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PubMed1980

Inhibition of experimental blood-borne lung metastasis by protease inhibitors.

The inhibitory effects of protease inhibitors on blood-borne metastasis in male Donryu rat lung were studied. Injection i.v. of 10(6) Yoshida ascites hepatoma AH7974 cells induced about 118 +/- 92 (S.D.) metastatic foci in rat lung after 3 weeks. Leupeptin (50 mg/kg body weight twice a day), injected i.p. from 2 days before to 4 days after the inoculation of tumor cells, reduced the number of metastatic foci to about 49 +/- 45 (p less than 0.005). Leupeptin also suppressed the formation of metastatic foci of Yoshida ascites hepatoma AH100B cells (p less than 0.001). Elastatinal (100 mg/kg body weight twice a day) and chymostatin (100 mg/kg body weight once a day) did not inhibit formation of metastatic foci of AH7974 cells. Injection i.v. of 10(6) AH7974 cells induced pulmonary thrombi within 1 hr. Leupeptin (50 mg/kg body weight twice a day) reduced the number of thrombi from 1298 +/- 395 to 646 +/- 218, when injected i.p. for 2 days before the inoculation of the cells (p less than 0.005). Chymostatin and elastatinal did not significantly change the number of pulmonary thrombi. These results indicate that leupeptin inhibited metastasis formation and suggest that this effect may be due to the inhibition of thrombus formation after the arrest of circulating tumor cells.

Animals↗
PubMed1987

Circulating lymphoma cells in patients with B & T non-Hodgkin's lymphoma detected by immunoglobulin and T-cell receptor gene rearrangement.

We studied peripheral blood mononuclear cells from 50 patients with active B- and T-cell non-Hodgkin's lymphoma by DNA hybridisation. Nineteen patients (38%) had circulating clones of cells detected by immunoglobulin gene rearrangement (17 patients) or T-cell receptor gene rearrangement (2 patients) with JH and J beta 2 probes. Lymphoma tissue and peripheral blood were studied simultaneously in 22 patients, 9 of which had a circulating clone of cells in peripheral blood. In 7 patients the gene rearrangement in lymphoma tissue and peripheral blood mononuclear cells was identical. However, in 2 patients both heavy chain and light chain gene rearrangements were different in tissue and peripheral blood. The incidence of peripheral blood involvement was commonest in advanced CSIII & IV disease (54%) compared to CSI & II disease (18%) (P less than 0.05), and in low grade (45%) compared to intermediate and high grade lymphoma (31%) (difference not statistically significant). Only 4 patients had definite lymphoma cells seen on peripheral blood smear. The presence of circulating lymphoma cells correlated with conventional assessment of bone marrow involvement although circulating clones were detected in 30% (12/40) of patients with apparently normal bone marrow.

B-Lymphocytes↗
PubMed1990

Detection of neuroblastoma cells in blood.

Tumor surveillance tests are used to detect relapse and to guide the clinician in making therapeutic changes. Identification of relapse early in the course of disease may improve long-term survival. For patients with neuroblastoma, current conventional surveillance methods include radiologic testing, bone marrow analysis, physical examination, and measurement of urinary catecholamine metabolites. Immunocytologic analysis of blood for the detection of circulating tumor cells is a highly specific and sensitive method, which may prove useful in monitoring patients with neuroblastoma. In our study, circulating tumor cells were detected in seven of 10 patients with known disseminated disease at diagnosis and in six of 13 patients during therapy. In some patients, as few as two tumor cells were identified among 100,000 normal hematopoietic cells. The presence or absence of circulating neuroblasts in the 13 patients evaluated during therapy was significantly correlated with tumor relapse (P = .002). We conclude that immunocytologic analysis of blood is a sensitive method for the detection of circulating neuroblasts. We recommend that this technique be used in conjunction with other conventional methods for improved tumor detection and subsequent monitoring of neuroblastoma patients.

Antibodies, Monoclonal↗
PubMed1981

[The determination of myeloid-committed stem cells in systemic disorders of myelopoiesis: implications for physiopathology, diagnosis and prognosis].

Myeloid committed stem cells belong to a subpopulation of small nucleated cells, which are defined by their capacity to form colonies of mature myeloid cells in agar-medium. They are termed "Colony forming Unit, CFUC", and such cells are detectable in bone marrow and peripheral blood. Bone marrow cells from 15 control patients with regular myelopoiesis contained 86 +/- 46 CFUC/10(5) bone marrow cells and 23 +/- 14 CFUC/ml blood. In 10 patients with aplastic anemia, only 0-10, 5 CFUC/10(5) BM-cells were found and no CFUC were detectable in the peripheral blood. 17 patients with chronic myeloid leukaemia showed a moderate elevation of bone marrow CFUC (X = 105), while the circulating CFUC were markedly elevated (105-42.000/ml). The circulating CFUC were closely correlated with the number of leukocytes (p less than 0,001). In 12 patients with primary osteomyelofibrosis, the number of circulating CFUC was also (raised (325-22.199/ml) and again, a correlation with the number of leukocytes was observed (p less than 0,05). As, on the other hand, there was no difference in the leukocyte count between the control group and patients with osteomyelosclerosis, the simultaneous assessment of circulating leukocytes and CFUC proves a diagnostic tool. Pancytopenia with a hypercellular bone marrow results from either neoplastic or metabolic alterations of haemopoiesis; in pancytopenia with neoplastic infiltration or transformation, the number of CFUC was lowered, whereas it was slightly elevated in pancytopenia due to metabolic alterations. In patients with acute leukaemia, only a minority of cells was capable of proliferation in vitro. The growth of leukaemic cells in culture, their prolonged survival along with the expression of functional properties may be clinically used for a more subtle classification of blast populations. The data on patients with acute leukaemia indicate, that basic mechanisms of normal blood cell regulation operate in leukaemic haemopoiesis as well.

Adolescent↗
PubMed1985

Involvement of platelet-released 5-HT in tumor cell lodgement.

Adherence of circulating tumor cells in the microvasculature (lodgement) is a key event in metastasis formation, once cells released from a primary neoplasm have reached the bloodstream. Platelets appear to be involved in tumor cell lodgement, since thrombocytopenia significantly reduces the number of lodged tumor cells. Platelets activated by tumor cells are known to release 5-HT, and therefore the present study was focused on the influence of this amine on hepatic lodgement of intraportally injected fibrosarcoma cells in rats. Lodgement was quantified by labeling of tumor cells with [5-125I]iodo-2-deoxy-uridine. 5-HT levels were simultaneously determined using liquid chromatography with electrochemical detection (LCED) from hepatic areas with lodged tumor cells, as seen in vital microscopy. Injection of tumor cells increased hepatic 5-HT levels twofold (614 +/- 64 micrograms/g) compared with the levels found in rapidly killed (284 +/- 22 micrograms/g) or saline-injected animals (231 +/- 75 micrograms/g). In thrombocytopenia lodgement was reduced by 47% and hepatic 5-HT levels by 52% in comparison with tumor cell-injected controls. Peripheral blockade of 5-HT2 receptors with ketanserin (0.6 mg/kg sc) also reduced lodgement by 28% and hepatic 5-HT levels by 36%, strongly indicating involvement of platelet-released 5-HT in the lodgement process. Immunocytochemical studies of hepatic tissue in untreated rats demonstrated presence of 5-HT only in mast cells, while as early as 15 min after tumor cell injection 5-HT immunopositive nerves were also seen. This implies an active uptake of circulating 5-HT by neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Acute non-immunological inflammation inhibits natural killer (NK) activity in the lungs and enhances metastatic development.

A non-immunological acute inflammatory reaction, induced in rats by intrapleural injection of calcium pyrophosphate microcrystals, decreased natural killer activity of lung intracapillary leucocytes (LICL), and, to a lesser extent, of spleen cells. NK activity was significantly depressed as early as 2 h after pleurisy induction, maximally suppressed at 72 h (when the inflammation had resolved) and returned to near-normal values by day 10. This decrease in NK activity was demonstrated using YAC-I lymphoma cells and syngeneic fibrohistiocytoma (P77) cells, as targets. The inhibition of NK activity of LICL was accompanied by an impaired destruction of P77 tumour cells injected i.v. 3 days after the onset of pleurisy. This was shown by an increase in the number of radiolabelled tumour cells surviving in the lungs at 24 h and of tumour nodules developed in lung parenchyma at 3 weeks. Suppression of NK activity was not due to a decrease in the proportion of large granular lymphocytes (LGL) in the LICL population. Suppressor macrophages may be involved, at least in part, since a partial restoration of LICL cytotoxicity was obtained after depletion of plastic-adherent cells. The effect of inflammation was reproduced in normal rats by injecting inflammatory serum or PGE2, suggesting a possible role of circulating mediators.

Animals↗
PubMed2003

Detection of circulating tumor cells in blood using an optimized density gradient centrifugation.

The aim of the study was to compare the new density gradient centrifugation system OncoQuick with the standard density gradient centrifugation system Ficoll for improved tumor cell enrichment in blood of tumor patients. Evaluation of OncoQuick and Ficoll density gradient centrifugation was performed by flow-cytometry and immunocytochemistry using 10 ml unspiked and tumor cell-spiked blood samples of tumor-free probands. From 10 ml blood, OncoQuick density gradient centrifugation separated a cell fraction which consisted of a mean cell number of 9.5x10(4) mononuclear cells compared to 1.8x10(7) cells by Ficoll. Density gradient centrifugation of tumor cell-spiked blood samples with OncoQuick and Ficoll led to similar tumor cell recovery rates, between 70% and 90% for both methods. The improved depletion of mononuclear blood cells by OncoQuick simplified further immunocytochemical evaluation of the enriched cell fraction, which could be spun onto 1-2 glass slides by cytocentrifugation. In comparison, the mononuclear cells separated by Ficoll had to be spun onto more than 50 glass slides for complete immunocytochemical evaluation. Consequently, tumor cell density on each cytospin was higher after OncoQuick preparation compared to Ficoll. Density gradient centrifugation with OncoQuick results in higher relative tumor cell enrichment than Ficoll density gradient centrifugation. This simplifies further immunocytochemical tumor cell detection and is a promising tool for the detection of circulating tumor cells in blood of tumor patients.

Centrifugation, Density Gradient↗
PubMed1999

Sensitive detection of rare Ewing's sarcoma cells in peripheral blood by reverse transcriptase polymerase chain reaction.

Disseminated disease is very important in the clinical assessment of pediatric sarcomas. Several reports suggest that reverse transcriptase polymerase chain reaction (RT-PCR) holds great promise in the early staging of cancer patients in general. However, the complexities of these protocols hamper adequate standardization, and their application as routine diagnostic tools has been difficult. The aim of this study is to assess the actual minimal number of tumor cells that may be detected by RT-PCR in a blood sample. Specific tumor cell dilutions from a Ewing's sarcoma cell line reconstituted in peripheral blood from healthy individuals were "ficolled" and submitted to RNA extraction for cDNA preparation and PCR amplification of the t(11-22) (q24;q12) fusion transcript. After PCR amplification, we were able to detect the EWS/FI-1 chimeric gene product at a dilution of 10 tumor cells per 1 or 2 mL of blood. Our simple method supports a role for routine clinical use of RT-PCR in the detection of circulating Ewing's sarcoma cells.

Artificial Gene Fusion↗
PubMed1999

Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.

The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases.

Biomarkers, Tumor↗
PubMed2006

Case study of the morphologic variation of circulating tumor cells.

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.

Adult↗