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Clinical Cancer Research Vol. 8, 1057-1064, May 2002
© 2002 American Association for Cancer Research

Clinical Trials

A Phase I/Pilot Study of Sequential Doxorubicin/Vinorelbine

Effects on p53 and Microtubule-associated Protein 41

Judy Bash-Babula2, Deborah Toppmeyer2, Marie Labassi, Janice Reidy, Michelle Orlick, Rachelle Senzon, Elizabeth Alli, Thomas Kearney, David August, Weichung Shih, Jin-Ming Yang and William N. Hait3

Departments of Medicine [D. T., W. N. H.], Pharmacology [E. A., J-M. Y., W. N. H.], Biostatistics [W. S.], and Surgical Oncology [T. K., D. A.], University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School and The Cancer Institute of New Jersey [J. B-B., D. T., M. L., J. R., M. O., R. S., E. A., T. K., D. A., W. S., J-M. Y., W. N. H.], New Brunswick, New Jersey 08901


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Few molecular determinants of sensitivity to cancer chemotherapy exist. In experimental systems, p53 regulates the sensitivity to antimicrotubule drugs through its effect on microtubule-associated protein 4 (MAP4). MAP4 is the major microtubule-associated protein in nonneuronal tissues and promotes microtubule polymerization. We reported that wild-type p53 induction by doxorubicin in C127 breast cancer cells repressed MAP4, decreased microtubule polymerization, and increased Vinca alkaloid sensitivity. The goals of this Phase I/pilot clinical trial were to determine: (a) the safety of delivering a DNA-damaging agent (doxorubicin) followed in sequence by treatment with an antimicrotubule drug (vinorelbine); and (b) the feasibility of detecting activation of p53 and repression of MAP4 in patients’ tissues.

Experimental Design: Peripheral blood mononuclear cells (PBMNCs) and tumor were obtained from 16 women with locally advanced (stage IIIb) or metastatic (stage IV) breast cancer before doxorubicin treatment and immediately before treatment with vinorelbine 24 or 48 h later.

Results: After doxorubicin treatment, p53 increased in 12 of 14 PBMNC and 4 of 10 tumor samples. Changes in MAP4 were variable; however, in samples in which p53 was induced, MAP4 decreased in 7 of 12 PBMNC and 3 of 4 breast cancer specimens. Immunohistochemistry confirmed lower MAP4 expression in tumor cells after doxorubicin treatment. Seven of 16 patients had a partial response, and treatment was well tolerated.

Conclusions: These data demonstrate the ability to detect the activation of p53 and the repression of MAP4 in normal and malignant tissues in patients treated with a DNA-damaging agent, and that an antimicrotubule drug can be administered safely at a time when cells may be more sensitive to treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The choice of cancer chemotherapy is often empirical, based more on the histological appearance of the tumor than on an understanding of the molecular determinants of drug sensitivity. In patients with metastatic breast cancer, the complete response rate is <20%, the median duration of response is 9 months, and the median survival is 12–24 months. Similarly, the prognosis for inflammatory or locally advanced breast cancer is dismal, with <50% of diagnosed patients alive at 5 years (1) . Yet virtually all patients treated with chemotherapy suffer significant side effects. Therefore, we have focused on defining molecular markers of drug sensitivity to help identify those patients who are most likely to respond to therapy and to develop more effective therapeutic strategies.

p53 can regulate the fate of cells injured by radiation and chemotherapy through its effects on cell cycle kinetics and programmed cell death (2 , 3) . In response to DNA damage, wtp534 protein is stabilized and transcriptionally activates a series of genes having p53-responsive elements in their promoter regions (4, 5, 6) . p53 can also repress transcription through an indirect mechanism requiring the cooperation between mSin3a, p53, and histone deacetylase 1 (7) . Several p53-repressible genes have been identified including the antiapoptotic protein bcl-2, which may account for the propensity of cells with wtp53 to undergo apoptosis in response to DNA damage (3 , 8) . Murphy et al. (7 , 9) demonstrated that MAP4 is repressed by wtp53. MAP4 is the major microtubule-associated protein in nonneuronal tissues and promotes polymerization of microtubules through binding to the COOH termini of {alpha}- and ß-tubulin (10) . We postulated and demonstrated that repression of MAP4 by wtp53 increased the sensitivity to Vinca alkaloids by favoring the binding of these drugs to the greater mass of depolymerized tubulin heterodimers. In these experiments, cells in which DNA was damaged with irradiation, bleomycin, or doxorubicin increased wtp53, decreased MAP4, decreased tubulin polymerization, and increased binding of and sensitivity to Vinca alkaloids (11) .

Because p53 is wild type in ~50% of breast cancers (12, 13, 14) , we sought to determine whether patients with breast cancer treated with doxorubicin, a DNA-damaging drug, would also demonstrate induction of p53 and repression of MAP4, thereby creating a background of enhanced sensitivity to vinorelbine, a Vinca alkaloid used commonly in the treatment of this disease (15, 16, 17) . We administered doxorubicin to patients with locally advanced or metastatic breast cancer and studied the effects on p53 and MAP4 expression in PBMNCs and in breast cancer biopsies. In addition, we studied whether it would be safe to administer vinorelbine either 24 or 48 h after doxorubicin and whether there would be detectable changes in p53 activation and MAP4 reduction during the postdoxorubicin interval.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Eligibility Criteria
Patients with histologically confirmed metastatic (stage IV) or locally advanced (stage IIIb) breast cancer without prior chemotherapy other than in the adjuvant setting were eligible for study. Eligible patients were 18 years of age or older, had an Eastern Cooperative Oncology Group performance status of 0–2, had a life expectancy of >12 weeks, and provided written informed consent in accordance with institutional review board guidelines. Patients who had received doxorubicin previously were eligible if the total dose had not exceeded 300 mg/m2. Prior hormonal therapy and localized radiotherapy for metastatic disease were permitted but must have been completed 3 weeks before entry. Measurable disease was defined as follows: (a) any lesion with two measurable perpendicular diameters; and (b) computed axial tomography demonstrating defects >1.5 cm in diameter. Stage IIIb disease was assessed by measuring skin changes as well as tumor size. All patients were required to have adequate renal, hepatic, bone marrow, and cardiac function. Pretreatment evaluation performed within 2 weeks of initiation of therapy included a complete history and physical examination, complete blood cell count, platelet count, serum chemistries, chest X-ray, electrocardiogram, site-specific imaging, and tumor measurements.

Patients were ineligible if they had a past history of cancer with the exception of nonmelanoma skin cancer or treated in situ cervical carcinoma. Other exclusions included evidence of visceral crisis (lymphangitic pulmonary metastases or liver or marrow replacement sufficient to cause significant dysfunction) or a pre-existing medical condition that would preclude the safe administration of chemotherapeutic drugs.

Treatment Plan
Patients received doxorubicin (40 mg/m2) on day 1 and vinorelbine (20 mg/m2) on days 2 and 9 (odd-number enrollees) or on days 3 and 10 (even-number enrollees). If only one of the first six patients experienced a dose-limiting toxicity, the dose was increased to 50 mg/m2 doxorubicin and 25 mg/m2 vinorelbine, which were the Phase II doses established previously. The second cycle began on day 22. In patients with stage IIIb breast cancer, doxorubicin and vinorelbine were administered to maximal response defined as stability of disease for two cycles after an initial response. Patients with stage IV disease received doxorubicin and vinorelbine until progression. A multigated acquisition scan to assess left ventricular cardiac ejection fraction was obtained before treatment and after a total dose of 400 mg/m2 of doxorubicin. PBMNCs and biopsies of accessible tumor tissue were obtained before treatment and 24 or 48 h after doxorubicin, i.e., immediately before receiving vinorelbine.

Response and Toxicity Assessment
All treated patients with measurable disease were assessed for response. Tumor measurements were done every cycle by physical examination and every other cycle by tumor imaging. Responses were scored according to WHO criteria. Stable disease was defined as a reduction of 50% and increase of 25% in the sum of the products of the perpendicular diameters of all measurable lesions, for longer than 8 weeks, during which time no new lesions appear. Physical examination and serum chemistries were repeated before each cycle, and a complete blood cell count was obtained weekly. Toxicity was assessed using the National Cancer Institute Common Toxicity Criteria. Dose-limiting toxicity was defined as either: (a) <500 neutrophils/µl or <25,000 platelets/µl for >5 days or febrile neutropenia; or (b) irreversible grade 2 or any grade 3–5 nonhematological toxicity.

Evaluation of MAP4, p53, and p21/WAF1
PBMNCs were isolated from 8 ml of heparinized blood by Ficoll-Hypaque gradient centrifugation using Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ). Cells were washed with PBS by centrifugation at 700 x g and lysed in radioimmunoprecipitation assay buffer [10 mM sodium phosphate (pH 7.2), 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% aprotinin, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 50 µg/ml leupeptin]. Protein was measured by the method of Bradford (Ref. 18 ; Bio-Rad, Hercules, CA). Proteins (30 µg) were resolved by 7.5 and 15% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The expression of MAP4, p53, and p21/WAF1 was assayed by Western blotting using the following monoclonal antibodies: MAP4, clone 18 (Transduction Laboratories, Lexington, KY); p53, clone DO-7 (Dako Corp., Carpinteria, CA); and p21/WAF1, clone EA10 (Calbiochem, San Diego, CA). ß-Actin was used to control for protein loading and transfer using clone AC-15 (Sigma Chemical Co., St. Louis, MO). A 1:1000 dilution of horseradish peroxidase-linked mouse IgGs (Dako) in PBST (PBS, 0.05% Tween 20) plus 1% nonfat milk was used as the secondary antibody in an enhanced chemiluminescence system (Amersham, Piscataway, NJ). Laboratory personnel were blinded to the results of treatment.

Core needle biopsies were used to obtain tumor tissue using a 14-gauge, spring-loaded biopsy gun (Bard, Inc., Covington, GA) after the area was cleaned with povidone-iodine and anesthetized with 1% lidocaine. Three to four cores were harvested at each time point to account for tumor heterogeneity. Skin lesions were biopsied using a 4-mm punch. One portion of each biopsy was embedded in paraffin; a second portion of the biopsy was immediately frozen in liquid nitrogen for Western blots. This portion was homogenized on ice in 20 mM HEPES (pH 7.4), 100 mM NaCl, 20 mM sodium pyrophosphate, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.1 mM sodium orthovanadate with a Dounce homogenizer. Protein was quantified by the Bradford assay, subjected to 7.5 and 15% SDS-PAGE gels, and transferred to nitrocellulose for Western blot analysis of MAP4, p53, p21/WAF1, and ß-actin as described above. Intensity of staining on Western blots was scored by densitometry; a 1.5-fold change in intensity of the bands, normalized to ß-actin, was defined as significant.

Immunohistochemistry
The monoclonal p53 antibody DO-7 (Dako) was used for immunohistochemistry as reported previously by our laboratory (19) . Staining for MAP4 was performed using the anti-MAP4 antibody clone 18 (Transduction Laboratories). The anti-p53 antibody was diluted 1:10 in 1% BSA-PBS, and anti-MAP4 antibody clone 18 was diluted 1:2000 in 1% BSA-PBS. Staining with mouse ascites fluid was used as a negative control. Staining with H&E was performed as described previously (19) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We carried out a Phase I/pilot clinical study to determine the safety of delivering a DNA-damaging agent (doxorubicin) followed in sequence with an antimicrotubule drug (vinorelbine) and the feasibility of detecting activation of p53 and repression of MAP4 in patients’ tissues. Patients received doxorubicin on day 1 and vinorelbine either 24 or 48 h later.

Patient Characteristics.
The characteristics of the 16 enrolled patients are listed in Table 1Citation . Six of 16 patients had received adjuvant chemotherapy, and two had received hormonal therapy for metastatic disease. There was a nearly equal number of patients with locally advanced (stage IIIb) and metastatic (stage IV) disease. All patients had an Eastern Cooperative Oncology Group performance status of 0–1. Four of 16 patients had involvement of the viscera as the dominant site of disease. A total of 82 cycles of treatment were delivered with a range of 2–10 per patient. Doses of doxorubicin and vinorelbine were escalated in this pilot study to the Phase II doses defined previously (16) in the last 10 patients after the first six patients were enrolled, because only one of six patients developed a dose-limiting toxicity (febrile neutropenia).


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  		Table 1 Patient characteristics

 
Induction of p53 and Repression of MAP4.
To determine whether treatment with i.v. doxorubicin increased p53 in patients, tumor and PBMNCs were assayed before and 24 or 48 h after receiving doxorubicin. As shown in Figs. 1Citation and 2Citation , we found an induction of p53 in the PBMNCs of 12 of 14 patients (95% CI, 68–100%) and in 4 of 10 breast cancer biopsies (95% CI, 10–50%) after doxorubicin treatment. In addition, we detected increased expression of p21/WAF1, a transactivation target for wtp53 (20) , in 7 of 12 PBMNCs (95% CI, 30–86%) and in 4 of 4 tumors (95% CI, 90–100%) in which p53 was induced.



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  		Fig. 1. Western blot of MAP4, p53, and p21 expression in PBMNCs before and after doxorubicin (post dox). Equal amounts of protein (30 µg) were loaded onto 7.5 and 15% SDS-PAGE gels, and proteins were detected by enhanced chemiluminescence as described in "Patients and Methods." Dashed-line boxes, patients with increased p53 after doxorubicin. Black boxes, patients with both increased p53 and decreased MAP4 after doxorubicin as determined by densitometry normalized to actin. Blood was not available from patient 8, and insufficient material precluded analysis of p53 and p21 in patient 1. The blots are representative of two experiments.

 


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  		Fig. 2. Western blot of MAP4, p53, and p21 expression in tumor samples before and after doxorubicin (post dox). Equal amounts of protein (30 µg) were loaded onto 7.5 and 15% SDS-PAGE gels, and proteins were detected by enhanced chemiluminescence as described in "Patients and Methods." Dashed-line boxes, patients with increased p53 after doxorubicin. Black boxes, patients with both increased p53 and decreased MAP4 after doxorubicin as determined by densitometry normalized to actin. The blots are representative of two experiments.

 
We next asked whether the induction of p53 after DNA damage with doxorubicin led to repression of MAP4. As shown in Fig. 1Citation , we observed repression of MAP4 in PBMNCs from 7 of 12 patients in which p53 was induced (95% CI, 30–86%). Additionally, we found repression of MAP4 in 3 of 4 breast biopsies (95% CI, 33–100%) in which p53 was induced (Fig. 2)Citation . The majority of patients showing both increased p53 and repressed MAP4 also had increased expression of p21/WAF1 [5 of 7 PBMNC samples (95% CI, 38–100%) and 3 of 3 tumor specimens (95% CI, 89–100%)]. In two patients who provided blood and tumor samples at both 24 and 48 h (patients 2 and 3, respectively), we found maximal induction of p53 at 24 h and maximal repression of MAP4 at 48 h (Figs. 1Citation and 2Citation ). However, not all patients exhibited the anticipated changes in protein expression after treatment, e.g., patient 5 demonstrated no change in p53 in tumor, whereas patient 15 showed decreased p53 expression in PBMNCs and MAP4 repression. In addition, increased MAP4 was observed in four PBMNCs and one tumor biopsy after doxorubicin treatment.

Immunohistochemistry.
To confirm that changes seen on Western blots represented changes in cancer cells rather than contaminating stroma, we analyzed p53 and MAP4 expression by immunohistochemistry in three patients. Fig. 3Citation demonstrates increased expression of p53 at 24 h and repression of MAP4 expression at 48 h after doxorubicin treatment in the tumor cells of a representative patient (no. 3). H&E staining revealed the presence of infiltrating lobular carcinoma (Fig. 3, A, C, E, and G)Citation . In agreement with the short half-life of p53 of ~30 min in undamaged cells (21) , staining for p53 was weak in untreated cancer cells (Fig. 3B)Citation , whereas MAP4 stained strongly positive in the cytoplasm of untreated cancer cells (Fig. 3F)Citation . After doxorubicin treatment, staining for p53 increased in the nucleus (Fig. 3D)Citation , whereas cytoplasmic MAP4 decreased in tumor cells (Fig. 3H)Citation . Control staining with mouse ascites fluid was negative. Similar results were obtained in two other patients (patients 10 and 14, data not shown).



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  		Fig. 3. Immunohistochemical staining of p53 and MAP4 in breast tumor tissue. Patient 3 tumor samples were either untreated (A and B) and 24 h after doxorubicin treatment (C and D), or untreated (E and F) and 48 h after doxorubicin treatment (G and H). Tissues were stained with H&E (A, C, E, and G) or immunostained with p53 (B and D) and MAP4 antibodies (F and H) as described in "Patients and Methods." Tissue was counterstained with toluidine blue and photographed at x60. Arrows, tumor cells. The photomicrographs are representative of three patients.

 
Toxicities.
The number of patients experiencing toxicities as defined by National Cancer Institute criteria from the 82 cycles of sequential doxorubicin and vinorelbine are listed in Table 2Citation . There were no significant differences in toxicities between the 24- and 48-h sequences; therefore, these data were pooled. Neutropenia was the most common grade 3 and grade 4 toxicity; febrile neutropenia was the most common grade 3 toxicity. Two patients without previous anthracycline exposure developed congestive heart failure at cumulative doxorubicin doses of 350 and 400 mg/m2. One of these patients entered the study with a baseline ejection fraction of 50%. Four patients experienced grade 3–4 fatigue; one of these patients required a 1-week delay in treatment. Three patients developed a thromboembolic event. One patient was treated for an asymptomatic pulmonary embolism diagnosed on a restaging computed axial tomography scan. Two additional patients developed a deep venous thrombosis with one complicated further by a pulmonary embolism. Twenty-six episodes of mild (grade 1–2) nausea and vomiting were observed. Only two episodes of grade 3 nausea and vomiting were reported. One patient developed grade 2 mucositis that prevented the administration of vinorelbine on day 9 in two of four cycles. There was no significant neuropathy.


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  		Table 2 Incidence and grade of toxicities

 
Responses.
In the current pilot trial, 7 of 16 patients had a partial response to treatment (Table 3)Citation . There were no complete responses. Two patients progressed while on treatment; four of eight patients with decreased MAP4 after doxorubicin responded to treatment, three had disease stabilization, and one progressed.


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  		Table 3 Response data as a function of MAP4 expression in tumor biopsies

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The treatment of breast cancer improved when molecular markers were identified that would determine an optimal treatment plan and predict response, e.g., the ability to measure estrogen and progesterone receptors identified a patient population more likely to respond to hormonal therapies such as tamoxifen (22) . Similarly, the measurement of HER-2/neu overexpression identified a subset of patients more likely to respond to trastuzumab (Herceptin; Ref. 23 ). Yet, we have found few markers of sensitivity to most chemotherapeutic agents.

In preclinical studies, we found that induction of wtp53 after DNA damage by doxorubicin predicted sensitivity to subsequent treatment with Vinca alkaloids (11) . This response was attributed to the ability of p53 to transcriptionally repress MAP4 (7 , 9 , 24) , a protein that regulates the polymerization state of microtubules (25, 26, 27) . These results raised important opportunities for the choice and sequence of chemotherapeutic drugs in the clinic. Therefore, we have begun to translate our laboratory observations into the more complex clinical setting by administering doxorubicin, a drug that damages DNA (through its effects on topoisomerase II, generation of free radicals, and creating structural changes that interfere with DNA and RNA synthesis; Ref. 28 ), followed by the Vinca alkaloid, vinorelbine.

Doxorubicin produces single- and double-strand DNA breaks through poisoning of topoisomerase II (28) . Although the mechanism by which DNA damage leads to increased p53 protein stability has yet to be elucidated fully, it is known that phosphorylation of p53 by several kinases (e.g., ATM, DNA-dependent protein kinase) diminishes the interaction of p53 with MDM2, a protein that promotes p53 degradation (5 , 6 , 29 , 30) . In this Phase I/pilot clinical study, we determined the safety of sequencing doxorubicin followed by vinorelbine and whether treatment of patients with doxorubicin activated p53 in blood and tumor. Figs. 1Citation and 2Citation demonstrate that standard doses of doxorubicin increase p53 in both PBMNCs (12 of 14 patients) and breast cancer biopsies (4 of 10 patients). These percentages are consistent with the expectation that PBMNCs contain wtp53 and show increased p53 protein stability in the majority of samples and that only 50% of breast cancers retain wtp53 (12, 13, 14) . This is in general agreement with our finding that only 40% of the tumor samples demonstrated increased p53 protein stability after treatment with doxorubicin. To our knowledge, this is the first time p53 in PBMNCs was measured in patients with cancer before and after doxorubicin treatment. The detection of p53 in untreated blood cells is not unexpected because low levels of wtp53 protein are often detectable in cell lines grown in vitro (11 , 31) .

We also determined whether doses of doxorubicin that increased p53 could also result in repression of MAP4. Figs. 1Citation and 2Citation demonstrate that the induction of p53 led to the repression of MAP4 in the majority of PBMNCs (7 of 12 patients) and breast cancer biopsies (3 of 4 patients). Immunohistochemistry verified that the repression of MAP4 after treatment is found in tumor cells rather than surrounding stroma (Fig. 3)Citation . Consistent with these observations, the majority of patients showing both increased p53 and repressed MAP4 also increased expression of p21 (5 of 7 PBMNC samples and 3 of 3 tumor specimens). Although the presence of tumor with wtp53 was not a requisite for this pilot study, the increased p21 protein expression in tumor tissue after doxorubicin treatment most likely reflects a wtp53 genotype because doxorubicin treatment fails to induce p21 in the presence of mutant p53 in cells and breast tumors (32 , 33) . Our finding that p21 increased in 4 of 4 tumor samples in which p53 was increased agrees with this assumption.

Not all patients exhibited the anticipated changes in response to doxorubicin, e.g., four patients with induced p53 in PBMNC samples after doxorubicin had increased rather than decreased expression of MAP4 (patients 4, 12, 13, and 14). Furthermore, MAP4 repression was observed in five tumor samples in which we failed to detect induction of p53 (patients 5, 6, 9, 13, and 16). There are several possible explanations for these results:

(a) The failure to detect increased p53 in certain specimens may be attributable to the timing of sampling, i.e., the transient induction of p53 at an earlier time point may have been missed (29 , 30) . Furthermore, repression of genes such as MAP4 is dependent on both the amount of p53 induced and the duration of time that p53 remains elevated in the target tissue (34) .

(b) The response of p53 to DNA damage may differ in normal cells from that of cancer cells. To date, the relationship between p53 and MAP4 has been studied in cycling immortal or transformed tumor cells (9 , 11 , 24) , whereas PBMNCs are terminally differentiated, quiescent cells. In this regard, Siu et al. (35) reported that quiescent Swiss 3T3 cells had lower levels of p53 and p21 in response to doxorubicin and had longer survival than proliferating cells.

(c) The failure to detect the induction of p53 or repression of MAP4 may also be attributable to the drug concentration and extent of DNA damage at the time of sampling. However, we found that treatment of PBMNCs in vitro with concentrations of doxorubicin equal to those achieved in patients (36) was able to induce p53 and p21 and repress MAP4 in the majority of but not all cases.5

(d) Differences in proteins that degrade p53 such as MDM2 could account for interpatient variations. In addition, repression of MAP4 by p53 can be blocked by cellular proteins that can be differentially expressed such as bcl-2 and WT-1 (9 , 37 , 38) . However, we have not found changes in MDM-2 and bcl-2 to correlate with the alterations in p53 or MAP4 in this study (data not shown).

In this study, we also compared 24 and 48 h after doxorubicin for the injection of vinorelbine based on preclinical models demonstrating maximal repression of MAP4 during this time period (11) . Although all patients agreed to two biopsies of their tumors (i.e., before and after doxorubicin treatment), it was far more difficult to get patients to volunteer for a third biopsy (i.e., before and 24 and 48 h after treatment). In two patients who provided blood and tumor samples before treatment and at both 24 and 48 h after treatment (patients 2 and 3, respectively), the induction of p53 peaked at 24 h, and the repression of MAP4 was most pronounced at 48 h (Figs. 1Citation and 2Citation ). This result is consistent with the long half-life of MAP4 and the negative feedback on p53 by MDM2 (9 , 29 , 30) .

The induction of p53 and repression of MAP4 in normal tissues could potentially increase the toxicity of Vinca alkaloids, thereby limiting the therapeutic index of this combination. Anticipating increased toxicity with sequential versus combined treatment, the first six patients were treated with 40 and 20 mg/m2 of doxorubicin and vinorelbine, respectively. Doses were escalated to the Phase II established maximum tolerated dose of the doxorubicin/vinorelbine combination of 50 and 25 mg/m2 in subsequently enrolled patients after only one of the first six patients experienced significant toxicity. The toxicities seen with the sequential use of doxorubicin and vinorelbine do not appear to be in excess of those seen when doxorubicin and vinorelbine are given together (15, 16, 17) . As was observed in Phase II studies reported previously, febrile neutropenia and neutropenia were the most common grade 3 and grade 4 toxicities, respectively (15, 16, 17) . The incidence of cardiac dysfunction observed in this study (12.5%) was higher than anticipated. Anthracycline-induced cardiotoxicity is dose dependent and cumulative (39) and is unusual at a cumulative dose of <450 mg/m in the absence of other risk factors (40) . The two patients who developed congestive heart failure were at cumulative doses of doxorubicin at 350 and 400 mg/m2. Both had a history of hypertension and were >65 years of age. One of the two patients entered the study with a baseline ejection fraction of 50%, and her ejection fraction obtained at 300 mg/m2 was unchanged from baseline. The occurrence of thromboembolic events in three patients is consistent with the 2–18% incidence in patients with advanced-stage cancers treated with chemotherapy (41) .

The responses to sequential doxorubicin and vinorelbine in this trial (44%) were in the range for those reported previously for this combination. This was observed despite the lower dose of doxorubicin and vinorelbine used to treat the first six patients in comparison with the established Phase II doses used in the subsequent 10 patients (15, 16, 17) . Six of 10 patients treated with the established Phase II doses responded. The results of this pilot Phase I study will be used to investigate whether the response rate to sequential doxorubicin/vinorelbine is enhanced in patients with p53 induction and MAP4 repression.

In summary, this trial demonstrates the relative safety of delivering a DNA-damaging agent (doxorubicin) followed in sequence by an antimicrotubule drug (vinorelbine) and the ability to detect the activation of p53 in patients’ tissues. These data illustrate that the molecular changes seen in tissue culture can be seen in patients treated with doxorubicin, albeit with significant variability. This study has provided important information for future trials in patients with wtp53 breast cancer to determine whether activation of p53 and repression of MAP4 are predictive of response.


   ACKNOWLEDGMENTS
 
We thank Julie Friedman and Peter Amenta for immunohistochemical analyses and expertise and Dr. Maureen Murphy for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grant CA82607, NIH/National Cancer Institute Grant CA78695, NCI CA72720 and Department of Defense Contract DAMD17-98-1-8043. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at UMDNJ-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901. Phone: (732) 235-8064; Fax: (732) 235-8094; E-mail: haitwn{at}umdnj.edu Back

4 The abbreviations used are: wtp53, wild-type p53; MAP4, microtubule-associated protein 4; PBMNC, peripheral blood mononuclear cell; CI, confidence interval. Back

5 J. Bash-Babula and W. N. Hait, unpublished observations. Back

Received 11/ 2/01; revised 1/25/02; accepted 2/ 1/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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