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Published OnlineFirst February 15, 2010; DOI: 10.1158/1078-0432.CCR-09-1192

Cancer Therapy: Clinical Clinical Cancer Research Tipifarnib Plus Tamoxifen in Tamoxifen-Resistant Metastatic Breast Cancer: A Negative Phase II and Screening of Potential Therapeutic Markers by Proteomic Analysis

Florence Dalenc1,2, Sophie F. Doisneau-Sixou1,2,3, Ben C. Allal1,4, Sabrina Marsili1, Valérie Lauwers-Cances5, Karima Chaoui3,6, Odile Schiltz3,6, Bernard Monsarrat3,6, Thomas Filleron1, Nicole Renée7, Emilie Malissein1,2, Elise Meunier1,2,3, Gilles Favre1,2,3, and Henri Roché1,2

Abstract Purpose: Tipifarnib, a farnesyltransferase inhibitor, has antitumor activity in heavily pretreated metastatic breast cancer patients. Preclinical data suggest that FTIs could restore tamoxifen responsiveness in tamoxifen-resistant disease. Thus, combining FTIs and tamoxifen may be a promising clinical approach after relapse or progression on tamoxifen. Experimental Design: Postmenopausal patients with measurable estrogen receptor– and/or progesterone receptor–expressing metastatic breast cancers were enrolled. Only patients with disease progression on tamoxifen were eligible, but there was no limitation regarding prior chemotherapy or hormone therapy regimens. Patients were immediately treated with 300 mg (n = 12) or 200 mg (n = 10) tipifarnib twice daily for 21 of 28-day cycles plus tamoxifen once daily. Serum was collected at baseline and after 8 weeks of treatment to enable proteomic comparison and identify possible predictive response markers. Results: Twenty patients were enrolled and evaluated for efficacy: one patient had an objective response (liver metastasis) and nine had stable disease after 6 months for a clinical benefit rate of 50%; median duration of benefit was 10.3 (range, 7.4-20.2) months. The proteomic analysis by SELDI-TOF and LTQ-FT-Orbitrap identified a known peptide of fibrinogen α, the intensity of which was significantly increased in patients with progression compared with patients who benefited from the combined treatment after 8 weeks. Conclusions: Because the primary end point of efficacy (three objective responses) was not achieved, the study is negative. Nevertheless, the identified peptide could be of interest in discriminating, at 8 weeks of treatment, responders from nonresponders. Clin Cancer Res; 16(4); 1264–71. ©2010 AACR.

The use of antihormone therapies has improved the cross-talk between growth factor and estrogen (E2) signaling survival of women with breast carcinoma and causes min- pathways (3). The activation of tyrosine kinase receptors has imal side effects. However, for patients with metastatic dis- multiple consequences that may cause tamoxifen resistance. ease, resistance to treatment, especially to tamoxifen, Ras and some Rho proteins are overexpressed in hu- eventually occurs. Multiple mechanisms have been identi- man tumors, including breast carcinoma, with possible fied to explain this resistance in preclinical and in clinical correlations with clinical outcome (4, 5). Major compo- studies (1, 2). A major mechanism is the alteration of the nents of the growth factor receptor pathways are GTPase proteins, such as Ras or Rho, which require posttransla- tional prenylation with farnesyl or geranylgeranyl lipids. Authors' Affiliations: 1Institut Claudius Regaud; 2Institut National de la Santé et de la Recherche Medicale U563; 3Université Paul Sabatier; A farnesyltransferase catalyzes the covalent attachment of 4EA 3035; 5Institut National de la Santé et de la Recherche Medicale a farnesyl group to the carboxy-terminal cysteine of pro- U588; and 6Centre National de la Recherche Scientifique, IPBS, teins (6, 7). We previously showed the involvement of Toulouse, France; and 7Centre Antoine Lacassagne, Nice, France prenylated proteins in E2 actions, with contrasting effects; Note: Supplementary data for this article are available at Clinical Cancer indeed, prenylation inhibitors on the one hand block E - Research Online (http://clincancerres.aacrjournals.org/). 2 mediated proliferation and on the other hand stimulate F. Dalenc and S.F. Doisneau-Sixou are co-first authors of this work. the transcriptional activity of estrogen receptor (ER)α in Previous presentation of part of the work: Part of this work was presented MCF-7 cells (8). at the San Antonio Breast Cancer Symposium 2005 (28th San Antonio, TX, December, Abstract 5098). Tipifarnib (Zarnestra, R115777) is an orally bioavail- Corresponding Author: Florence Dalenc, Institut Claudius Regaud, 20-24 able, potent, selective farnesyltransferase inhibitor (FTI; rue du pont St. Pierre, 31052 Toulouse, France. Phone: 00-33-5-67-69-63- ref. 9). It inhibits the growth of several human breast tumor 54; Fax: 00-33-5-61-42-46-23; E-mail: [email protected]. cell lines in vitro and in xenografts (9, 10). It has been recently doi: 10.1158/1078-0432.CCR-09-1192 published that tipifarnib significantly enhances the breast ©2010 American Association for Cancer Research. pathologic complete response rate associated with standard

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The protocol was approved by the French Ethics Committee Translational Relevance (Comité Consultatif de Protection des Personnes se prêtant à des Recherches BioMédicales). Patients provided written Farnesyl transferase inhibitors have been initially devel- informed consent. oped to block the development of the numerous human Initial assessment of patients included a medical history, tumors presenting an activation of the Ras pathway. physical examination, biological tests, and imaging of dis- Nonetheless, these compounds also exhibit antiprolifera- ease sites with tumor measurements within 4 wk of starting ras tive effects on wild-type tumors and especially in breast therapy. During the first 2 mo, complete blood counts were tumors. It seems that they clearly can lead to additive or done weekly. Patients were reassessed every 4 wk for adverse synergistic effects with other cancer treatments and have events, and a physical exam and metabolic panel was done. to be considered to avoid the resistance to hormone ther- Tumor lesions were measured using the Response Evaluation apies of breast cancers. At present, another major issue is Criteria in Solid Tumors (17) at baseline and every 8 wk there- to identify biological markers that may predict which sub- after. Adverse events were graded according to the WHO scale. populations of patients may benefit from treatment. The primary end point was to determine the OR rate Based on preclinical data, we conducted a phase II clin- [complete response + partial response (PR)] produced by ical trial with combined tipifarnib/tamoxifen as a rescue the tipifarnib/tamoxifen combination. Secondary end therapy for acquired tamoxifen resistance in metastatic points included the estimation of CB [defined as the OR plus breast cancers. In parallel, we performed a serum profiling. stable disease (SD) for at least 6 mo], median duration of ben- The trial was negative with only one objective response. efit, median time to progression (time from enrollment until Nonetheless, one of the secondary objectives of the study progression of disease or death due to disease progression), was met: the combination resulted in a clinical benefit rate and determination of biological predictive factors of efficacy. of 50%. In addition, we identified a peptide of fibrinogen α peptide as a possible therapeutic response marker. Patient population Women with histologically proven, metastatic, or locally advanced inoperable breast cancer were eligible. Tumors preoperative doxorubicin-cyclophosphamide chemotherapy were considered potentially hormone sensitive if they ex- (11). Regarding hormone-dependent metastatic breast can- pressed ER and/or progesterone receptor in >10% of the cers, a phase II trial was conducted with single agent tipifar- primary tumor cells or at a metastatic site by immunohis- nib, at two dosing regimens (continuous dosing of 400 or tochemistry. Tipifarnib was given to patients when pro- 300 mg on the one hand and 300 mg twice daily in a cyclical gression, according to Response Evaluation Criteria in regimen on the other hand), in advanced endocrine-resistant Solid Tumors criteria, was observed on tamoxifen treat- cancers. This trial showed good tolerability and objective re- ment, given either as an adjuvant treatment or for ad- sponse (OR) rates of 10% and 14%, respectively, with an advanced/metastatic disease. There was no limit on the ditional 15% and 9% clinical benefit (CB) rate in 41 and 35 number of prior chemotherapy regimens, with one line patients, respectively (12). We (13, 14) and other (9) showed for two patients, two lines for one patient, and three lines that FTIs combined with endocrine therapy have additive or for one patient. At least one measurable lesion according to synergistic effects on the antiproliferative effects on MCF-7 the Response Evaluation Criteria in Solid Tumors criteria cells and in xenografts (15). We then hypothesized that the was required at study entry. For patients who had only bone combination of tamoxifen and tipifarnib may benefit meta- metastases, a detectable nonirradiated lytic lesion was re- static breast cancer patients. Moreover, phase I studies of ti- quired. Other eligibility criteria included age ≥18 y, perfor- pifarnib and tamoxifen in metastatic breast cancer showed mance status (WHO) of ≤2, adequate bone marrow that the combination has minimal toxicity and the clinical (absolute neutrophil count of ≥2×109 cells/L, platelets of data were encouraging (16). ≥100 × 109 cells/L, hemoglobin of ≥10 g/dL), and adequate Based on the preclinical data, we conducted a phase II hepatic (aspartate amino transferase and alanine amino study with combined tipifarnib/tamoxifen as a rescue thera- transferase of ≤2.5 times the upper limit of normal or py for acquired tamoxifen resistance in ER and/or progester- 5 times in cases of liver metastasis) and renal (serum creat- one receptor (PgR)–positive metastatic breast cancers. We inine of ≤1.5 times the upper limit of normal) function. also used serum proteomic profiling to look for markers that Patients were excluded if they were pregnant, taking cyto- may predict which subpopulations of patients may benefit chrome P450–inducing drugs, presented with a sensory from treatment. We identified a peptide of fibrinogen α neuropathy ≥grade 1, a contraindication to antiestrogens, peptide (p5900) as a possible therapeutic response marker. had life-threatening lesions (such as hepatic lesion of 1/3 of liver volume, pulmonary lymphangitis, uncontrolled Patients and Methods cerebral metastases, and carcinomatous meningitis), or any other malignancy within the past 5 y. Study design This phase II, single-arm, open-label, multicenter study Drug delivery and dose adjustments was conducted through the Grand Sud Ouest (GSO) can- Tipifarnib was taken immediately after a meal twice ceropole in France (Toulouse, Bordeaux, and Limoges). dailyat12-hintervalsin28-dcyclesconsistingof21d

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of treatment followed by a 7-d rest period. When the study as described by the supplier (Bio-Rad). Then, 1 μL of sina- was initiated, before publication of the results of the phase pinic acid solution (50% v/v) was applied twice to each I studies (16) and according to Ortho-Biotech recommen- spot as an energy-absorbing matrix. Arrays were read on a dation, preliminary data indicated that 300 mg tipifarnib Protein Biological System IIc ProteinChip reader (SELDI- twice daily (level 0) was a tolerable. However, during the TOF, Bio-Rad) and the spectra were analyzed using the study, it became clear that the maximum tolerated dose Ciphergen Express software. was 200 mg twice daily (level −1) in combination with ta- Statistics and bioinformatics. Peaks were detected auto- moxifen. Thus, after inclusion of 12 patients, the protocol matically and the clusters' P values were calculated using was amended to prescribe 200 mg tipifarnib twice daily. the Mann-Whitney Test on the Ciphergen Express Cluster Tamoxifen (20 mg) was given once daily in the morning. Wizard. Then, a more advanced statistical analysis was In case of toxicity, tipifarnib dose adjustments were al- done using a Wilcoxon-Rank test. lowed by the protocol (Supplementary Table S1). Study Biomarker enrichment. Purification was done on an treatment was continued until progression or unaccept- anion exchange column (Bio-Rad) followed by 10- then able toxicity. 3-kDa cutoff ultrafiltration columns (Millipore) and followed through the SELDI-TOF profiling. Statistical considerations Biomarker identification. The fraction containing the Patients in this study had progressed on tamoxifen ther- protein of interest was analyzed by nanoliquid chromatog- apy. The response rate of tipifarnib alone in hormone-resis- raphy tandem MS using an Ultimate3000 system (Dionex) tant, advanced breast carcinoma is ∼14% (12). To evaluate coupled to an LTQ-Orbitrap mass spectrometer (Thermo the benefit for the combination, the response rate should be Fisher Scientific) as previously described (20). The tandem greater than that of tipifarnib alone and should be at least MS data were searched against human sequences in the equivalent to that of aromatase inhibitors after antiestrogen public database UniProt version 13.4, which consists of therapy (10-24%). As per Simon, the study had an optimal Swiss-Prot Protein Knowledgebase Release 55 and TrEMBL two-stage design with a significance level of 0.05, had a Protein Database Release 38.4 (72 400 entries), using the power of 0.80, and assumes a target response rate of 25% Mascot search engine (Mascot Daemon, version 2.2.0; and a lowest level of interest of 10%. The study plan was to Matrix Science). The search was done with no enzyme include 40 patients, with 22 patients in stage 1 with an in- specificity; mass tolerances in MS and tandem MS was terim analysis; if 3 or more OR were observed, an additional set to 10 ppm and 1 Da, respectively; and oxidation of 18 patients would be enrolled in stage 2. methionines, histidines, tryptophans, and protein NH2- terminal acetylation was set as variable modifications. Pharmacokinetic assessment Protein identification was confirmed by the manual inter- A volume of 5 mL of blood was collected at baseline, pretation of corresponding tandem MS data. Protein mass before the first tipifarnib dose was administered (i.e., ta- was determined by raw data deconvolution using the inte- moxifen only), and on day 14, before tipifarnib was admin- grated Xcalibur Extract software (Thermo Electron). istered (i.e., tamoxifen and tipifarnib), and 1 h after tipifarnib administration (i.e., tipifarnib only). Tipifarnib serum levels were measured with a validated Results liquid chromatography tandem mass spectrometry (MS) assay developed by Ortho-Biotech Oncology Research Patient characteristics. From September 2003 to June and Development (18). Serum levels of tamoxifen and 2005, 22 patients were enrolled. Patient and main tumor its metabolites were measured by high performance liquid characteristics are reported in Table 1. The median age was chromatography with fluorescence detection (19). 64 years, with 14 patients with a performance status of 0 A Wilcoxon test was used to compare the paired data of and 8 patients with a performance status of 1. One patient initial versus final measurements. had breast carcinoma with HER2 overexpression, 82% of patients had visceral metastases (liver and/or lung), and Serum proteomic analysis 17 (77%) and 2 patients (9%) had received tamoxifen Protein profiling. A volume of 10 mL of blood was collect- or aromatase inhibitors, respectively, in the adjuvant set- ed before the first tipifarnib dose, again at 8 wk (first disease ting. Two patients (9%) received aromatase inhibitors evaluation of each patient), and at the time of OR or progres- for metastatic disease. They all had disease progression sion. Serum aliquots were frozen and stored at −80°C within while receiving tamoxifen in either the adjuvant (n = 11) 60 min of blood withdrawal. We followed best experimental or metastatic setting (n = 11), with median times of non- design practices, specifically, uniform blood sample collec- progression of 48 months (range, 16-60) and 18 months tion, processing, and freezing. (range, 7-54), respectively. The overall median time of Samples were fractionated on an anion exchange frac- nonprogression (n = 22) was 30.5 months (range, 9-60). tionation kit according to the manufacturer's instructions Moreover, 14 patients (64%) received chemotherapy (Bio-Rad). A volume of 5 μL of each serum fraction was as adjuvant treatment, one of whom received high-dose applied and incubated with a metal affinity ProteinChip chemotherapy and stem cell transplantation, and 4 array (IMAC30) loaded with copper sulfate and washed (18%) received chemotherapy for metastatic disease.

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Table 1. Patient characteristics at baseline Table 3. Efficacy (n = 20) (n = 22) No. of patients (%) Median age, years (range) 64 (39-74) OR 1 (5%) No. of patients (%) CB (SD + OR) 10 (50%) Hormone receptor status Median time of CB; months (range) 10.3 (7.4-20.2) ER positive 20 (91%) PD at 6 mo or before 10 (50%) PgR positive 19 (86%) TTP median; mo (range) 5.7 (1.9-20.2) HER2 status Abbreviations: PD, progressive disease; TTP, time to IHC (0 or +) 21 (91%) progression. IHC (+++) 1 (5%) Sites of metastases Bone only 3 (14%) rapidly recovered to grade 1 neuropathy and could be treated Visceral (liver, lung) 18 (82%) with docetaxel after this study. The marker analysis at 8 weeks Lymph nodes 1 (4%) could therefore have been influenced by the dose reduction Prior adjuvant endocrine therapy at 4 weeks for the first two patients and by the difference of Tamoxifen/aromatase inhibitors 17 (77%)/2 (9%) dose (300 mg versus 200 mg twice daily) for all six of them. Progression under tamoxifen in: The initial tipifarnib dose was decreased to 200 mg Adjuvant setting 11 (50%) twice daily for the next 10 patients and was well tolerated, Metastatic setting 11 (50%) except for one case of venous thrombosis leading to the Median of duration of tamoxifen 30.5 (7-60) patient's removal from the study after 6 months (with treatment, mo (range) SD). The most common nonhematologic grade 1 toxicities Aromatase inhibitor for metastatic 2 (9%) were diarrhea, asthenia, and nausea. One patient pre- disease sented with a cutaneous rash and was discontinued from Prior adjuvant chemotherapy 16 (64%) the study after a few days. One patient developed an acute Previous chemotherapy in metastatic 4 (18%) myeloblastic leukemia a few months after the end of treat- setting ment, with no apparent correlation with any previous treatment. No analysis of the results could include the tox- Abbreviations: PgR, progesterone receptor; IHC, icity because of the low number of events for each type of immunohistochemistry. toxicity. Efficacy results. From the 22 patients, those 2 with cutaneous rash or venous thrombosis at 4 months were ex- Safety profile. All patients were assessable for toxicity. At cluded after a few days and at 4 months, respectively, the 300 mg tipifarnib twice daily dose, 6 of 12 patients because this toxicity appeared very early in the treatment. required dose reductions due to hematologic or nonhema- Moreover, at 4 months, the patient was considered neither tologic toxicities (Table 2). Four patients had grade 3 or 4 as being in progression nor having an OR. Twenty patients neutropenia or thrombocytopenia, one had grade 3 muco- were then assessed for efficacy (Table 3). At the end of the sitis, and two developed a venous thrombosis. Moreover, first step, only one patient (5%) had a partial response at one patient experienced grade 3 sensitive and painful neu- 6 months in a liver metastasis, but she was discontinued rotoxicity requiring her removal from the study at 6 from the trial because of neurotoxicity. Because the primary months. This patient had received neurotoxic agents in end point of at least three OR was not achieved at the end of the adjuvant and metastatic settings, including vinorelbine, the first step, the study was discontinued. The CB rate was and had grade 1 neurotoxicity upon entry to the trial. She 50% (n = 10) with a median duration of benefit of 10.3 (range, 7.4-20.2) months; the median time to progression was 5.7 months. Of note, one patient with liver metastasis Table 2. Toxicities reported at 300 mg n had SD for 20.2 months. tipifarnib twice daily ( = 12) Pharmacokinetic results. We observed an expected significant accumulation of tipifarnib in plasma 1 hour after No. of patients (%) administration on day 14. This showed the variation in ti- Grade 3-4 hematologic pifarnib concentration after repeated daily dosing (Table 4). Neutropenia 2 (16%) The statistical analysis from 16 patient sera showed a signif- Thrombopenia 2 (16%) icant but modest increase in the median tamoxifen concen- Mucositis, grade 3 1 (8%) tration (from 108-124.5 nmol/L) after 14 days of tipifarnib P Neurotoxicity, grade 3 1 (8%) treatment ( < 0.02). This suggested that the effect of tipifar- Venous thrombosis 2 (16%) nib on tamoxifen pharmacokinetics may be biased because of the small number of patients evaluated, or is not relevant

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Table 4. Tipifarnib (ng/mL) and tamoxifen or metabolites (nmol/L) sera concentrations (n = 16)

Serum sample 1 Serum sample 2 P Median Range Median Range

Trough concentration before day Trough concentration 1 h after day 14 tipifarnib administration (cycle1) 14 tipifarnib administration (cycle 1) Tipifarnib 55.8 (13.1; 222) 551 (137; 1,980) Trough concentration before day 0 Trough concentration before day 14 tipifarnib administration (cycle 1) tipifarnib administration (cycle 1) Tamoxifen 108 (0; 215) 124.5 (66; 275) 0.02 NDT 1,999.5 (165; 4,607) 2,043.5 (977; 6,669) 0.38 Endoxifen 6.5 (0; 190) 4 (0; 215) 0.68 NDDT 276.5 (76; 664) 306 (118; 550) 0.51 Sum 2,404 (247; 5,159) 2,464.5 (1,376; 7,534) 0.32

NOTE: The sum is the addition of the tamoxifen concentration and of its three metabolite concentrations (NDT or N-desmethyl- tamoxifen, endoxifen, or 4-hydroxy-N-desmethyl-tamoxifen, and NDDT or N-desdimethyl-tamoxifen).

because neither the tamoxifen metabolites (especially en- To identify this proteomic signature, and after several endoxifen; refs. 21, 22) nor the sum of tamoxifen and of its richment protocols, the fraction was analyzed by the LTQ- metabolites showed any significant difference. Orbitrap MS (Supplementary Fig. S2). We identified the The early identification of any therapeutic response bio- peptide with a high probability score as a COOH-terminal marker would be very valuable as it is likely to be detected much earlier than clinical symptoms. Due to the very short half-life of tipifarnib (estimated 2.8 hours; ref. 16) and the twice daily administration, the steady-state concentrations are already reached at day 14. However, no statistically significant difference was found in tipifarnib or tamoxifen concentrations between the patients who progressed and those who achieved CB (data not shown). Proteomic results. Detection of biomarkers was expected at either at the initiation of treatment or after 8 weeks. Sera from 19 patients were analyzed. Among several protein peaks, three had statistically significant differential expression (Supplementary Fig. S1). One of these was detected at baseline (before administration of tipifarnib) and two were detected after 8 weeks of treatment. In a multivariate Cox re- gression model taking into account the three proteins, only one was strongly and statistically significantly associated with clinical outcome (area under receiver operating characteristic, curve 0.88; 95% confidence interval, 0.70-1.00). The molecular mass of this protein was determined at ∼5,900 Da. After 8 weeks of treatment, the immediate risk of progression at 6 months for patients whose p5900 intensity was higher than a threshold value of 0.84 (chosen as the value on the receiver operating characteristic curve closest to the upper left-hand corner of the graph) was 8.2-fold higher (95% confidence interval, 1.0-66.4; P < 0.05) than the risk for patients whose p5900 intensity was <0.84. Two profile examples are shown in patients who either benefited or progressed at 6 months (Fig. 1A). Finally, a Kaplan-Meier plot of the probability according to the expression of p5900 at 8 weeks is shown in Fig. 1B, clearly Fig. 1. Differential expression of P5900 predicts time to progression in illustrating the significantly different immediate risk of patients from groups B (CB) and P (progressed) in sera after 8 wk of treatment with tamoxifen and tipifarnib. A, examples of protein profiles. progression after 8 weeks of treatment between the two B, proportion of patients with P5900 intensity inferior (dashed line) or populations. superior (solid line) to 0.84 who were without progression at 6 mo.

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Fig. 2. Mass spectrum of the protein of interest acquired using an LTQ-FT-Orbitrap showing several multicharged (z charges) protein states. The amino acid sequence corresponds to the identified 576 to 629 peptide of fibrinogen α chain precursor. peptide (amino acid residues 576-629) of fibrinogen α On the contrary, another recently reported phase II ran- chain precursor (Fig. 2). domized trial of letrozole with or without tipifarnib in 121 patients with tamoxifen-resistant, advanced breast cancers did not show an increased response rate with the Discussion combination compared with letrozole alone (24). The absence of an additive effect for tipifarnib in this trial could Signal transduction pathways involving prenylated proteins be because tamoxifen, like fulvestrant, acts through an ER may be activated in endocrine-resistant tumors. Tipifarnib intracellular signaling pathway, whereas letrozole inhibits monotherapy in pretreated advanced breast cancer patients E2 synthesis. Moreover, we are in the situation of patients never produced a >25% CB rate (12). In our study, the pri- who are in progression on tamoxifen therapy at the ini- mary end point of at least three OR before proceeding to tiation of the combination treatment, whereas the pa- stage 2 was set too high and was not met, as only one OR tients of the former study already showed resistance to was observed. A 50% CB rate (10 patients) was found with tamoxifen but could respond to letrozole alone. We minimal toxicity with 200 mg tipifarnib twice daily. A larger, and others previously showed the additive or synergistic randomized study is then needed to confirm the CB observed action of a combination of tamoxifen with a FTI to inhibit in this study with the tamoxifen/tipifarnib combination with proliferation and promote apoptosis in vitro and in vivo that of tipifarnib alone. The CB rate or the progression-free (9, 13–15). survival, and not the OR rate, should be the primary objective If preliminary results from studies combining hormone in future nonrandomized phase II studies that combine en- therapy and FTIs or other signal transduction inhibitors docrine therapy and tailored treatments because the trial had failed to show high potency, it may be due to the inability to be discontinued despite the achievement of a 50% CB rate. to identify the target population who would best benefit A phase II trial of tipifarnib plus fulvestrant was con- from this treatment (25). The challenge for future thera- ducted in women with hormone receptor–positive, inoper- peutic development is to identify those patients who will able, and locally advanced or metastatic breast cancer after benefit from a specific treatment combination. The median first-line endocrine therapy (23). Preliminary results in time to progression of the 20 progressing metastatic pa- 31 patients showed a 47.6% CB rate and a median time tients in the present study is 5.7 months with the tipifarnib/ to progression of 7.2 months. tamoxifen combination. The absence of modification of

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tipifarnib pharmacokinetics and pharmacodynamic in the lated to the therapeutic targets but to the pathology presence of tamoxifen was reported previously (16). We ob- signature. Scientifically, our data set is small with no inde- served that the concentration of endoxifen, the active metab- pendent validation of the fibrinogen peptide as a phar- olite of tamoxifen present at concentrations 10-fold higher macodynamic response marker. After this first step of than 4-OH-tamoxifen (22), are maintained in the presence screening, further work includes the development of spe- of tipifarnib. However, none of these concentrations corre- cific monoclonal antibodies to analyze the benefit of lated with the risk of progression. p5900 in larger cohorts of patients and determining if an No valuable peptide could be identified as a therapeutic- increased concentration of this peptide in sera determines response marker in the pretreatment sera, but we found that which patients benefit from combination therapy. high expression of p5900 was associated with an 8.2-fold The role of FTIs in breast cancer continues to be ex- higher risk of progression at 6 months after 8 weeks of treat- plored in four ongoing studies of tipifarnib or lonafarnib ment. This 8-week period might be relevant to stop the alone or in combination with chemotherapy or herceptin. combination treatment before a clinical progression is ob- In conclusion, this study is negative regarding the pri- served. In breast cancers, the SELDI-TOF technique has mary end point. An interesting finding of the exploratory identified serum biomarkers (e.g., ubiquitin, ferritin light analysis was to identify a potential pharmacodynamic, ther- chain, fibrinogen α peptide) that discriminate those with apeutic-response biomarker in breast cancer. Translational breast cancer from those with benign disease or healthy pa- studies should become an integral part of future clinical trial tients (26, 27). designs by analyzing the tumor and blood genotypes and/or We identified p5900 as a new 54-amino acid fibrinogen phenotypes at the initiation of the treatment using proteomic α peptide (576-629, COOH terminal end). We expected serum profiling or other approaches. p5900 to be a protein or a peptide degradation product, whose concentration increases before clinical progression Disclosure of Potential Conflicts of Interest and therefore expected it to be associated with either inva- sion and/or the inflammation and/or the action of tipifar- No potential conflicts of interest were disclosed. nib. Indeed, fibrinogen is a circulating multidomain protein consisting of two pairs of three polypeptide chains, Acknowledgments α, β and γ. After formation of a fibrin clot, plasmin de- grades the clot into many peptides with various biological We thank Dr. A. Howes and Prof. E. Chatelut for critically reading the activities, including a protumorigenic one in breast cancer. manuscript. The presence of smaller fibrinogen α peptides in plasma, but not sera, has been associated with the absence of breast Grant Support cancer (27, 28). One of them, a 25-amino acid peptide identified as declining in HER2-positive breast cancer pa- Ortho-Biotech Oncology Research and Development (Belgium), who tients (encompassing residues 605-629, COOH terminal provided tipifarnib and financial support, by the Institut National du Cancer, Canceropole Grand Sud-Ouest, Institut National de la Santé et de la Recherche end; ref. 27) overlaps the 54 amino acids of the fibrinogen Medicale, and University of Toulouse. A Programme Hospitalier de Recherche α peptide we identified. On the contrary, a serum profile Clinique was obtained in 2004 by H. Roché to conduct the study. The costs of publication of this article were defrayed in part by the identified one of those 25-amino acid peptides as a breast payment of page charges. This article must therefore be hereby marked cancer–specific marker that increases in cancer patients' advertisement in accordance with 18 U.S.C. Section 1734 solely to sera, unselected for HER-2 status (29). The specificity of ei- indicate this fact. ther sera or plasma profiling is of high importance and we Received 5/11/09; revised 11/12/09; accepted 12/13/09; published have to be aware that those markers may not be directly re- OnlineFirst 2/9/10.

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www.aacrjournals.org Clin Cancer Res; 16(4) February 15, 2010 1271

Tipifarnib Plus Tamoxifen in Tamoxifen-Resistant Metastatic Breast Cancer: A Negative Phase II and Screening of Potential Therapeutic Markers by Proteomic Analysis

Florence Dalenc, Sophie F. Doisneau-Sixou, Ben C. Allal, et al.

Clin Cancer Res 2010;16:1264-1271. Published OnlineFirst February 15, 2010.

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