Promoting evidence-based practices for breast cancer care through web-based collaborative learning.

The Knowledge Summaries for Comprehensive Breast Cancer Control (KSBCs) are a series of 14 publications aligned with World Health Organization guidance on evidence-based breast cancer control and accepted frameworks for action. To study utilization of the KSBCs in the development of locally relevant breast cancer control policies and programs in limited resource settings, the National Cancer Institute Center for Global Health, the University of Washington and the Fred Hutchinson Cancer Research Center developed the Project ECHO® for KSBCs (KSBC ECHO). Project ECHO is an online model which employs case-based learning, while promoting multi-directional learning and network-building. The program was evaluated using a pre-post study design to assess if this online collaborative learning platform can be an effective model for dissemination and utilization of the KSBCs to inform breast cancer control programs and policy advocacy in limited resource settings. A total of 28 KSBC ECHO participants (57%) responded to the baseline and endpoint program evaluation surveys. Across all 28 respondents, analysis of the data indicates that knowledge increase was statistically significant overall: average knowledge gain was 0.77, 95% CI [0.44 - 1.08] and p value < 0.0001. A majority of responding team leads reported that the core ECHO components (case/didactic presentations, discussion) contributed to a great extent to strengthening their project proposal/goals. Program evaluation survey responses indicate that utilization of this online platform provided an opportunity for individual knowledge gain, multi-directional information exchange, network-building, and strengthening of the proposed breast cancer control projects based in limited resource settings.

Characterisation of the HLA-DRB1*07:01 biomarker for lapatinib-induced liver toxicity during treatment of early-stage breast cancer patients with lapatinib in combination with trastuzumab and/or taxanes.

HLA-DRB1*07:01 allele carriage was characterised as a risk biomarker for lapatinib-induced liver injury in a large global study evaluating lapatinib, alone and in combination with trastuzumab and taxanes, as adjuvant therapy for advanced breast cancer (adjuvant lapatinib and/or trastuzumab treatment optimisation). HLA-DRB1*07:01 carriage was associated with serum alanine aminotransferase (ALT) elevations in lapatinib-treated patients (odds ratio 6.5, P=3 × 10-26, n=4482) and the risk and severity of ALT elevation for lapatinib-treated patients was higher in homozygous than heterozygous HLA-DRB1*07:01 genotype carriers. A higher ALT case incidence plus weaker HLA association observed during concurrent administration of lapatinib and taxane suggested a subset of liver injury in this combination group that was HLA-DRB1*07:01 independent. Furthermore, the incidence of ALT elevation demonstrated an expected correlation with geographic HLA-DRB1*07:01 carriage frequency. Robust ALT elevation risk estimates for HLA-DRB1*07:01 may support causality discrimination and safety risk management during the use of lapatinib combination therapy for the treatment of metastatic breast cancer.

Effect of raloxifene on bone mineral density in premenopausal women at increased risk of breast cancer.

CONTEXT: Raloxifene is a promising breast cancer prevention agent in postmenopausal women at increased risk for breast cancer. The effects of raloxifene in premenopausal women are unknown. OBJECTIVE: We evaluated the effect of raloxifene in premenopausal women at increased risk for breast cancer on bone mineral density (BMD). DESIGN: This was a phase II clinical trial. SETTING: This study was conducted at an academic medical center. PARTICIPANTS: Thirty-seven premenopausal women at increased risk for breast cancer enrolled in the trial. Thirty subjects began treatment and 27 were evaluable. INTERVENTION: Raloxifene (60 mg daily) and elemental calcium (500 mg daily) were given for 2 yr. Subjects were followed up off medications for 1 yr. MAIN OUTCOME MEASURE: The primary end point was the intrasubject percent change in BMD at 1 yr measured by dual-energy x-ray absorptiometry. RESULTS: The mean baseline lumbar spine density was 1.027 g/cm(2). Lumbar spine density decreased 2.3% at 1 yr (P < 0.00001) and 3.5% at 2 yr (P < .00001). Percent change from yr 2 to 3 was +1.4%. The mean baseline total hip bone density was 0.905 g/cm(2). Total hip density decreased 0.3% at 1 yr and 1.0% at 2 yr (P = 0.033). Percent change from yr 2 to 3 was +1.7%. CONCLUSIONS: Raloxifene use is associated with a decrease in BMD in premenopausal women at increased risk for breast cancer. The clinical significance of this decrease is unknown and is attenuated with stopping raloxifene.

Thromboembolism following removal of femoral venous apheresis catheters in patients with breast cancer.

BACKGROUND: Apheresis catheters have simplified collection of peripheral blood stem cells (PBSC), but may be associated with thrombosis of the instrumented vessels. We performed a retrospective analysis to study the prevalence of thromboembolism associated with the use of femoral apheresis catheters in patients with breast cancer. PATIENTS AND METHODS: Patients were participants in clinical trials of high-dose chemotherapy with autologous PBSC rescue. They underwent mobilization with either high-dose cyclophosphamide (n = 21) or cyclophosphamide/paclitaxel (n = 64), followed by filgrastim. Double lumen catheters (12 or 13 Fr) were placed in the femoral vein and removed within 12 h of the last apheresis procedure. Apheresis was performed using a continuous flow cell separator and ACD-A anticoagulant. Thromboembolism was diagnosed by either venous ultrasonography or ventilation-perfusion scan. RESULTS: Nine of 85 patients (10.6%) undergoing large volume apheresis with use of a femoral catheter developed thromboembolic complications. Pulmonary embolus (PE) was diagnosed in five and femoral vein thrombosis in four patients. Four of the five patients who developed PE were symptomatic; one asymptomatic patient had a pleural-based, wedge-shaped lesion detected on a staging computed tomography scan. The mean number of apheresis procedures was 2.4 (range one to four) and the mean interval between removal of the apheresis catheter and diagnosis of thrombosis was 17.6 days. In contrast, none of 18 patients undergoing apheresis using jugular venous access and none of 54 healthy allogeneic donors undergoing concurrent filgrastim-mobilized PBSC donation (mean 1.7 procedures/donor) using femoral access experienced thromboembolic complications. CONCLUSIONS: Thromboembolism following femoral venous catheter placement for PBSC collection in patients with breast cancer may be more common than previously recognized. Healthy PBSC donors are not at the same risk. Onset of symptoms related to thrombosis tended to occur several weeks after catheter removal. This suggests that the physicians not only need to be vigilant during the period of apheresis, but also need to observe patients for thromboembolic complications after the catheter is removed. The long interval between the removal of apheresis catheter and the development of thromboembolism may have a potential impact on prophylactic strategies developed in future, such as the duration of prophylactic anticoagulation. Avoidance of the femoral site in breast cancer patients, and close prospective monitoring after catheter removal, are indicated.

Phase I clinical trial of alitretinoin and tamoxifen in breast cancer patients: toxicity, pharmacokinetic, and biomarker evaluations.

PURPOSE: To determine the overall and dose-limiting toxicities (DLTs) of alitretinoin (9-cis-retinoic acid) in combination with tamoxifen and the pharmacokinetics of alitretinoin alone and when combined with tamoxifen in patients with metastatic breast cancer. The effect of tamoxifen and alitretinoin on MIB-1, a marker of proliferation, in unaffected breast tissue was explored. PATIENTS AND METHODS: Eligible patients had metastatic breast cancer. Previous tamoxifen therapy was allowed. Planned dose levels for alitretinoin ranged from 50 to 140 mg/m2/d with 20 mg/d tamoxifen in all patients after 4 weeks of alitretinoin as a single agent. Plasma concentrations of alitretinoin and retinol were measured at baseline and after 1, 2, and 3 months. Breast core biopsies were obtained at baseline and after 2 months of therapy. RESULTS: Twelve patients with metastatic breast cancer received a total of 86 cycles of therapy. At 90 mg/m2/d, three of five patients experienced a DLT: grade 3 headache, grade 3 hypercalcemia, and grade 3 noncardiogenic pulmonary edema. At 70 mg/m2/d, one of six patients experienced a DLT (headache), and this level was considered the maximal tolerated dose in this study. Three toxicities occurred that had not been reported previously with alitretinoin: an asymptomatic delay in dark adaptation, a marked decrease in high-density lipoprotein cholesterol, and the occurrence of enthesopathy. Two of the nine assessable patients had a durable clinical response: one partial response and stable disease for 18 months and one complete response in continuous remission for 48+ months. Both responding patients were estrogen receptor-positive and had had previous tamoxifen therapy. There was a high degree of interpatient variability of plasma alitretinoin concentrations, although a significant decline in alitretinoin plasma levels over time was observed. MIB-1 scores declined in four of the eight paired breast specimens obtained. CONCLUSION: The combination of tamoxifen and alitretinoin is well tolerated and has antitumor activity in metastatic breast cancer. The recommended phase II dose is 70 mg/m2/d with 20 mg/d tamoxifen.

Transforming growth factors-beta are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system.

Using a carcinogen-initiated rat model of mammary tumorigenesis, we tested the hypothesis that transforming growth factor (TGF)-betas are useful biomarkers of chemopreventive efficacy in the breast. The chemopreventive agents tested were tamoxifen and the retinoids 9-cis-retinoic acid (9cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR), because both antiestrogens and retinoids have previously been shown to upregulate TGF-betas in vitro. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-betas in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-betas are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.

Effect of tamoxifen on mammographic density.

There are strong data showing that increased breast cancer risk is associated with increased mammographic density. Tamoxifen has been shown to decrease the risk of invasive breast cancer and decrease breast density. We sought to demonstrate and calculate the extent of change in mammographic density in women who have taken tamoxifen for up to 2 years. We evaluated mammograms from 28 high-risk women who were taking tamoxifen. Four different methods of evaluation were used: (a) two qualitative methods (Wolfe criteria and the American College of Radiology Breast Imaging and Reporting Data System criteria); (b) one semiquantitative method (mammograms were assigned one of five semiquantitative scores by visual inspection); and (c) one quantitative method (computer-aided calculation of fibroglandular area from digitized mammograms). The Wolfe criteria showed a 0.03 category decrease per year (P = 0.50). The American College of Radiology Breast Imaging and Reporting Data System criteria showed a 0.1 category decrease per year (P = 0.12). Semiquantitative criteria showed a 0.2 category decrease per year (P = 0.039). Digitized scores showed a 4.3% decrease per year (P = 0.0007). In conclusion, tamoxifen causes a decrease in mammographic density with use, an effect that is better quantitated with semiquantitative criteria or digitized images. Density change might become useful as a surrogate end point for the effect of tamoxifen and other chemopreventive measures, although our data do not predict an individual's degree of risk reduction.

Phase I and pharmacokinetic study of farnesyl protein transferase inhibitor R115777 in advanced cancer.

PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetic profile of the farnesyl protein transferase inhibitor R115777 when administered orally bid for 5 days every 2 weeks. PATIENTS AND METHODS: Twenty-seven patients with a median age of 58 years received 85 cycles of R115777 using an intrapatient and interpatient dose escalation schema. Drug was administered orally at escalating doses as a solution (25 to 850 mg bid) or as pellet capsules (500 to 1300 mg bid). Pharmacokinetics were assessed after the first dose and the last dose administered during cycle 1. RESULTS: Dose-limiting toxicity of grade 3 neuropathy was observed in one patient and grade 2 fatigue (decrease in two performance status levels) was seen in four of six patients treated with 1,300 mg bid. The most frequent clinical grade 2 or 3 adverse events in any cycle included nausea, vomiting, headache, fatigue, anemia, and hypotension. Myelosuppression was mild and infrequent. Peak plasma concentrations of R115777 were achieved within 0.5 to 4 hours after oral drug administration. The elimination of R115777 from plasma was biphasic, with sequential half-lives of about 5 hours and 16 hours. There was little drug accumulation after bid dosing, and steady-state concentrations were achieved within 2 to 3 days. The pharmacokinetics were dose proportional in the 25 to 325 mg/dose range for the oral solution. Urinary excretion of unchanged R115777 was less than 0.1% of the oral dose. One patient with metastatic colon cancer treated at the 500-mg bid dose had a 46% decrease in carcinoembryonic antigen levels, improvement in cough, and radiographically stable disease for 5 months. CONCLUSION: R115777 is bioavailable after oral administration and has an acceptable toxicity profile. Based upon pharmacokinetic data, the recommended dose for phase II trials is 500 mg orally bid (total daily dose, 1, 000 mg) for 5 consecutive days followed by 9 days of rest. Studies of continuous dosing and studies of R115777 in combination with chemotherapy are ongoing.

Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer.

PURPOSE: N-(4-hydroxyphenyl) retinamide (¿4-HPR, Fenretinide; R.W. Johnson Pharmaceutical Research Institute, Springhouse, PA) and tamoxifen (TAM) have synergistic antitumor and chemopreventive activity against mammary cancer in preclinical studies. We performed a pilot study of this combination in women at high risk for developing breast cancer. PATIENTS AND METHODS: Thirty-two women were treated with four cycles of 4-HPR, 200 mg orally (PO) for 25 days of each 28-day cycle, and TAM, 20 mg PO once daily for 23 months beginning after 1 month of 4-HPR alone. Tolerability, dark adaptometry, tissue biopsies, and retinoid plasma concentrations (Cp) were evaluated. RESULTS: Symptomatic reversible nyctalopia developed in two patients (6%) on 4-HPR, but 16 (73%) of 22 patients had reversible changes in dark adaptation, which correlated with relative decrease in Cp retinol (P

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Tamoxifen and fenretinide in women with metastatic breast cancer.

BACKGROUND: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factor-beta (TGF-beta) levels and serum lipids was also examined. PATIENTS AND METHODS: Thirty-one patients were treated with tamoxifen (20 mg p.o. daily), and fenretinide (400 mg p.o. daily with a 3-day drug holiday each month). Plasma TGF-beta testing was performed using isoform specific sandwich ELISA. RESULTS: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ER-negative patients, and three hormone therapy naive ER-positive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of -13.5 mg/dl; p = 0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18 mg/dl, p = 0.0001, a 35% increase) with tamoxifen and fenretinide therapy. TGF-beta1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated. CONCLUSIONS: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400 mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells.

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.

Effects of fenretinide (4-HPR) on dark adaptation.

OBJECTIVES: To assess the alterations in dark adaptation induced by low (200 mg/d) doses of fenretinide (4-HPR), to assess whether these effects were cumulative and whether they were reversible, and to attempt to elucidate the mechanism underlying the changes in night vision. DESIGN: Case series. SETTING: Outpatient eye clinic. PATIENTS: Twenty-two women enrolled in a breast cancer chemoprevention trial, and 18 normal control subjects. INTERVENTION: Measurements of absolute luminance thresholds during dark adaptation. MAIN OUTCOME MEASURES: Parameters of an exponential model of the dark-adaptation function before, during, and after administration of fenretinide. RESULTS: The most conspicuous effect of fenretinide on dark adaptation was a significant delay in the timing of the rod-cone break (P<.001). A minimal elevation of the final cone threshold was also observed. These effects were reversible after fenretinide therapy was discontinued and did not seem to be cumulative. An inverse relationship between delay of the rod-cone break and plasma retinol concentration was found. CONCLUSION: The dose of fenretinide used in this study produced clearly measurable, but not severe, changes in night vision, which were rarely symptomatic.

Selection of local therapy after neoadjuvant chemotherapy in patients with stage IIIA,B breast cancer.

BACKGROUND: Stage IIIA,B breast cancer is commonly treated with neoadjuvant chemotherapy because of high objective response rates and improved operability. Criteria for subsequent selection of local therapy--mastectomy, radiotherapy, or both--are not well defined. We adopted a policy of selective local therapy based on rebiopsy of the breast and clinical axillary lymph node status at the time of best response to chemotherapy. METHODS: Between 1980 and 1993, 126 patients with stage IIIA,B breast cancer were treated with neoadjuvant chemotherapy and definitive local therapy. The long-term incidence of locoregional failure (in-breast, chest wall, axilla, supraclavicular, neck), relapse-free survival, and overall survival was determined. RESULTS: The overall clinical objective response rate to chemotherapy was 95.2%. Eighty-three patients underwent mastectomy, with negative margins achieved in 91.6%. Forty-two patients had breast preservation; the overall in-breast recurrence rate was 19.0% (8 of 42 patients). The overall locoregional recurrence rate by site was: chest wall-8.7% (11 of 126 patients), axilla-8.7% (11 of 126 patients), supraclavicular-5.6% (7 of 126 patients), and neck-4.0% (5 of 126 patients). The axillary recurrence rate was 6.6% (5 of 76 patients) for clinically negative axilla treated with radiotherapy only, and 12.0% (6 of 50 patients) for clinically positive axilla treated with surgery only. The overall long-term survival probabilities (6 years) according to stage were: stage IIIA-58.0%, stage IIIB(noninflam)-58.0%, stage IIIB(inflam)-36.0%. CONCLUSIONS: These findings support a selective approach to local therapy in patients with stage IIIA,B breast cancer. This approach provides local control in most patients, and allows for breast preservation and elimination of axillary dissection in selected patients.

Much ado about not...enough data: high-dose chemotherapy with autologous stem cell rescue for breast cancer.

High-dose chemotherapy with autologous bone marrow or stem cell rescue (HDC/ASCR) has been proposed as a promising treatment strategy for breast cancer. Despite the frequency with which this procedure is performed, the role of HDC/ASCR in the treatment of breast cancer remains undefined. The purpose of this review is to examine the rationale for the procedure, the research progress to date, and the limitations of available data. A literature search of Medline from January 1966 through May 1997, CancerLit from January 1983 through May 1997, and Current Contents through May 1997 identified more than 600 English language papers or abstracts on this topic. Our review focuses on the preclinical and clinical data that explore the concept of chemotherapy dose intensity and the role of dose intensity in treating breast cancer. HDC/ASCR is based on the hypothesis that high-dose chemotherapy will overcome drug resistance, eradicate metastatic disease, and increase the proportion of women with breast cancer who are "cured." To date, results from only one phase 3 trial of HDC/ASCR compared with more conventional therapy have been published. Phase 2 and some phase 3 data on HDC/ASCR in the treatment of high-risk primary breast cancer and metastatic breast cancer are discussed. However, the results are inconclusive. The completion of national and international randomized trials is urgently needed to establish definitively the role of HDC/ASCR in the treatment of breast cancer.

Constraints on CD4 recovery postchemotherapy in adults: thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells.

To examine the mechanisms of CD4 reconstitution in an adult population, lymphocyte repopulation was assessed following dose-intense chemotherapy in 25 breast cancer patients, ages 33 to 69 years. Chemotherapy resulted in a greater than 60% reduction in total CD4 T cells and, in particular, a greater than 90% loss of the CD45RA+ CD4 cells. CD4 recovery was protracted, achieving less than 50% of pretreatment levels after 12 to 14 months. Two facets of the CD4 recovery were notable. First, generation of CD45RA+ CD4 cells played only a minor role in the first year, suggesting that thymic production was not the main route of CD4 regeneration. Indeed, recovery of CD45RA+ CD4 cell levels remained limited in half of the patients even after 2 years. Second, expansion of the mature peripheral CD4 cells (CD45RO+) remaining after chemotherapy was the main source of early CD4 repopulation, peaking at 3 to 6 months postchemotherapy. This expansion was limited in duration, however, and was followed by a secondary decline, such that the total CD45RO+ CD4 levels at 9 to 12 months were lower than at 6 months. When stimulated by mitogens, an increased susceptibility to apoptosis was observed in postchemotherapy CD4 cells as compared with those from normal donors. The elevated expression of markers such as HLA-DR during chemotherapy and for several months postchemotherapy is consistent with the presence of an activated T-cell population. CD4 apoptotic frequency correlated with the frequency of HLA-DR expression on T cells. Thus, CD4 recovery is constrained in adults by a limited thymic regenerative capacity and by an increased susceptibility to apoptosis within the expanding peripheral CD4 population.

Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.