Personalized therapy for breast cancer.

Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies.

Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging.

To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells. Following infection of breast cancer cells with an adenovirus expressing beta-galactosidase gene, high levels of beta-galactoside activity were observed. No beta-galactosidase activity was observed in low-density human bone marrow cells. A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing beta-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency. In contrast, human bone marrow cells treated under the same conditions expressed very low levels of transfected beta-galactosidase DNA. Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments. A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells. Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells. However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine. On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.

Negative regulators may mediate some of the inhibitory effects of HIV-1 infected stromal cell layers on erythropoiesis and myelopoiesis in human bone marrow long term cultures.

This report presents results concerning the potential role of negative regulators in hematopoietic suppression observed in human immunodeficiency virus (HIV)-infected long-term cultures (LTC) of human bone marrow cells. Confluent stromal cell layers established from human bone marrow cells were exposed to HIV-1ADA, a monocytotropic strain of HIV-1. A progressive increase in the concentration of HIV-1 p24 antigen in cultures exposed to HIV-1ADA demonstrated that there was a productive infection. Cells from both noninfected and HIV-infected stromal cell layers produced factors that stimulated the proliferation of colony-forming units for granulocytes and macrophages (CFU-GM) from non-infected CD34+ cells. In contrast, when noninfected CD34+ cells were directly cocultured on intact stromal cell layers fewer CFU-GM and burst-forming units for erythroid cells (BFU-E) were detected in HIV-infected LTC than in noninfected LTC. One week after the addition of CD34+ cells, the number of CFU-GM in HIV-infected LTC in six of nine experiments was reduced compared to noninfected control LTC. In those six experiments, the number of CFU-GM was only 53 +/- 5% (SEM) of the number in noninfected LTC. The number of BFU-E in HIV-1-infected LTC was only 46 +/- 5% of the number in noninfected LTC (n = 5). There were fewer BFU-E in HIV-1-infected LTC, whether or not there was a reduced number of CFU-GM. Neutralizing antibody to tumor necrosis factor alpha (TNF-alpha) had no effect on the number of BFU-E in HIV-infected LTC. The number of BFU-E, however, was 2.1 +/- 0.2-fold greater (n = 3) in HIV-infected LTC incubated with neutralizing antibody to interferon-alpha. In HIV-infected LTC with decreased numbers of CFU-GM, the number of CFU-GM was approximately 2-fold greater after incubation of HIV-infected LTC with anti-interleukin-4 (IL-4). The effect of anti-TNF-alpha was variable, and anti-transforming growth factor-beta had no effect on the number of CFU-GM in HIV-infected LTC. After 2 weeks, the number of CFU-GM in HIV-infected LTC incubated with anti-IL-4 and anti-TNF-alpha was 2- to 4-fold greater than in untreated HIV-infected LTC. Antibody treatment did not promote an increase in the number of CFU-GM in noninfected LTC or in LTC in which CFU-GM numbers were not reduced after HIV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycosylated insulin-like growth factor II promoted expansion of granulocyte-macrophage colony-forming cells in serum-deprived liquid cultures of human peripheral blood cells.

This report presents the results of studies investigating the effect of a glycosylated form of insulin-like growth factor II with an apparent molecular weight of 15,000 (appM(r) = 15K IGF-II) and one with a molecular weight of 7500 (M(r) = 7.5K IGF-II) on the expansion of granulocyte-macrophage colony-forming cells (GM-CFC) in human peripheral blood cells. Blood cells were enriched for GM-CFC and other CFCs, and liquid cultures of these cells were established in serum-deprived medium supplemented with either interleukin-3 (IL-3) alone (no insulin or IGF added to the medium) or with IL-3 plus M(r) = 7.5K recombinant (r) IGF-II or appM(r) = 15K IGF-II. After incubation for 3 days or 1 week, the blood cells were subcultured in plasma clots, and the number of colonies detected at 7 days (D7) and at 14 days (D14) was used to calculate the number of D7 GM-CFC and D14 GM-CFC. The number of GM-CFC in liquid cultures of blood cells incubated for 3 days with IL-3 alone was similar to the number added at day 0. By 1 week, the number of D14 GM-CFC and D7 GM-CFC had increased to 3.5 +/- 0.9-fold (p = .03) and two- to 50-fold (p = .04) of the number at day 3, respectively. There were 1.5- to six-fold more D7 GM-CFC in cultures of blood cells incubated for 1 week with IL-3 plus either 100 ng/mL M(r) = 7.5K IGF-II or 200 ng/mL appM(r) = 15K IGF-II than after incubation with IL-3 alone. appM(r) = 15K IGF-II also promoted a two-fold increase in the number of D14 GM-CFC. appM(r) = 15K IGF-II promoted a greater increase in D14 GM-CFC than incubation with IL-3 alone even for blood samples in which M(r) = 7.5K IGF-II did not promote such an increase. The results of these studies demonstrate that physiologic concentrations of appM(r) = 15K IGF-II and M(r) = 7.5K IGF-II increased the number of GM-CFC more than IL-3 alone and suggest that appM(r) = 15K IGF-II was more potent than M(r) = 7.5K IGF-II in augmenting IL-3-induced expansion of GM-CFC in serum-deprived liquid cultures of peripheral blood cells.

Recovery of hematopoietic colony-forming cells in irradiated mice pretreated with interleukin 1 (IL-1).

Data in this report determined the effect of a single injection of recombinant interleukin 1 alpha (rIL-1) prior to irradiation of B6D2F1 mice on the recovery of colony-forming cells (CFC) at early and late times after sublethal and lethal doses of radiation. Injection of rIL-1 promoted an earlier recovery of mature cells in the blood and CFC in the bone marrow and spleen. For example, 8 days after 6.5 Gy irradiation, the number of CFU-E (colony-forming units-erythroid), BFU-E (burst-forming units-erythroid), and GM-CFC (granulocyte-macrophage colony-forming cells) per femur was approximately 1.5-fold higher in rIL-1-injected mice than in saline-injected mice. Also, 5, 9, and 12 days after irradiation, the number of both day 8 and day 12 CFU-S (colony-forming units-spleen) was almost twofold greater in bone marrow from rIL-1-injected mice. The earlier recovery of CFU-S in rIL-1-injected mice was not associated with an increase in the number of CFU-S that survived immediately after irradiation. Also, 7 months after irradiation, the number of CFU-S per femur of both saline- and rIL-1-injected mice was still less than 50% of normal values. Data in this report demonstrate that a single injection of rIL-1 prior to irradiation accelerates early hematopoietic recovery in irradiated mice, but does not prevent expression of radiation-induced frontend damage or long-term damage to hematopoietic tissues.

The rhesus monkey: a primate model for hemopoietic stem cell studies.

Two heterogeneous cell populations (CP 1-7 and CP 8-10) were separated from rhesus monkey bone marrow using counterflow centrifugation-elutriation (CCE). These two cell populations were distinct with respect to morphological composition, expression of cell surface antigens, hemopoietic progenitor cell activity, and concentration of hemopoietic stem cells (HSC). The hemopoietic progenitor cell activity and HSC were concentrated in CP 8-10. In autologous transplantation studies, CP 8-10 reconstituted the lymphohemopoietic system of lethally irradiated monkeys in a manner similar to that of monkeys transplanted with unfractionated bone marrow cells. CP 1-7 was lymphocyte rich and depleted of progenitor cell activity. Transplantation of CP 1-7 led to eventual lymphohemopoietic reconstitution of irradiated monkeys; however, complete engraftment was delayed by as much as 14 days compared to either the transplantation of CP 8-10 or to unfractionated bone marrow. Thus, a presence of the HSC in the lymphocyte-rich progenitor-cell-depleted population can be detected in the rhesus model.

Countercurrent centrifugal elutriation (CCE) recovery profiles of hematopoietic stem cells in marrow from normal and 5-FU-treated mice.

Data presented in this report describe countercurrent centrifugal elutriation (CCE) recovery profiles of hematopoietic colony-forming cells (CFC) in marrow from normal and 5-fluorouracil-(5-FU) treated mice. Of the total nucleated cells, 75%-95% were recovered, and up to 80% of CFC were recovered after CCE of bone marrow from normal mice. Red blood cells and the majority of lymphocytes were collected in fractions well separated from the CFC. In addition, the CCE recovery profiles of populations of CFC (i.e., BFU-E, CFU-E, GM-CFC, and HPP-CFC) were distinct. The distribution of recovered day 8 CFU-S was different from the distribution of day 12 CFU-S. The CCE recovery profiles of CFC in regenerating marrow from 5-FU-treated mice were shifted to fractions of larger cells, presumably in cell cycle. These data demonstrate that CCE is useful as a method of further characterizing qualitative and quantitative changes in populations of CFC occurring after various hematopoietic-influencing regimens.

Inhibitory effects of HIV-1-infected stromal cell layers on the production of myeloid progenitor cells in human long-term bone marrow cultures.

This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.

The clinical development of paclitaxel and the paclitaxel/carboplatin combination.

Paclitaxel and carboplatin have nonoverlapping toxicities with a broad range of clinical activity. The combination of escalating dose paclitaxel and carboplatin dosed to a fixed area under the curve (AUC) was explored in a series of phase I studies. 76 patients were treated with paclitaxel over three hours followed by a 30 min carboplatin infusion, dosed by the Calvert formula to a target AUC of 4.0 or 4.5 mg/min/ml-1. The maximum tolerated dose of paclitaxel was 270 to 290 mg/m2, with a dose limiting toxicity of peripheral sensory neuropathy. Activity was seen in lung cancer, with a paclitaxel dose at or above 230 mg/m2. Neuropathy correlated with paclitaxel AUC due to nonlinear pharmacokinetics at higher doses. Ongoing studies include the use of amifostine as a neuroprotectant and phase II studies of the paclitaxel/carboplatin regimen in head and neck cancer, small cell lung cancer and sarcomas.

A model to study drug effects on lymphoma and normal cell populations using the AKR/J mouse.

The existence of an AKR subline, AKR(Rb6.15)1A1d, with a chromosome marker provided a means to differentiate between proliferating lymphoma and normal cell populations within a single animal. An AKR(Rb6.15)1A1d lymphoma cell line has been maintained for 6 yr by serial passage in AKR/J recipients. The mice die in 7 +/- 2.0 days with evidence of extensive infiltration of the tissues by lymphoma cells. Cytogenetic analysis showed that approx. 1% of the metaphase cells in the bone marrow of mice at day 1 of the lymphoma passage were of the AKR(Rb6.15)1A1d donor-type. This increased to 54% by day 4 and 96% by day 6. The number of donor-type metaphase cells per humerus increased from 3.4 +/- 0.29 (X 10(3] at day 1 to 2.0 +/- 0.49 (X 10(5] at day 4 with a concomitant decrease in the number of non-lymphoma host-type metaphase cells. The population doubling time of donor-type metaphase cells per humerus was 12 +/- 1.4 h. At day 4, there was a significant decrease in the percentage of donor-type metaphase cells in mice that had been treated with BCNU (19.0 +/- 5.85%) or spirogermanium (38.6 +/- 5.85%) 24 h earlier. For BCNU treated animals, this also represented a decrease to 4.4 +/- 1.1 (X 10(4] donor-type metaphase cells per humerus.

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.

Oxygenation of tumors by a hemoglobin solution.

Tumor oxygen tensions were measured using a computer-controlled PO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: (a) during normal air breathing, (b) during carbogen breathing, (c) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and (d) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air-breathing conditions. Although carbogen breathing increased the oxygenation of the better-oxygenated portions of the tumor, it made no impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air-breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma. At 24 h after administration of the hemoglobin solution the 13672 mammary carcinoma showed greater hypoxia than before treatment, which was partially corrected with carbogen breathing.

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays.

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.

Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells.

This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units.

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.

Trisomy of chromosome 6.15 is not necessary for proliferation of AKR(Rb6.15)1Ald lymphoma cells.

By utilizing a unique AKR(Rb6.15)Ald (AKRb) mouse line we have been able to determine the incidence and frequency of cells with and without abnormalities of chromosome number (specifically trisomy 6.15) in spontaneous and transplanted lymphoma. The transplanted lymphoma cells were easily distinguished from the normal cells of AKR/J recipients by the presence in diploid cells of 2 metacentric and 36 acrocentric chromosomes. The majority of the cells from 17 normal control AKRb mice were diploid and trisomy 6.15 (3 metacentric and 36 acrocentric chromosomes) was not seen. In 73% of the 59 AKRb mice with spontaneous lymphoma, the majority of cells were diploid. In 54% of the mice none of the cells had trisomy 6.15, and in 22% of these mice a majority (greater than 50%) of cells had trisomy 6.15. In order to determine which of the cells from AKRb mice with spontaneous lymphoma were malignant, they were transplanted into normal young AKR/J recipient mice, and when lymphoma developed in recipients, their cells were analyzed. The majority of first passage lymphoma cells from 30 recipient mice were donor-type and the majority of these donor cells were diploid. Ten (17%) of these recipients had a majority of cells with trisomy 6.15. In order to further determine whether or not trisomic lymphoma cells had a different proliferative rate than diploid lymphoma cells, eight first passage recipient mice were used to start sequential passage lines. In two diploid lines and four trisomic lines, the same relative frequency of the trisomy was maintained in the sequential passages. However, in two trisomic lines, the frequency of lymphoma cells with three metacentric chromosomes diminished after passage. These studies suggest that aneuploidy is not necessary for proliferation of lymphoma and that diploid cells are a major part of the malignant cell population.

Modulation of antitumor alkylating agents by novobiocin, topotecan, and lonidamine.

Topoisomerase I and topoisomerase II allow a metabolically active cell to mobilize its supercoiled chromosomal DNA and undergo replication, transcription, recombination, and repair. Several topoisomerase inhibitors have recently been shown to be active in preclinical systems. Topotecan (SK&F 104,864), a water-soluble camptothecin analog, is an inhibitor of topoisomerase I. Novobiocin is an inhibitor of topoisomerase II. Lonidamine depletes cellular adenosine 5'-triphosphate (ATP) and may impede energy-dependent DNA repair, MCF-7 human breast-cancer cells were treated in vitro with topotecan, novobiocin, and lonidamine alone, in paired combinations, and in combination with CDDP and melphalan. The three enzyme inhibitors alone and in combination did not increase tumor cell sensitivity to CDDP. However, the combinations of topotecan/novobiocin and lonidamine/novobiocin did enhance the cytotoxicity of melphalan. Mice bearing the FSaII fibrosarcoma were treated in vivo with topotecan, novobiocin, and lonidamine alone, in paired combinations, and in combination with CDDP, melphalan, BCNU, and cyclophosphamide. The combination of topotecan/novobiocin had the greatest impact on tumor cell sensitivity to each cytotoxic agent tested in both tumor cell-survival and tumor growth-delay assays. This sensitization was greatest at the highest concentrations of the cytotoxic agent tested. Combinations of topoisomerase I and topoisomerase II inhibitors may be useful as modulators of antitumor alkylating agents.

Minocycline in combination with chemotherapy or radiation therapy in vitro and in vivo.

In the present study the potential of minocycline, a semisynthetic tetracycline that inhibits collagenase activity in vivo, as an adjuvant to standard anticancer therapies was explored in vitro and in vivo. In EMT-6 cells, minocycline proved to be only minimally cytotoxic, producing a 50% cell kill at concentrations of 132 and 220 microM in normally oxygenated and hypoxic cells, respectively, after 24 h exposure to the drug. In vitro, there appeared to be no interaction between minocycline and cisplatin (CDDP), melphalan, 4-hydroperoxycyclophosphamide, or radiation. In tumor-cell survival studies using the FSaIIC murine fibrosarcoma, short-term treatment with minocycline (5 x 5 mg/kg given over 24 h) was only minimally cytotoxic and did not alter the tumor response to a range of radiation doses. However, when minocycline (5 x 5 mg/kg given over 24 h) was added to treatment with cyclophosphamide, there was a 4-fold increase in FSaIIC tumor-cell killing across the dose range of cyclophosphamide doses tested, whereas the killing of bone marrow granulocyte macrophage colony-forming units (CFU-GM) remained unchanged. The Lewis lung carcinoma was used to assess the response of both the primary tumor and metastatic lung disease to treatment with minocycline (14 x 5 mg/kg) given alone or in combination with several cytotoxic anticancer drugs or with radiation delivered locally to the primary tumor. Of the various therapies tested, minocycline proved to be especially effective as an addition to treatment with cyclophosphamide both in increasing the response of the primary tumor and in reducing the number of lung metastases. The tumor growth delay produced by melphalan, radiation, Adriamycin, and bleomycin was also increased by the addition of minocycline to these therapies. These results indicate that minocycline given in clinically achievable doses may be an effective addition to some standard therapeutic regimens and that the mechanism of modulation by minocycline is likely to involve an effect of the drug on the host and not its direct interaction with other therapeutic modalities at the level of the tumor cell.

Is MUGA scan necessary in patients with low-risk breast cancer before doxorubicin-based adjuvant therapy? Multiple gated acquisition.

Doxorubicin-based chemotherapy in the adjuvant treatment of breast cancer has become standard. Use of doxorubicin is limited by cardiac dysfunction; however, the incidence is dramatically reduced by limiting the dose to less than 550 mg/m(2). Although the cumulative dose in breast cancer is typically 240 mg/m(2), multiple gated acquisition (MUGA) scans are still recommended for determining cardiac functional status in these patients. To examine the need for this practice, we reviewed 296 patients who underwent surgery for breast cancer at Roswell Park Cancer Institute between July 1997 and December 1998. Fifty-nine of 95 (62%) patients receiving doxorubicin-based regimens, and 3 of 39 (7%) receiving nondoxorubicin regimens had pretreatment MUGA scans. The MUGA scans showed normal results in 58 patients and low-normal in 4 (6.5%), with no wall motion abnormalities encountered. There were no cases where doxorubicin was not used because of an abnormal MUGA scan. There were no cardiac complications in the 59 women who received doxorubicin-based chemotherapy. MUGA will screen out few, if any, women under consideration for doxorubicin-based adjuvant therapy; the decision to avoid doxorubicin can be made based on age and preexisting comorbidity. Guidelines recommending routine use of MUGA before the administration of doxorubicin for adjuvant therapy for breast cancer should be reconsidered.

Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors.

This report presents the results of an investigation in which Gel-Well culture chambers were evaluated for their utility as a liquid culture assay system to measure the responses of hematopoietic colony-forming cells (CFC) to recombinant and cell-derived growth factors. Gel-Wells, designed for anchorage-independent cell growth and diffusion of media components, permitted the weekly replacement of media and growth factors without removing cells from the culture chambers. In these studies, changes in cellularity and CFC content in Gel-Well cultures of human umbilical cord blood cells induced by recombinant interleukin 3 (rIL-3) were quantified. After one week in culture without rIL-3, the number of erythroid burst-forming units (BFU-e) had decreased to 25 +/- 38% of pre-values. In contrast, addition of rIL-3 induced an increase in the number of BFU-e to 390 +/- 135% of pre-values. By three weeks with rIL-3, the number of granulocyte-macrophage colony-forming units (CFU-gm) had increased to 292 +/- 58% of pre-values. Also, the presence of a bone marrow stromal cell layer under the Gel-Well helped to maintain the survival of CFC in liquid culture. These studies demonstrated that Gel-Well culture chambers provide a useful liquid culture system for studying the responses of CFC to growth factors.