Effects of Granulocyte Colony-stimulating Factor (G-CSF), Trastuzumab, and Vinorelbine on Immune Cell Function

Study Overview
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Brief Summary
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Trastuzumab or Herceptin is an antibody directed against Her-2. Her-2 is a growth factor receptor which is present on the tumors of 25% of patients with breast cancer. The addition of trastuzumab to chemotherapy has been shown in a randomized clinical trial to increase the response rate to chemotherapy, the duration of response to chemotherapy, and to improve the duration of survival of patients with metastatic breast cancer. The anticancer mechanism of action of trastuzumab is unknown, but it is possible that trastuzumab acts by promoting antibody-dependent cell mediated cytotoxicity (ADCC), or direct killing of cancer cells by immune cells, triggered by antibodies bound to the surface of the cancer cell. G-CSF is a drug which is a growth factor for certain types of immune cells. G-CSF has two favorable effects on ADCC. G-CSF increases the pool of circulating cancer-killing immune cells, and G-CSF increases the strength of binding of cancer-killing immune cells to a specific part of the antibody. Therefore, priming with G-CSF significantly increases the efficiency of ADCC, and four days of treatment with G-CSF has been shown to optimize ADCC in some studies. Recent data from the investigators' laboratory indicates that chemotherapy can augment ADCC directed against tumor cells. The investigators' hypothesis is that pre-treatment with the drug G-CSF would increase the effectiveness of chemotherapy given with trastuzumab.

Detailed Description
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This is a randomized phase II study comparing trastuzumab with G-CSF against trastuzumab with placebo during the first two weeks of therapy. Twenty five patients with metastatic breast cancer will be randomized to receive weekly trastuzumab plus either G-CSF or placebo by subcutaneous (SQ) injection daily for five days weekly for two weeks. Subsequently, all patients will receive an additional 12 weeks of weekly trastuzumab, G-CSF by SQ injection daily for five days weekly for 12 weeks, and vinorelbine once weekly at a dose of 25 mg/m2 weeks 3, 4, 6, 7, 9, 10, 12, 13. Baseline evaluation will include a history and physical exam, comprehensive metabolic panel (CMP), complete blood count (CBC), serum pregnancy test, computerized tomography (CT) scan for disease measurements, and a Multiple Uptake Gated Acquisition (MUGA) scan. The CT scan and MUGA will be repeated upon completion of the study treatment. Blood will be drawn pre-trastuzumab, 2 hours post-trastuzumab, and 48 hours post-trastuzumab on weeks 1, 2, 3, 4, and 12 to measure whole blood ADCC activity. Two additional assays for whole blood ADCC activity will be drawn at baseline pre-treatment, and following completion of protocol treatment. These assays will measure chromium release from a Her-2 positive target cell exposed to the patient's effector cells. Measurement of soluble Her-2 in patient serum will also be measured at each ADCC time point.

Official Title
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A Phase II Trial of Trastuzumab, Neupogen, and Vinorelbine Investigating the Effects on Immune Function and Clinical Outcomes in Patients With Metastatic Breast Cancer Overexpressing Her-2/Neu

Conditions
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Metastatic Breast Cancer

Intervention / Treatment
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* Drug: G-CSF
* Drug: trastuzumab
* Drug: vinorelbine
* Drug: G-CSF
* Drug: saline placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: All patients must have pathological confirmation of carcinoma of the breast. Patients must have metastatic breast cancer by documented clinical or radiological assessment. Immunohistochemical analysis of HER-2/neu expression on paraffin-embedded specimens will be performed. HER-2/neu overexpression will be qualitatively scored as 0, 1+, 2+, or 3+, with 3+ indicating the strongest positivity. Fluorescence In Situ Hybridization (FISH) analyses will also be performed on these patients. Patients with 2+ to 3+ overexpression of HER-2/neu (membranous staining) are eligible, regardless of the results of the FISH analysis. Age ≥18 years. Karnofsky performance status ≥ 60%. Adequate hepatic, renal, and hematologic function. Prior treatment with trastuzumab will be allowed. All patients must have adequate cardiac function (defined as left ventricular ejection fraction ≥ 45%) documented by echocardiogram or MUGA scan. Premenopausal women will be required to have a negative urine or serum pregnancy test and to use an effective form of contraception. Patients with a history of brain metastases are permitted as long as it has been at least 30 days since definitive treatment, they are clinically stable and a magnetic resonance imaging scan of the brain demonstrates control of the lesion(s). All patients must give written informed consent indicating they are aware of the investigational nature of this treatment, as well as the risks and benefits of this protocol. Exclusion Criteria: No treatment with chemotherapy or trastuzumab will be allowed within four weeks of study entry. Prior therapy with vinorelbine. Known history of hypersensitivity to trastuzumab, Chinese hamster ovary (CHO) cell proteins, or any component of these products. History of current unstable angina, symptomatic congestive heart failure, or myocardial infarction within the last 6 months. Pregnant women are excluded. History of a known hypersensitivity to E. coli-derived proteins, filgrastim, or any component of the product.

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: 1<br>Subjects will receive ten doses of G-CSF at a dose of 5 mcgm/kg daily Monday through Friday for the first two weeks of the trial. All patients will also receive trastuzumab at 4 mg/kg week 1, and 2 mg/kg week 2 during the first two weeks of the trial. All patients will then receive 12 weeks of trastuzumab at 2 mg/kg IV weeks 3 through 14, vinorelbine at 25 mg/m2 IV weekly weeks 3,4,6,7,9,10,12,13 and G-CSF at 5 mcgm/kg SQ daily Monday through Friday weeks 3-14. | Drug: G-CSF<br>* 5 mcgm/kg daily Monday through Friday weeks 3-14<br>* Other names: Neupogen;Drug: trastuzumab<br>* 4 mcgm/kg intravenously (IV) over 90 minutes week 1, then 2 mg/kg IV over 30 minutes weeks 2-14<br>* Other names: Herceptin;Drug: vinorelbine<br>* 25 mg/m2 over 6 minutes IV weekly, weeks 3, 4, 6, 7, 9, 10, 12, 13<br>* Other names: Navelbine;Drug: G-CSF<br>* 5 mcgm/kg SQ daily for ten days, Monday through Friday of the first two weeks of the study<br>* Other names: Neupogen;|
| Placebo Comparator: 2<br>Subjects will receive ten doses of a placebo injection SQ daily Monday through Friday for the first two weeks of the trial. All patients will also receive trastuzumab at 4 mg/kg week 1, and 2 mg/kg week 2 during the first two weeks of the trial. All patients will then receive 12 weeks of trastuzumab at 2 mg/kg weeks 3-14, vinorelbine at 25 mg/m2 IV weekly weeks 3,4,6,7,9,10,12,13, and G-CSF at 5 mcgm/kg SQ daily Monday through Fridays weeks 3-14. | Drug: G-CSF<br>* 5 mcgm/kg daily Monday through Friday weeks 3-14<br>* Other names: Neupogen;Drug: trastuzumab<br>* 4 mcgm/kg intravenously (IV) over 90 minutes week 1, then 2 mg/kg IV over 30 minutes weeks 2-14<br>* Other names: Herceptin;Drug: vinorelbine<br>* 25 mg/m2 over 6 minutes IV weekly, weeks 3, 4, 6, 7, 9, 10, 12, 13<br>* Other names: Navelbine;Drug: saline placebo<br>* Saline will be given SQ daily for ten days, Monday through Friday of the first two weeks of the study<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Antibody Dependent Cell-mediated Cytotoxicity of Effector Cells Isolated From Subjects Receiving Trastuzumab With Either G-CSF or a Saline Placebo Against a Her-2 Overexpressing Target in Vitro | Buffy coat effector cells were isolated by centrifugation from heparinized blood, and washed and counted. SKBR3 target cells were labeled with 51-Cr at 100 uCi per 5 x 105 cells for 1 hour at 37 C, washed and effector cells and target cells were plated at a ratio of 70:1 in 96 well microtiter plates, with trastuzumab 2 ug/ml and with no antibody. After addition of the target cells, the plate was centrifuged gently at 1200 rpm to pellet cells, the plate was incubated for 4 hours at 37C, and 100 uL of supernatant was measured on a gamma counter set for 51Cr counting for 1 minute per tube. Specific lysis in % is defined as follows: % specific lysis = (counts released into the supernatant under experimental conditions - spontaneous counts released into the supernatant) / (maximum counts released into the supernatant - spontaneous counts released into the supernatant) Specific lysis at Day 12 was compared to specific lysis at baseline between the G-CSF group and the placebo group. | Baseline and 12 days | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Antibody Dependent Cell-mediated Cytotoxicity of Effector Cells Isolated From Subjects Receiving Chemotherapy, Trastuzumab, and G-CSF Against a Her-2 Overexpressing Target in Vitro | Buffy coat effector cells were isolated by centrifugation from heparinized blood, washed, and counted. SKBR3 target cells were labeled with 51-Cr at 100 uCi per 5 x 105 cells for 1 hour at 37 C, washed and effector cells and target cells were plated at a ratio of 70:1 in 96 well microtiter plates, with trastuzumab 2 ug/ml and with no antibody. After addition of the target cells, the plate was centrifuged gently at 1200 rpm to pellet cells, the plate was incubated for 4 hours at 37C, and 100 uL of supernatant was measured on a gamma counter set for 51Cr counting for 1 minute per tube. Specific lysis in % is defined as follows: % specific lysis = (counts released into the supernatant under experimental conditions - spontaneous counts released into the supernatant) / (maximum counts released into the supernatant - spontaneous counts released into the supernatant) Specific lysis at Week 14 was compared to specific lysis at baseline for 17 patients with week 14 samples available. | Baseline and 14 weeks | 
| Clinical Response Rate of the Combination of Trastuzumab, G-CSF, and Vinorelbine in Subjects With Her-2 Overexpressing Metastatic Breast Cancer | 19 subjects (11 on the G-CSF arm and 8 on the placebo arm) completed 14 weeks of treatment and completed restaging at that time. Responses were evaluated by RECIST criteria. | 14 weeks | 
| Safety of the Combination of Trastuzumab, G-CSF, and Vinorelbine in Subjects With Her-2 Overexpressing Metastatic Breast Cancer | Adverse events were graded per RECIST v4.0. There were two severe adverse events: Grade 3 mental status changes in one subject on the G-CSF arm, and Grade 3 febrile neutropenia in one subject on the Placebo arm. Refer to Adverse Events Table for specifics. | 14 weeks | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Breast cancer, Her-2/neu, Granulocyte Colony Stimulating Factor, Trastuzumab, Vinorelbine, Antibody-dependent cellular cytotoxicity, Effector cells