced assembly provokes an irreversible polymerization, increasing micro-tubule diameter and inhibiting tubulin function.6,17,26 In the present study, we screened all parts of the beta-tubulin gene encoding the GTP- and paclitaxel-binding sites. Beta-tubulin mutations were found in 16 of the 49 paclitaxel-treated NSCLC patients (Table 2). Fifteen of the 16 patients who harbored mutations in their tumors had mutations in exon 4. One of these mutations was located at amino acid 260, mirroring alterations at amino acids 250 to 300 found in a paclitaxel-dependent mutant beta-tubulin CHO cell line.22,23 Cabral et al22 showed that paclitaxel-dependent mutants have normal arrays of cytoplasmic microtubules, but in the absence of paclitaxel, these mutants are unable to complete assembly of the mitotic spindle apparatus. When deprived of the drug, these cells cannot progress through the cell cycle; they become large and multinucleated, losing cell viability. However, in the presence of paclitaxel, these cells can complete mitosis. Some of these resistant sublines had an internal deletion of about 270 bp (approximately 90 amino acids) encompassing amino acids 255 to 345. In a more recent study, Giannakakou et al25 identified acquired mutations at amino acid 270 in paclitaxel-resistant human ovarian cancer cells; this finding is relevant to the mutation we observed at amino acid 260 in patient V6. Patients V1 and A1 had mutations at amino acid 180 and patients B2 and B21 at 183; interestingly, photoaffinity labeling studies show that amino acids 178 to 181 are involved in the ribose-binding site of beta-tubulin.14 Patients M5, A7, and A8 had mutations at amino acid 147, a region close to amino acids involved in the phosphate-binding site of beta-tubulin.14,15 A cluster of mutations was found in amino acids involved in the guanine-binding site (Table 2). All of these mutations were in exon 4 GTP-binding regions and amino acids that are phylogenetically highly preserved13 and lead us to hypothesize that alterations in exon 4 are crucial to the alteration of microtubule function.
It is interesting to note that patients V4 and B16 had mutations in the N-terminal portion of beta-tubulin, which is where Shivanna et al16 described a striking photoreaction occurring between the exchangeable site guanine moiety and cysteine 12 of beta-tubulin. Moreover, Rao et al18 observed a close correlation between the beta-tubulin reactive residues of exons 1 and 2 and a paclitaxel analog. In our study, patient V4 had a mutation at amino acid 4 (Ile Beta-tubulin mutations do not explain all resistance to antimicrotubule agents, and the identification of alternate molecular pathways of resistance is ongoing. A growing body of knowledge indicates that several other mechanisms need to be further explored. First, the possible role of alpha-tubulin in the tubulin-paclitaxel interaction and in chemoresistance must be investigated.27 In the study of Giannakakou et al,25 the total tubulin content of the resistant cells was similar to that of the parental cells, and no differences in the acetylation of purified alpha-tubulin were observed. Second, differential expression of tubulin isotypes in cell lines that are resistant to microtubule-disrupting drugs may also be a mechanism of paclitaxel resistance,19,28 although Giannakakou et al25 found beta-tubulin point mutations in the class I isotype M40, which comprises the majority of beta-tubulin mRNA. Further research is warranted using reverse transcriptase PCR analysis of beta-tubulin and cDNA sequencing. Indeed, if an acquired mutation confers a growth advantage, prolonged selection might lead to its overexpression. Giannakakou et al25 postulate that the manifested altered isotype expression may be the effect of a selective advantage conferred by the mutation and not by the native isotype. We were unable to rule out the possibility that increased expression of specific beta-tubulin isotypes in paclitaxel-resistant tumors occurs and that the role of different tubulin isotypes is related to the binding of distinct microtubule-associate proteins. Third, posttranslational modifications of beta-tubulin have been described in the carboxy-terminal domain,19 and the intricate process of protein folding in the C terminus often requires the assistance of preexisting proteins collectively known as molecular chaperones.29 The influence of these molecules on the tubulin folding process has not been fully established and needs to be investigated. Finally, other alterations may well play a pivotal role in paclitaxel chemoresistance. For instance, overexpression of Bcl-2 or Bcl-xL inhibits paclitaxel-induced apoptosis without affecting its antimicrotubule or cell cycle effects.30 In conclusion, decreased sensitivity to antitubulin drugs is associated with beta-tubulin mutations, although the degree of this sensitivity may also depend on cell type and/or the tissue-specific genetic background of the tumor cells. Such is the case with GML expression, which has been shown to increase sensitivity to paclitaxel.31 Beta-tubulin mutations clearly do occur, albeit primarily in more aggressive tumors; in a large number of tumors, however, they do not occur. There are two possible explanations for this: either there are as yet unidentified mechanisms that have a more fundamental role in resistance, or the true incidence of mutations has been underestimated. Other investigators have ruled out intronless tubulin pseudogenes (RNA with or without digestion by DNase was tested by reverse transcriptase PCR). These experiments did not generate PCR products with these primers, ruling out the presence of pseudogenes in the MES-SA cell line.32 Finally, paclitaxel is secreted into bile by still unknown transporters. It has been documented that class I P-glycoproteins (Pgp) prevent the entry of paclitaxel, digoxin, and doxorubicin across the blood-brain barrier and promote the excretion of these drugs through the gut because accumulation in these tissues was increased in knockout mice.33 Along these lines, a recently cloned member of the Pgp family of genes (Spgp, sister gene of Pgp) mediates low-level resistance to paclitaxel but not to other drugs of the MDR phenotype.34 Further exploratory analysis of mutations on certain residues in class I Pgp might explain their drug resistance profile. For instance, mutation of Gly 185 to Ala in MDR1 has been found to increase colchicine and decrease vinblastine resistance.35 Similarly, in the mouse, mutation of Ser 941 to Phe reduces the ability of MDR1 to confer adriamycin and colchicine resistance while maintaining vinblastine resistance.36 Missense mutations, as evidenced by altered migration of alpha- or beta-tubulin on two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis, were described by Cabral et al.21 Our study provides the first evidence of beta-tubulin missense mutations in chemotherapy-naive NSCLC patients and their dramatic impact on paclitaxel response. We speculate that the presence of missense mutations in beta-tubulin provides cells with a survival advantage when they are exposed to a stabilizing drug such as paclitaxel. The frequency and occasional multiplicity of beta-tubulin mutations in some tumors supports the hypothesis that such mutations confer generalized primary resistance to antitubulin drugs in NSCLC patients. Further research may well validate beta-tubulin as a prognostic marker of response and survival in NSCLC.
Supported in part by Fondo de Investigaciones Sanitarias de la Seguridad Social grant no. 97/1064 and Bristol-Myers Squibb, Madrid, Spain. We thank Paul Gumerlock and David Gandara of University of California at Davis for their helpful discussions during the preparation of the manuscript. William K. Murphy, MD, generously facilitated access to clinical material regarding the American patients included in the study. Maura O'Sullivan and Renée O'Brate provided editing assistance, and Dolores Fuster provided technical assistance.
This work was performed at Laboratory of Molecular Biology of Cancer, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain. R.R. is a Foreign Research Fellow of the Foundation for Promotion of Cancer Research, Tokyo, Japan.
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Gros P, Dhir R, Croop J, et al: A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps. Proc Natl Acad Sci U S A 88:7289-7293, 1991 Submitted November 17, 1998; accepted February 26, 1999. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 analysis with Yates' correction or Fisher's exact test was used to compare discrete variables. Survival curves were estimated according to the Kaplan-Meier method from the date of the first paclitaxel treatment to the time of progression or death. The differences in survival curves were compared with the log-rank test. The Brookmeyer-Crowley method was used to calculate the 95% confidence intervals (CIs) for the median period of survival; statistical significance was defined as two-tailed P < .05. All computations were carried out with the SPSS for Windows software package (SPSS, Inc, Chicago, IL). 
Thr). In patient B16, sequencing analysis revealed a polymorphism at amino acid 11, both in the tumor and in the patient's normal tissue 
