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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC Apr 15, 2010.
Published in final edited form as:
PMCID: PMC2785076
NIHMSID: NIHMS105425
Phase II trial of the farnesyl transferase inhibitor tipifarnib plus neoadjuvant doxorubicin-cyclophosphamide in patients with clinical stage IIB-IIIC breast cancer
Joseph A. Sparano,1 Stacy Moulder,2 Aslamuzzaman Kazi,3 Domenico Coppola,3 Abdissa Negassa,1 Linda Vahdat,4 Tianhong Li,1 Christine Pellegrino,1 Susan Fineberg,1 Pam Munster,2 Mokenge Malafa,3 David Lee,1 Shira Hoschander,1 Una Hopkins,1 Dawn Hershman,5 John J. Wright,6 Celina Kleer,7 Sofia Merajver,7 and Said M. Sebti3
1New York Cancer Consortium, including the Montefiore- Einstein Cancer Center, Montefiore Medical Center, Bronx, NY
2Breast Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL
3Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL
4Weill Cornell Medical Center, New York, NY
5Columbia Presbyterian Medical Center, New York, NY
6Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD
7University of Michigan Ann Arbor, MI
Address for correspondence: Joseph A. Sparano, MD, Montefiore Medical Center-Weiler Division, Dept. of Oncology, 2 South, Room 47−48, 1825 Eastchester Road, Bronx, New York 10461; Phone 718−904−2555, Fax 718−904−2892; Email: jsparano/at/montefiore.org
Purpose
Tipifarnib is a farnesyl transferase inhibitor (FTI) that has activity in metastatic breast cancer and enhances the efficacy of cytotoxic agents in preclinical models. We evaluated the biological effects of tipifarnib in primary breast cancers in vivo, whether adding tipifarnib to preoperative chemotherapy increased the pathologic complete response rate (pCR) at surgery, and determined whether biomarkers predictive of pCR could be identified.
Experimental Design
Forty-four patients with stage IIB-IIIC breast cancer received up to 4 cycles of neoadjuvant doxorubicin-cyclophosphamide (AC) every two weeks plus tipifarnib and filgrastim followed by surgery. Enzymatic assays measuring FTase activity and western blotting for pSTAT3, pERK, pAKT, and p27 were performed in 11 patients who agreed to optional tissue biopsies before therapy and 2 hours after the final dose of tipifarnib during the first cycle, and predictive biomarkers were evaluated by immunohistochemistry in 33 patients. The trial was powered to detect an improvement in breast pCR rate of 10% or less expected for AC alone to 25% for AC-tipifarnib (alpha 0.05, beta 0.10).
Results
Eleven patients had a breast pCR (25%; 95% C.I. 13%, 40%). FTase enzyme activity decreased in all patients (median 91%, range 24%, 100%), and p-STAT-3 expression decreased in 7 of 9 patients (77%). Low tumor Ki67 expression (below the median of 60%) therapy was significantly associated with resistance to therapy (p=0.01).
Conclusion
Tipifarnib inhibits FTase activity in human breast tumors in vivo, is associated with downregulation of p-STAT-3, and enhances the breast pCR rate, and merits further evaluation. .
Keywords: Farnesyl transferase inhibitor, Ras, breast cancer, neoadjuvant therapy
Ras proteins are low molecular weight guanosine nucleotide binding GTPases (G protein) that play a critical role in cell growth and regulation.1 Oncogenic mutations of the three known human ras genes are found in 30% of all human cancers; these mutations lead to hyperactivation of Ras protein. Although the frequency of ras mutations in breast cancer is very low (<2%)2, 3, hyperactivation of Ras protein and its downstream effectors is very common due to either overexpression of upstream components such as EGFR and HER-2/neu4, 56 In addition, Ras protein overexpression is associated with poor prognosis7, and RhoC overexpression (a downstream effector of Ras) is associated with regional and/or distant metastases8, and with inflammatory carcinoma.9
Posttranslational modification with a 15-C farnesyl lipid at the carboxyl terminus of Ras is essential for mediation of its downstream signaling effects.10, 11 This modification is catalyzed by farnesyl transferase (FTase), a heterodimeric zinc metalloenzyme. FTase inhibitors (FTIs) cause accumulation of cells in G2/M phase or G1 phase11 12 13, 14, induce apoptosis of a variety of tumor cell lines15, inhibit angiogenesis.16, inhibit growth of MCF-7 human breast cancer xenografts (which have wild type Ras)17, induce tumor regression in breast cancer transgenic mouse models18 19, and revert the RhoC GTPase-induced inflammatory breast cancer phenotype.9 Increased Ras/Raf-1/MEK/MAPK activity has been implicated in doxorubicin resistant MCF-7 cell line20, paclitaxel-resistant cells21, and the expression of the P-glycoprotein (P-gp) extrusion pump.22 Objective responses have been observed in about 10% of patients with metastatic breast cancer treated with tipifarnib (formerly R115777; Zarnestra™, Johnson & Johnson, PRD, LLC, Raritan, NJ & Tibotec Therapeutics, Raritan, NJ), an orally available FTase inhibitor (FTI).23
Based upon these considerations, we initiated a phase I/II trial of tipifarnib in combination with preoperative doxorubicin and cyclophosphamide (AC) in patients with stage IV breast cancer (for the phase I trial) and clinical stage IIB-IIIC breast cancer (for the phase II trial), and previously identified the recommended phase II dose of tipifarnib (200 mg PO BID on days 2−7 of therapy) that could be safety used with dose-dense AC plus granulocyte colony stimulating factor.24 Furthermore, we also reported that 7 of the first 21 patients with LABC treated with up to 4 cycles of the combination had a pathologic complete response (pCR) in the breast (33%; 95% C.I. 15%, 55%), providing a sufficient level of activity to proceed to the second stage of the phase II trial. We herein report the final results of the completed phase II trial in 44 patients with clinical stage IIB-IIIC breast cancer. Our primary objectives were to determine whether tipifarnib enhanced the breast pCR rate associated with standard preoperative AC chemotherapy, to determine the biological effects of tipifarnib in vivo, and to determine whether we could identify biomarkers predictive of breast pCR.
Patient Selection
Patients were required to have histologically or cytologically confirmed adenocarcinoma of the breast, and clinical stage IIB-IIIC disease and meet. other requirements as previously described.24 The study was reviewed, approved, and sponsored by the Cancer Therapy Evaluation Program of the National Cancer Institute (NCI study number P5598, Clinical Trials.gov identifier NCT00049114). The protocol was reviewed by the local institutional review board at each participating institution, and all patients provided written informed consent.
AC Chemotherapy and Tipifarnib
All patients received “dose-dense AC”, consisting of doxorubicin (60 mg/m2 by slow intravenous push over 10−15 minutes) and cyclophosphamide (600 mg/m2 by intravenous infusion over 30−60 minutes) given on day 1 every 2 weeks for up to 4 cycles, preceded by standard antiemetic therapy. Tipifarnib was given at a dose of 200 mg BID on days 2−7 of each treatment cycle. Treatment cycles were repeated if the neutrophil count was at least 1500/uL, platelet count at least 100,000/uL, and if there was adequate recovery from nonhematologic toxicity (to grade 0 or 1). All patients also received granulocyte-colony stimulating factor (G-CSF); 5 mg/kg subcutaneously on days 2−13 of each cycle (pegfilgrastim was not used).
Surgery and Additional Therapy
All patients with an operable primary breast cancer who were candidates for surgery underwent mastectomy or lumpectomy plus axillary dissection about 4 weeks after completion of 4 cycles of AC. After surgical resection, patients received additional chemotherapy, hormonal therapy or radiation therapy as clinically indicated.
Centralized Pathology Review
Pathological response was assessed by the local pathologist using procedures normally utilized for evaluation of surgical breast cancer specimens; pCR was defined as no evidence of invasive carcinoma in the specimen. In 35 of 44 cases, pathologic responses were reviewed by two of the coauthors who were breast pathologists for cases at Moffitt Cancer Center (DC) and for cases at Montefiore Medical Center plus other centers (SF); the specimens were evaluated for “residual cancer burden” (RCB) as described by Symmans et al25; in 5 cases RCB was determined by review of the pathology report, and the remaining 6 cases were not evaluable (received non-protocol therapy before surgery or specimens not available).
Estimation of Predicted Pathologic Complete Response Rate
We performed a post-hoc analysis to estimate the expected pCR rate in breast and lymph nodes for each patient enrolled on the trial using the nomogram developed by Rouzier and colleagues (http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert2) 26, which had not been published at the time that the trial was initiated. For each patient, an expected pCR rate was calculated using information required by the nomogram.
Optional Tumor Biopsy and FTase Enzyme Analysis
Patients who consented to an optional biopsy had paired tumor biopsies performed before treatment and during cycle 1, day 6 or 7, two hours after the last tipifarnib dose. Three core biopsies were obtained from the tumor using a 14-gauge needle after local anesthesia. Specimens were placed in a sterile container, placed on dry ice, and transported to the pathology laboratory where they were wrapped in foil and placed in liquid nitrogen. After freezing in liquid nitrogen, specimens were stored at −700C until they were analyzed using methods that have been previously described for FT ase and GGTI ase enzyme activity18, and by Western blots for p-STAT-3, p-ERK, p-AKT and p27. Frozen breast tumor tissues were mechanically homogenized with TISSUEMISER in RIPA buffer (150mM NaCl, 10mM Tris at pH 7.4, 0.1% SDS, 1.0% Triton X-100, 5mM EDTA, 0.1% Sodium deoxycholate) containing protease inhibitors. The homogenates were incubated on ice for 30min, and then spun at 10,000g for 5min at 4° to pellet cell debris. The resulting lysates were normalized for total protein content (50 μg per lane), resolved on 12% SDS-PAGE gels, transferred onto PVDF membranes, and incubated with anti-p-STAT3, p-Erk1/2, total Erk1/2 and p-Akt (Cell Signaling Technologies, Danvers, MA), total STAT3 and total Akt (Santa-Cruz Inc., Santa Cruz,CA), p27 (BD Biosciences, San Jose, CA) and beta actin (Saint Louis, MO) followed by HRP-conjugated secondary antibody and enhanced chemiluminescence (ECL)-based detection. The same blot was reprobed with anti-β-actin polyclonal antiserum (Sigma) as a loading control. The immunoblot data were quantified by scanning densitometry using the AlphaEaseFC software and normalized by β-actin.
Predictive Biomarker Analysis
Primary tumor specimens from pre-treatment biopsies were available in 33 patients, including 10 of 11 patients who had a breast pCR For Ki-67, p27, p21, p-STAT3, p-ERK, p-AKT, pretreatment paraffin embedded tumor specimens were evaluated by immunohistochemistry by a single pathologist (DC) without knowledge of clinical patient characteristics or response to therapy. This sample included 32 of 44 patients in the phase II trial (including 9 patients with inflammatory carcinoma), and one patient with inflammatory carcinoma treated in the phase I portion of the study who did not have a breast pCR but who had a baseline specimen available.
Immunohistochemistry was performed on a Ventana BenchMark XT (Tucson, AZ, USA) automated slide stainer, using the avidin-biotin complex method, and the following antibodies: Ki-67 (mouse mAb, clone K2, Ventana, proprietary dilution); p27 (mouse mAb, clone Sx53G8, Cell Marque, proprietary dilution); p21 (mouse mAb, clone EA10, Calbiochem, dilution 1:50); p-STAT3 (rabbit polyclonal Ab, clone 9145, Cell Signaling, dilution 1:900), p-ERK (mouse mAb, clone 4376, Cell Signaling, dilution 1:100); pAKT (mouse mAB, clone Ser473 587F11, Cell Signaling, dilution 1:20). Antigen retrieval, CC1 Standard for Ki-67, p27, p21, p-STAT3, and protease for p-AKT was used. The detection of p-ERK did not require antigen retrieval. The immunostains were evaluated considering the percent of tumor cells staining positive (nuclear staining), and p-AKT staining was evaluated by scoring the staining intensity (0, 1+, 2+ and 3+) and percent of positive cells {(0%), 1+ (1−33%), 2+ (34−66%), 3+ (more than 66%), with the product of intensity and percentage scores used to assigned a composite score.
For RhoA, B, and C, paraffin embedded specimens were likewise evaluated by another pathologist (CK) without knowledge of the clinical patient characteristics or response to therapy. Cytoplasmic RhoA, B, and C protein expression was scored from 0−3+ by comparison to the positive internal controls by immunohistochemistry using antibodies against RhoA, RhoB and RhoC GTPases (which was previously developed, validated, and described by CK).27 A mouse monoclonal anti-RhoA antibody at 1:50 dilution, without pretreatment (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number SC-418), a rabbit polyclonal anti-RhoB antibody at 1:40 dilution, without pretreatment (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number SC-180), and a chicken polyclonal anti-RhoC antibody at 1:9000 dilution, with microwave citrate buffer (pH 6.0) pretreatment were used. The proteins were expressed in the cytoplasm of myoepithelial cells and vascular smooth muscle cells, which served as consistent internal positive controls. Using this schema, strong diffuse staining was considered score of 3+, moderate diffuse staining as 2+, low diffuse staining as 1+, and no staining as 0.
Statistical Considerations
The study was designed to detect an increase in the breast pCR rate from ≤ 10% to at least 25% (alpha =0.05, beta = 0.10) using Simon's two-stage design. Although the breast pCR rate was 13% in NSABP B18 and B27 trials after AC given for 4 cycles, 28-30we assumed a slightly lower breast pCR of 10% or less for standard therapy; our trial required to have palpable axillary nodes or a tumor larger than 5 cm (or inflammatory carcinoma), whereas B18 and B27 included patients with less advanced disease (T1c-N3 and N0−1 disease).28-30 If ≤ 2 breast pCRs (< 10%) were observed among the initial 21 patients, the study would be terminated early and declared negative; if at least three breast pCRs were observed, accrual would continue to a total of 50 evaluable patients. If at least eight pCRs (≥ 16%) were observed among the 50 evaluable patients, this regimen would be considered worthy of further testing. This design yields at least .90 probability of a positive result if the true pCR rate is at least 25%. It yields at least .90 probability of a negative result if the true pCR rate is 10% or less, with at least .65 probability of early negative stopping.
With regard to the predictive biomarker analysis, the relationship between each marker and either sensitivity to therapy (as indicated by breast pCR) or resistance to therapy (as measured by an RCB score of 3) was evaluated by Fisher's extact test with a 2-sided p value of <0.05.
Patient Characteristics
Forty-four patients were enrolled, and their characteristics are shown in Table 1. The median age was 51 years, 70% had clinical stage III disease, 52% had hormone receptor positive disease, and 34% had Her2/neu positive disease. The median breast and nodal pCR rate projected for this population was 12% (range 1%-44%) using the nomogram reported by Rouzier et al26, confirming that a predicted 10% breast pCR rate based upon historical data was an accurate estimation for the patient population included in this study.
Table 1
Table 1
Patient characteristics
Pathological and Clinical Response
With regard to pathologic response, 11 patients (25%; 95% C.I. 13%, 40%) had a pathologic complete response (pCR) in the breast, including eight patients who had a pCR in the breast and lymph nodes (18%; 95% C.I. 8%, 33%). The number of breast pCRs observed exceeded the prespecified number required to distinguish between a 10% rate expected for historical data and a 25% rate targeted for the AC-tipifarnib combination. When analyzed by the residual cancer burden (RCB) score as described by Symmans et al25, the RCB score was 0 in 8 patients (18%), 1 in one patient (2%), 2 in 13 patients (30%), 3 in 17 patients (39%), and not evaluable in five patients (11%).
With regard to clinical response by RECIST criteria31, of the 44 evaluable patients, eight had a clinical CR (18%) and 26 had a clinical PR (59%), yielding an overall clinical response rate of 77% (95% CI 62%, 88%). Two of nine patients (22%) with a clinical CR had microscopic residual disease, whereas four of 26 patients (15%) with a clinical PR had no microscopic evidence of disease in the breast.
An exploratory analysis was performed to evaluate pathologic response by phenotypic subsets defined by hormone receptor (HR) and HER2/neu expression. Breast pCR occurred in five of 24 patients (21%; 95% C.I. 7%, 42%) with HR-positive disease, six of 21 patients (29%; 95% C.I. 11%, 52%) with HR-negative disease, five of 15 patients (33%; 95% C.I.12%, 62%) with HER2/neu positive disease, two of 11 patients (18%; 95% CI 2%, 52%) with triple-negative disease, and two of 12 patients (17%; 95% CI 2%, 48%) with inflammatory carcinoma.
Biological Effects of Tipifarnib in Vivo
Twelve patients consented to an optional biopsy before treatment and 2 hours after the final tipifarnib dose in cycle 1, of whom 11 patients had evaluable specimens, including two patients who had a breast pCR and RCB score of 0 (patient number 31 and 55). The effect of tipifarnib on tumor FT ase and (geranylgeranyl tranferase-I) GGT ase enzyme activity is shown in Figure 1. GGT ase and FT ase are similar proteins that consist of two subunits, including an α-subunit which is common to both enzymes and the β-subunit with 25% identity, and which have different isoprenoid substrates. There was consistent inhibition of FTase enzyme activity after tipifarnib administration in all patients (median 91%, range 21%-100%). The effect of tipifarnib on GGTase I enzyme activity was variable, being as decreased in six patients, increased in two patients, and unchanged in three patients. Regarding the effects of tipifarnib on expression of signaling proteins, there was consistent inhibition of p-STAT3 that was observed in seven of nine evaluable patients (77%), but there were inconsistent effects on p-ERK, p-AKT, and p27 expression. Representative results from two patients are shown in Figure 2, including one patient (number 31) who had a pCR and a second patient (number 30) who had a posttreatment RCB score of 3 (indicating extensive persistent disease and resistance to therapy). In summary, although tumor FT ase enzyme activity was substantially reduced by tipifarnib in most patients, and p-STAT3 decreased in the majority of patients, there was no correlation between FT ase enzyme inhibition or p-STAT3 inhibition and breast pCR.
Figure 1
Figure 1
Percent change in post-treatment FT ase and GGT ase enzyme activity in tumor samples obtained two hours after the last tipifarnib dose obtained during cycle 1 compared with a pretreatment sample. Results are shown for patients 16, 17, 23, 30, 31, 33, (more ...)
Figure 2
Figure 2
Representative Western blot analyses from two patients (number 30 and 31) before therapy and two hours after the last 200 mg tipifarnib dose given in cycle 1. The figures demonstrate downregulation of p-STAT3, p-ERK 1−2, p-AKT, and p27 after tipifarnib (more ...)
Predictive Biomarker Analysis
Biomarker data for the pretreatment tumor specimen was available for 33 patients, of whom 11 had inflammatory carcinoma and 10 had a breast pCR. The median value (and range) for each marker expressed as percent of positive tumor cells are shown in Table 2. for Ki-67, p-STAT3, p-ERK, p21, and p27; the median marker values (and range) expressed as a score for RHEB and AKT are also shown. There was no relationship between any marker (evaluated as a dichotomous variable above or below the median) and either sensitivity to therapy (ie, breast pCR) or resistance to therapy (ie, RBC score 3) with the exception of Ki67; low Ki67 score (below the median of 60%) was significantly associated with resistance to therapy (P=0.01).
Table 2
Table 2
Relationship between biomarker expression and sensitivity and resistance to therapy
For Rho A, B, and C, the proportion of samples that were graded as 0, 1+, 2+, and 3+ are represented in Figure 3. There was no significant association between expression of RhoA, B, and C protein (evaluated as a dichotomous variable comparing 2+/3+ vs. 1+/0 expression) with either response or resistance to therapy. There was no association between Rho A, B, or C expression and inflammatory phenotype. Three of 10 evaluable cases (30%) with inflammatory disease were 3+ for RhoC compared with four of 18 (22%) evaluable non-inflammatory cases.
Figure 3
Figure 3
Proportion of tumors exhibiting 0, 1+. 2+, or 3+ immunostaining for RhoA, RhoB, and RhoC.
Treatment Information
A total of 158 cycles of AC-tipifarnib combination were given. Thirty-five of 44 patients (80%) received all four cycles of the combination. Nine patients (20%) received less than four cycles of the AC-tipifarnib combination, including three patients who received one cycle, four who received two cycles, and two who received three cycles. Reasons for discontinuing the tipifarnib included gastrointestinal side effects of nausea, vomiting, and/or dyspepsia in five patients (11%), patient preference in two patients (5%), persistent neutropenia and thrombocytopenia in one patient (2%), and death due to pneumonitis in one patient (2%; described in next paragraph). The dose of AC was reduced in four patients (9%) due to toxicities including febrile neutropenia (N=2), thrombocytopenia (N=1), and anemia (N=1). Of 114 second or subsequent cycles of therapy given to 41 patients who received at least two treatment cycles of the AC-tipifarnib combination, all cycles were given on schedule in 30 patients (73%). Twelve treatment cycles were delayed one week or more in ten patients (24%) due to adverse events including three patients with grade 2 skin infection (7%) and one patient each with grade 4 chest pain resulting in hospitalization (2%), grade 1−2 thrombocytopenia (2%), grade 2 stomatitis (2%), grade 2 anemia (2%), febrile neutropenia (2%), and persistent sinus tachycardia (2%).
Overall Toxicity
The worst grade toxicity observed at the RPTD is shown in Table 3 (using the National Cancer Institute Common Terminology for Adverse Events, Version 3). Neutropenia and leukopenia were the most common grade 3−4 toxicities. Neutropenia occurred in 50% of all patients (including 32% who had grade 4 neutropenia). The duration of neutropenia was brief, however, resulting in only one patient (5%) developing grade 3 febrile neutropenia and a second patient (5%) developing grade 3 infection (cellulitis) unassociated with neutropenia. The incidence of grade 3 toxicity was 5% or less for all other categories.
Table 3
Table 3
Worst grade toxicity
With regard to other serious, unusual, or treatment-limiting toxicities, three patients (7%) had grade 3 gastrointestinal side effects (eg, nausea, vomiting, dyspepsia, gastritis) that prompted discontinuation of tipifarnib. One patient was hospitalized with grade 4 cardiac pain associated by dyspnea, migraine headache, and vomiting, which resolved spontaneously, with no cardiac or pulmonary etiology identified. Additionally, one patient was hospitalized and expired during cycle 1 due to pneumonitis associated with severe neutropenia. The patient had a several week history of cough and exertional dyspnea that was not reported to her treating physician. Physical exam prior to beginning therapy revealed bibasilar rales, and computerized tomography of the chest revealed bilateral pulmonary infiltrates. She developed rapidly progressive pulmonary symptoms several days after beginning therapy, developed acute respiratory distress syndrome in association with neutropenia, and died 8 days after initiating AC-tipifarnib.
Previous studies have shown that pathological complete response in the breast after preoperative chemotherapy correlates strongly with improved disease-free and overall survival, indicating that breast pCR may be a useful short-term surrogate for predicting improved long-term outcomes.28 Since most patients with locally advanced breast cancer require preoperative chemotherapy, and some patients with operable breast cancer may require preoperative therapy in order to facilitate breast conservation, these settings represent an appropriate model to determine whether the addition of targeted therapies enhance the effectiveness of standard cytotoxic therapy. We hypothesized that the addition of tipifarnib might enhance the effectiveness of standard AC chemotherapy, and designed this trial to determine whether the addition of tipifarnib improved the breast pCR rate from the approximately 10−15% historical rate to 25%.or higher28 The study design required observing at least 8 breast pCRs among 50 evaluable patients; we observed 11 breast pCRs among 44 (25%) evaluable patients, and therefore met our primary prespecified endpoint. Although it is possible that these improved results may be attributed in part to the dose-dense administration of AC, this seems unlikely given the particularly efficacy of the combination in ER-positive disease, a subset that has not been clearly shown to benefit from adjuvant dose-dense therapy.32 We also demonstrated that most tumors exhibited near complete inhibition of the arget enzyme, farnesyl transferase, when biopsied on the sixth and final day of tipifarnib therapy approximately two hours after the last 200 mg tipifarnib dose. Notably, there were variable changes in GGTase I enzyme activity, indicating a specific effect of tipifarnib on FTase. The inhibition of FTase was also associated with reduction in p-STAT3 expression in the majority of samples evaluated, although there were variable effects on other signaling molecules. STAT3 may be an important therapeutic target in breast cancer and other tumors, and STAT3 inhibition potentiates the cytotoxicity of doxorubicin. 33, 34
We also evaluated a panel of biomarkers in order to determine whether markers predictive of breast pCR could be identified in pretreatment tumor specimens. The markers evaluated included Ki-67, p27, p21, p-ERK, p-STAT3 (and total STAT3), p-AKT (and total AKT), RhoA, RhoB, and RhoC, all of which were chosen because of evidence that they might identify tumors sensitive to cytotoxic therapy and/or FTI inhibition. The only marker found to be predictive was Ki-67, which is known to reflect cellular proliferation, and which also is more likely to be elevated in ER-negative tumors. This is consistent with previous reports that an elevated Oncotype DX Recurrence Score predicted response to preoperative doxorubicin-containing chemotherapy, since proliferation genes comprise a significant component of the algorithm used to compute Recurrence Score. 35 It is also consistent with previous studies demonstrating higher breast pCR rates in ER-negative disease, as this phenotype is usually associated with higher expression of proliferation associated genes than ER-positive disease.30 It is notable that tipifarnib appeared to augment the breast CR rate in patients with ER-positive and ER-negative disease, although the trial was not adequately powered to establish this with certainty.
The incremental improvement in breast pCR associated with AC-tipifarnib combination is comparable to the effect of administering a longer duration of chemotherapy. For example, in the NSABP B27 trial, the breast pCR was significantly higher in patients treated with four cycles of AC followed by four cycles of docetaxel compared with four cycles of AC alone (27% vs. 13%). Although our results are encouraging, it is noteworthy that the NSABP B27 trial failed to demonstrate an improvement in disease-free survival or overall survival for the docetaxel arm despite an significant improvement in the breast pCR rate30, indicating that a greater improvement in breast pCR rates may be required. In addition, although a higher breast pCR rate was observed when docetaxel was used in ER-positive (14% vs. 6%) and ER-negative (23% vs. 14%), the breast pCR rate was substantially lower in the ER-positive subgroup in docetaxel treated patients (14% vs. 23%), which is consistent with adjuvant trials demonstrating relatively greater benefit from adjuvant taxane therapy in ER-negative disease.36 In contrast to the modest improvement in breast pCR rate observed in B27, a randomized phase II trial comparing preoperative chemotherapy used alone or in combination with trastuzumab in Her2/neu positive breast cancer demonstrated significantly higher pCR rate in the trastuzumab containing arm (65% vs. 25%)37, which was consistent with several trials demonstrating a 50% reduction in the risk of recurrence associated with adjuvant postoperative trastuzumab. 38-40 Taken together, these finding suggest that if breast pCR rate is to be utilized as a short term surrogate to screen for promising strategies to subsequently test in phase III adjuvant or neoadjuvant trials, that the target breast pCR rates should be substantially higher than the 25% rate observed in the B27 trial, and that different thresholds may required for different phenotypic subsets (ie, HR-positive vs. HR-negative disease).
In conclusion, we found that the combination of dose-dense doxorubicin-cyclophosphamide (AC) was feasible and tolerable when combined with tipifarnib given orally at a dose of 200 mg twice daily for six days following each chemotherapy dose. We also found that tipifarnib inhibited tumor FTase in vivo, inhibited p-STAT3 activation in most patients, and produced a significantly higher breast pCR rate than expected for chemotherapy alone. Based upon these encouraging results, we have initiated a second trial combining tipifarnib plus paclitaxel followed sequentially by tipifarnib plus dose-dense AC chemotherapy (ClinicalTrials.Gov identifier NCT00470301). Addition of tipifarnib to the paclitaxel component of sequential dose dense therapy represents a logical continuation of our previous effort for two reasons. Firstly, compared with every 3 week paclitaxel, weekly paclitaxel has been shown to produce higher breast pCR rates when given preoperatively, and improved overall survival when given postoperatively, irrespective hormone receptor expression.41 42 Second, preclinical evidence suggests that FTIs synergistically augment the effect of anti-tubulin agents such as paclitaxel.43-46 This trial may establish whether FTIs such as tipifarnib merit further evaluation in definitive, large-scale phase III adjuvant trials
Acknowledgements
Supported by United States Department of Health and Human Service contract N01-CM-62204 (P.I. Joseph A. Sparano, MD) and grant RO1CA98473 (P.I. Said Sebti, PhD) Presented at the 2007 Breast Cancer Symposium and the 2007 San Antonio Breast Cancer Symposium.
Footnotes
Translational Relevance
Oncogenic mutations human ras genes are found in 30% of all human cancers and are associated with activation of the Ras pathway, which led to the development of farnesyl transferae inhibitors (FTIs) that were developed to inhibit this pathway, and have been shown to have antiproliferative effects in cell lines and human tumor with both mutated and wild type Ras genes. In this trial, we demonstrate evidence that the FTI tipifarnib inhibits farnesyltranserase enzyme activity in vivo in the primary breast cancers (which are only rarely associated with Ras mutations), suppresses p-STAT3 expression, and enhances the clinical efficacy of doxorubicin-cyclophosphamide when given preoperatively. Our findings suggest that this is a promising therapeutic strategy for adjuvant therapy in early stage disease, and also for the treatment of advanced disease.
1. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001;81:153–208. [PubMed]
2. Rochlitz CF, Scott GK, Dodson JM, et al. Incidence of activating ras oncogene mutations associated with primary and metastatic human breast cancer. Cancer Res. 1989;49:357–60. [PubMed]
3. Thor A, Ohuchi N, Hand PH, et al. ras gene alterations and enhanced levels of ras p21 expression in a spectrum of benign and malignant human mammary tissues. Lab Invest. 1986;55:603–15. [PubMed]
4. Smith CA, Pollice AA, Gu LP, et al. Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas. Clin Cancer Res. 2000;6:112–26. [PubMed]
5. Bunone G, Briand PA, Miksicek RJ, Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. Embo J. 1996;15:2174–83. [PubMed]
6. Kato S, Masuhiro Y, Watanabe M, et al. Molecular mechanism of a cross-talk between oestrogen and growth factor signalling pathways. Genes Cells. 2000;5:593–601. [PubMed]
7. Theillet C, Lidereau R, Escot C, et al. Loss of a c-H-ras-1 allele and aggressive human primary breast carcinomas. Cancer Res. 1986;46:4776–81. [PubMed]
8. Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD. Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol. 2002;160:579–84. [PubMed]
9. van Golen KL, Bao L, DiVito MM, Wu Z, Prendergast GC, Merajver SD. Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor. Mol Cancer Ther. 2002;1:575–83. [PubMed]
10. Zhu K, Hamilton AD, Sebti SM. Farnesyltransferase inhibitors as anticancer agents: current status. Curr Opin Investig Drugs. 2003;4:1428–35. [PubMed]
11. Crespo NC, Ohkanda J, Yen TJ, Hamilton AD, Sebti SM. The farnesyltransferase inhibitor, FTI-2153, blocks bipolar spindle formation and chromosome alignment and causes prometaphase accumulation during mitosis of human lung cancer cells. J Biol Chem. 2001;276:16161–7. [PubMed]
12. Crespo NC, Delarue F, Ohkanda J, Carrico D, Hamilton AD, Sebti SM. The farnesyltransferase inhibitor, FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status. Cell Death Differ. 2002;9:702–9. [PubMed]
13. Ashar HR, James L, Gray K, et al. The farnesyl transferase inhibitor SCH 66336 induces a G(2) --> M or G(1) pause in sensitive human tumor cell lines. Exp Cell Res. 2001;262:17–27. [PubMed]
14. Sepp-Lorenzino L, Rosen N. A farnesyl-protein transferase inhibitor induces p21 expression and G1 block in p53 wild type tumor cells. J Biol Chem. 1998;273:20243–51. [PubMed]
15. Le Gouill S, Pellat-Deceunynck C, Harousseau JL, et al. Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells. Leukemia. 2002;16:1664–7. [PubMed]
16. Han JY, Oh SH, Morgillo F, et al. Hypoxia-inducible factor 1alpha and antiangiogenic activity of farnesyltransferase inhibitor SCH66336 in human aerodigestive tract cancer. J Natl Cancer Inst. 2005;97:1272–86. [PubMed]
17. Kelland LR, Smith V, Valenti M, et al. Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer. Clin Cancer Res. 2001;7:3544–50. [PubMed]
18. Sun J, Ohkanda J, Coppola D, et al. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. Cancer Res. 2003;63:8922–9. [PubMed]
19. Kohl NE, Omer CA, Conner MW, et al. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med. 1995;1:792–7. [PubMed]
20. Weinstein-Oppenheimer CR, Henriquez-Roldan CF, Davis JM, et al. Role of the Raf signal transduction cascade in the in vitro resistance to the anticancer drug doxorubicin. Clin Cancer Res. 2001;7:2898–2907. [PubMed]
21. Rasouli-Nia A, Liu D, Perdue S, Britten RA. High Raf-1 kinase activity protects human tumor cells against paclitaxel-induced cytotoxicity. Clin Cancer Res. 1998;4:1111–6. [PubMed]
22. Cornwell MM, Smith DE. A signal transduction pathway for activation of the mdr1 promoter involves the proto-oncogene c-raf kinase. J Biol Chem. 1993;268:15347–50. [PubMed]
23. Johnston SR, Hickish T, Ellis P, et al. Phase II study of the efficacy and tolerability of two dosing regimens of the farnesyl transferase inhibitor, R115777, in advanced breast cancer. J Clin Oncol. 2003;21:2492–9. [PubMed]
24. Sparano JA, Moulder S, Kazi A, et al. Targeted inhibition of farnesyltransferase in locally advanced breast cancer: a phase I and II trial of tipifarnib plus dose-dense doxorubicin and cyclophosphamide. J Clin Oncol. 2006;24:3013–8. [PubMed]
25. Symmans WF, Peintinger F, Hatzis C, et al. Measurement of residual breast cancer burden to predict survival after neoadjuvant chemotherapy. J Clin Oncol. 2007;25:4414–22. [PubMed]
26. Rouzier R, Pusztai L, Delaloge S, et al. Nomograms to predict pathologic complete response and metastasis-free survival after preoperative chemotherapy for breast cancer. J Clin Oncol. 2005;23:8331–9. [PubMed]
27. Kleer CG, Teknos TN, Islam M, et al. RhoC GTPase expression as a potential marker of lymph node metastasis in squamous cell carcinomas of the head and neck. Clin Cancer Res. 2006;12:4485–90. [PubMed]
28. Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol. 1998;16:2672–85. [PubMed]
29. Bear HD, Anderson S, Smith RE, et al. Sequential preoperative or postoperative docetaxel added to preoperative doxorubicin plus cyclophosphamide for operable breast cancer:National Surgical Adjuvant Breast and Bowel Project Protocol B-27. J Clin Oncol. 2006;24:2019–27. [PubMed]
30. Bear HD, Anderson S, Brown A, et al. The effect on tumor response of adding sequential preoperative docetaxel to preoperative doxorubicin and cyclophosphamide: preliminary results from National Surgical Adjuvant Breast and Bowel Project Protocol B-27. J Clin Oncol. 2003;21:4165–74. [PubMed]
31. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–16. [PubMed]
32. Citron ML, Berry DA, Cirrincione C, et al. Randomized trial of dose-dense versus conventionally scheduled and sequential versus concurrent combination chemotherapy as postoperative adjuvant treatment of node-positive primary breast cancer: first report of Intergroup Trial C9741/Cancer and Leukemia Group B Trial 9741. J Clin Oncol. 2003;21:1431–9. [PubMed]
33. Gariboldi MB, Ravizza R, Molteni R, Osella D, Gabano E, Monti E. Inhibition of Stat3 increases doxorubicin sensitivity in a human metastatic breast cancer cell line. Cancer Lett. 2007;258:181–8. [PubMed]
34. Timofeeva OA, Gaponenko V, Lockett SJ, et al. Rationally designed inhibitors identify STAT3 N-domain as a promising anticancer drug target. ACS Chem Biol. 2007;2:799–809. [PubMed]
35. Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol. 2005;23:7265–77. [PubMed]
36. Berry DA, Cirrincione C, Henderson IC, et al. Estrogen-receptor status and outcomes of modern chemotherapy for patients with node-positive breast cancer. Jama. 2006;295:1658–67. [PMC free article] [PubMed]
37. Buzdar AU, Ibrahim NK, Francis D, et al. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol. 2005;23:3676–85. [PubMed]
38. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005;353:1659–72. [PubMed]
39. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005;353:1673–84. [PubMed]
40. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, et al. Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer. N Engl J Med. 2006;354:809–20. [PubMed]
41. Green MC, Buzdar AU, Smith T, et al. Weekly paclitaxel improves pathologic complete remission in operable breast cancer when compared with paclitaxel once every 3 weeks. J Clin Oncol. 2005;23:5983–92. [PubMed]
42. Sparano JA, Wang M, Martino S, et al. Phase III study of doxorubicin-cyclophosphamide followed by paclitaxel or docetaxel given every 3 weeks or weekly in patients with axillary node-positive or high-risk node-negative breast cancer: results of North American Breast Cancer Intergroup Trial E1199. J Clin Oncol (Meeting Abstracts) 2007;25:6s. abstract 516.
43. Marcus AI, Zhou J, O'Brate A, et al. The synergistic combination of the farnesyl transferase inhibitor lonafarnib and paclitaxel enhances tubulin acetylation and requires a functional tubulin deacetylase. Cancer Res. 2005;65:3883–93. [PMC free article] [PubMed]
44. Loprevite M, Favoni RE, De Cupis A, et al. In vitro study of farnesyltransferase inhibitor SCH 66336, in combination with chemotherapy and radiation, in non-small cell lung cancer cell lines. Oncol Rep. 2004;11:407–14. [PubMed]
45. Wang EJ, Johnson WW. The farnesyl protein transferase inhibitor lonafarnib (SCH66336) is an inhibitor of multidrug resistance proteins 1 and 2. Chemotherapy. 2003;49:303–8. [PubMed]
46. Jin W, Wu L, Liang K, Liu B, Lu Y, Fan Z. Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells. Br J Cancer. 2003;89:185–91. [PMC free article] [PubMed]