A highly sensitive assay combining immunomagnetic enrichment with multiparameter f low cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10-20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by f low cytometry for the presence of circulating epithelial cells defined as nucleic acid ؉ , CD45 ؊ , and cytokeratin ؉ . Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1-10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.Evidence is accumulating that primary cancers begin shedding neoplastic cells into the circulation at an early stage (1-4); however, the natural history of these cells, their ability to establish metastases, and their bearing on future relapses are unclear. For instance, circulating tumor cells have been detected by PCR in a variety of patients with a good prognosis who are unlikely to develop metastatic disease (5-8). In addition, immunocytochemistry has detected cancer cells in the bone marrow in a proportion of patients with clinically localized disease (9-11). If tumor cell shedding is, in fact, an early event in tumorigenesis, it may be possible to detect cancer cells in the bloodstream before the primary tumor is large enough to be detected by standard screening examinations.To explore this possibility, we have developed a cellular assay that is more sensitive than PCR and that allows precise enumeration and characterization of circulating carcinoma cells. In model studies, the sensitivity of the technique is below 1 epithelial cell͞ml of blood regardless of the number of leukocytes present and the recovery is between 75 and 100%. The assay was used to study the blood of 30 patients with breast cancer, 3 with prostate cancer, and 13 control individuals. An excess of circulating epithelial cells was found in virtually all of the cancer patients unless they were being treated with chemotherapy. In addition, 8 patients with breast cancer undergoing chemotherapy were followed for 1-10 months to determin...

The CD19 antigen plays an important role in clinical oncology. In normal cells, it is the most ubiquitously expressed protein in the B lymphocyte lineage. CD19 expression is induced at the point of B lineage commitment during the differentiation of the hematopoietic stem cell, and its expression continues through preB and mature B cell differentiation until it is finally down-regulated during terminal differentiation into plasma cells. CD19 expression is maintained in B-lineage cells that have undergone neoplastic transformation, and therefore CD19 is useful in diagnosis of leukemias and lymphomas using monoclonal antibodies (mAbs) and flow cytometry. Interestingly, CD19 is also expressed in a subset of acute myelogenous leukemias (AMLs) indicating the close relationship between the lymphoid and myeloid lineages. Because B lineage leukemias and lymphomas rarely lose CD19 expression, and because it is not expressed in the pluripotent stem cell, it has become the target for a variety of immunotherapeutic agents, including immunotoxins. Treatment of non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL) with anti-CD19 mAbs coupled to biological toxins has proven to be effective in vitro and in animal models, and has shown some promising results in Phase I clinical trials. Recently, the analysis of anti-CD19 effects on lymphoma cell growth has highlighted a novel mechanism of immunotherapy. Engagement of cell surface receptors like CD19 by mAbs can have anti-tumor effects by the activation of signal transduction pathways which control cell cycle progression and programmed cell death (apoptosis).

Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximabinduced complement fixation, natural killer (NK)-cell activation, and NK cellmediated ADCC. Down-modulation of NKcell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heatinactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell-mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC. IntroductionMonoclonal antibodies (mAbs) are now a mainstay of therapy for a number of cancers. Rituximab was the first chimeric mAb to be approved for clinical use and remains the most extensively used mAb in cancer therapy. Rituximab binding of CD20 has been shown to signal apoptosis in a subset of lymphoma cell lines. 1 However, there is little evidence that signaling plays an important role in clinical responses to rituximab. Growing evidence suggests multiple interacting mechanisms, including complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC), play a role in the antitumor response of rituximab and other mAbs.Evidence is conflicting related to the role CMC plays in mediating the antitumor effects of rituximab. Van Meerten et al used target cells that express varying amounts of CD20 on their surface, and concluded that rituximab-mediated CMC depends on CD20 expression level and acts in a complementary manner to ADCC. 2 Target cell expression of the complement inhibitory proteins CD55 and CD59 correlates with the ability of rituximab to induce CMC in vitro, 3 and CMC is enhanced when these proteins are blocked. 4 However, no correlation was found between CD55/ CD59 expression by lymphoma cells and clinical response to therapy. 5 In mouse models using murine lymphomas expressing human CD20, Golay et al found that complement plays a key role in mediating rituximab's antitumor effects. 6,7 Cragg and Glennie reached similar conclusions from studies of human B-cell lines in severe combined immunodeficiency (SCID) mice. 8 Clinically, depletion of complement and evidence for complement fixation on target cells can be seen following rituximab...

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Purpose: Complement may play a role in the clinical response to rituximab and other monoclonal antibody^based therapies of cancer. The purpose of this study was to explore the relationship between the C1qA [276] polymorphism and the clinical response to rituximab in patients with follicular lymphoma. Experimental Design: Genotyping for C1qA [276A/G] was done in 133 subjects with follicular lymphoma treated with single-agent rituximab, and correlation with clinical response was done using Cox regression analysis. Results: Prolonged remission was observed among subjects that responded clinically to rituximab therapy and were carriers of the A allele compared with homozygous G subjects. Homozygous G subjects had a time to progression of 282 days, whereas A-allele carriers had a time to progression of 708 days [hazard ratio, (HR), 2.5; 95% confidence interval (95% CI), 2.0-3.1; P = 0.02]. Among subjects who achieved complete remission, homozygous G subjects had a time to progression of 250 days, whereas A-allele carriers had a time to progression of 1,118 days (HR, 4.5; 95% CI, 4.1-4.8, P = 0.04).The difference persisted after controlling for CD32 and CD16 polymorphisms. In patients who responded to rituximab used as first-line agent, a linear trend was observed among the C1qA [276] genotypes, with homozygous A subjects achieving complete response at a higher rate compared with heterozygous or homozygous G subjects. Conclusions: Our findings indicate that polymorphisms in the C1qA gene may affect the clinical response and duration of response to rituximab therapy of follicular lymphoma. These results could have direct implications on designing antibodies with improved efficiency and enhance our understanding of the role of complement in monoclonal antibody therapy.

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Interstitial lung disease (ILD) is a category of diffuse parenchymal lung diseases characterized by inflammation and/or fibrosis. The best characterized ILD is idiopathic pulmonary fibrosis (IPF). Acute exacerbation of IPF is a dreaded occurrence with grim prognosis and suboptimal treatment options. There have been recent reports that acute exacerbation can occur in other ILDs (AE-ILD). Of note, some of these acute exacerbations follow lung procedures. This review summarizes the available information on AE-ILD and discusses the procedures reported to cause AE-ILD. We also discuss proposed mechanisms, risk factors, treatment and prognosis. This review should help to inform decision-making about risks versus benefits of procedures that are commonly recommended to diagnose ILD.

Signal transduction initiated by binding of antibodis to cell surface molecules can have an important impact on the growth of tumor cells. (8), the effectiveness of anti-CD19 antibodies in preventing tumor growth probably results from a signal transduction phenomenon similar to that observed for anti-immunoglobulin (antiIg) on BCL1, rather than a classical antibody-mediated effector function, e.g., antibody-dependent cellular cytotoxicity.Here we show that anti-Ig induces an antiproliferative response in a subclone of BCL1, BCL1.3B3 (9), that includes the induction of cell cycle arrest and apoptosis. Treatment of BCL1.3B3 cells with specific antisense oligonucleotides results in the depletion of the Lyn tyrosine kinase protein.When depleted cells are challenged with anti-Ig, apoptosis is still induced, but cell cycle arrest is not. The same result is observed in anti-Ig-treated Daudi lymphoma cells. In addition, antisense lyn can prevent cell cycle arrest induced by the cross-linking ofCD19. These results indicate that the Lyn tyrosine kinase is required for transduction of the cell cycle arrest signal initiated by cross-linking of mlg or CD19. MATERIALS AND METHODSAntibodies and Antsens Olguclides. BCL1.3B3 cells were cultured in RPMI 1640 medium with 5% (vol/vol) fetal calf serum. Antibody preparations (7,8,10,11) were affinitypurified and included rabbit anti-ovalbumin (35 pg/ml), rabbit anti-BCL, idiotype (35 pg/ml), goat anti-human IgM (7.5 pg/ml), goat anti-ovalbumin (7.5 mg/ml), and anti-CD19 (HD37; 350 pg/ml).Cells were plated with antisense oligonucleotides 24 hr before the addition of antibody. Oligonucleotides were phosphorothioate derivatives synthesized by Oligos Etc. (Guilford, CT). For the BCL,.3B3-cell experiments, the sequences TG-