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Purpose: To elucidate the mechanism by which trastuzumab, a humanized monoclonal antibody against HER2 with proven survival benefit in women with HER2-positive metastatic breast cancer, mediates its antitumor activity. Experimental Design: A pilot study including 11 patients with HER2-positive tumors treated in a neo-adjuvant setting with trastuzumab was performed. Trastuzumab was administered i.v. at a dose of 4 mg/kg followed by three weekly i.v. doses of 2 mg/kg. The primary tumor was surgically removed 7 days after the last treatment. Surgical samples, tumor biopsies, and lymphocytes from these patients were collected for biological studies. Result: Clinical data indicated one complete pathological remission and four partial remissions using RECIST (Response Evaluation Criteria in Solid Tumors). Trastuzumab was well tolerated and neither serious adverse events nor changes in cardiac function were observed during this short-term treatment and after surgery. The biological data showed that, independent of response, (a) all patients showed high levels of circulating trastuzumab; (b) saturating level of trastuzumab was present in all of the tumors; (c) no down-modulation of HER2 was observed in any tumors; (d) no changes in vessel diameter was observed in any tumors; (e) no changes in proliferation was observed in any tumors; and (f) a strong infiltration by lymphoid cells was observed in all cases. Patients with complete remission or partial remission were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro antibody-dependent cellular cytotoxicity activity. Conclusions: The results of this pilot study argue against trastuzumab activity in patients through down-modulation of HER2 but in favor of antibody-dependent cellular cytotoxicity guiding efforts to optimize the use of trastuzumab in breast cancer patients.

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.

Background In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. Methods To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. Results Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. Conclusions This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC. Cytometry 45:124–132, 2001. © 2001 Wiley-Liss, Inc.

Nonhematopoietic malignant cells may express receptors for hematopoietic growth factors and respond to these peptides. The aim of the present study was to investigate whether small cell lung cancer (SCLC) cells may be stimulated to proliferate by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), which are currently used in clinical trials in combination with cytotoxic chemotherapy. We studied two SCLC cell lines, H69 and N417. The effects of GM-CSF and IL-3 were evaluated by studying clonal growth, 3H-thymidine incorporation, BUDR/DNA bivariate flow cytometry, c-myc and N-myc oncogene expression, and myeloid surface markers. Our experiments show that both GM-CSF and IL-3 can increase 3H-thymidine incorporation and cloning efficiency and reduce DNA synthesis time of H69 and, to a lesser extent, N417 cells, supporting the hypothesis that hematopoietic growth factors can stimulate the growth of some malignant nonhematopoietic cells in vitro. Further in vitro and in vivo studies are needed to determine whether clinical trials applying these factors for bone marrow recovery after chemotherapy of solid tumors may be hazardous by potentially stimulating growth of remaining tumor tissue.

Tamoxifen (TAM)-induced changes in proliferation kinetics of the human breast cancer cell line MCF-7 were investigated using dual parameter flow cytometry (FCM) of bromodeoxyuridine (BrdU) immunolabelling and of the expression of cell-cycle related proteins (the proliferating cell nuclear antigen, PCNA, and the non proliferation-specific protein, Statin), versus the DNA content. Single-parameter FCM DNA histograms confirmed that after 96 hours of treatment with 10(-7) M TAM the fraction of S-phase cells decreased significantly, with a simultaneous accumulation of cells in the G(0)/G(1) range of DNA content. In dual-parameter FCM cytograms, the fraction of BrdU-positive cells after TAM exposure was significantly lower than in the controls, and no unlabeled S-phase cells were found. The TAM-induced block in G(0)/G(1) phase was paralleled by a decrease in the number of cells with a DNA content typical of the S-phase expressing PCNA, and by an increase in Statin-positive (G(0)) cells. Upon readdition of 10(-9) M 17 beta-estradiol (E2) to the TAM-treated cultures, BrdU-labelling as well as PCNA expression levels increased significantly, whereas the fraction of Statin-positive cells remained higher than in the controls. The results obtained confirm that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide experimental evidence that two main mechanisms are operating: the accumulation of cells in G(1), before the onset of S-phase, and the exit of some cells from the cycling compartment; however, some of those that are blocked at G(0); cannot be totally reversed by estrogens and may be permanent; These data should be taken into account in the attempt to combine the antiestrogen treatment with chemotherapy more effectively.

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naive CD95− CD28+, memory CD95+ CD28+ and CD28− T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naive CD8+ CD95− CD28+ and CD4+ CD95− CD28+ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naive T cells, a striking diversion between putative memory T cells and CD28− T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3–5 years after treatment, naive T cells persisted at low levels, with expansion of CD28− T cells, suggesting that such alterations may extend further. These findings indicate that CD28− T cells were responsible for ‘blind’ T-cell homeostasis, but support the notion that memory and naive T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naive, memory and CD28− T cells.

Six biochemical parameters, recognized as being prognostic factors in breast cancer, were evaluated on fresh samples of human GI tract tumors to better define the biology and natural history of such neoplasms. Levels of expression of HER-2/neu oncogene and epidermal growth factor receptor (EGFr) protein products, ER, PgR, cathepsin-D and ploidy were determined in 16 gastric and 39 colorectal tumors and compared with normal samples from the same subjects. In 56% of gastric, 61% of colon and 35% of rectum carcinomas HER-2/neu gene product p185 was significantly overexpressed as compared to normal tissues. On the contrary colon and rectum tumors expressed significantly lower levels of EGFr than normal in 60% of cases. Very low levels of ER and PgR were detected in all the samples (normal and malignant) tested. 75% of tumour tissues showed a significant higher cathepsin-D content compared to the respective normal sample while an aneuploid DNA profile was documented in 72% of neoplasms. Overall, change of the markers evaluated seems to be a specific phenomenon of certain GI carcinomas. Higher EGFR levels in normal than malignant tissues suggest that EGFR can be implicated in the process of growth and differentiation of the normal gastrointestinal mucosa. Further studies on a larger number of cases along with an adequate follow-up of patients are needed to define the role of these markers in the pathogenesis of GI tract neoplasms and its prognostic significance when considered together with other major risk factors.

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.

To evaluate a new 'two step' method for purging T, B and neoplastic cells from hematopoietic progenitor cells (PC), PCs were collected by apheresis and some suspensions were deliberately contaminated with 2-5% breast cancer cells. PCs were first processed through CellPro columns for positive selection of cells that express CD34. After this first step, the mean CD34+ cell recovery was 68 +/- 12%, and CD34+ cell purity was 61 +/- 11%; CD3+ and neoplastic cell depletion were 2.1 +/- 0.4 and 1.9 +/0 0.4 logs, respectively. Cells were further processed through the StemSep device for direct depletion of T and B cells or of breast cancer cells. After the first and the second step, overall CD34+ cell recovery was 50 +/- 7%, T and B cell removal was 4.7 +/- 0.4 log and neoplastic cell purging was 4.4 +/- 0.3 log, ie significantly superior to methods described in the past.

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38−, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.

Tamoxifen is known to inhibit the growth of some human mammary carcinoma cells; this effect is accompanied by a decrease in the proportion of cells synthesizing DNA. In this work, flow cytometry of DNA and of bromodeoxyuridine labeling and the evaluation of the cell cycle-related antigens Ki-67, PCNA, and statin were used to investigate the changes in the proliferation kinetics of MCF-7 cells before and after treatment with 10(-7) M TAM. The treatment with TAM induced a significant decrease in the fraction of S-phase cells and an increase in those with a DNA content typical of G0/1 phase. The TAM-induced block in G0/1 is paralleled by a decrease in the frequency of cells expressing Ki-67 and PCNA, and by an increase in statin-positive (G0) cells. These results confirmed that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide the first experimental evidence that two main mechanisms are operating: the accumulation of cells in G1, before the onset of S-phase, and the exit of some cells from the cycling compartment.