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The influence of trauma, Dextran 1000, and Dextran 40 on the lodgement of circulating tumor cells.

Earlier studies have shown that trauma and infusion of Dextran 1000 increased the formation of pulmonary metastases after i.v. tumor cell injection in rats. Infusion of Dextran 40 enhanced the pulmonary metastasis formation in traumatized but not in non-traumatized animals. In this study tumor cells labelled with a radioactive isotope were used to register the lodgement circulating tumor cells after trauma and infusion of Dextran 1000 and Dextran 40. It was found that the pulmonary lodgement of tumor cells was increased after trauma and infusion of Dextran 1000. Dextran 40 did not influence the lodgement in a significant way either in normal or in traumatized animals. Thus, the increased formation of metastases after trauma and infusion of Dextran 1000 seems to depend upon an increased tumor cell lodgement.

Animals↗

The influence of antiserotonin treatment with ketanserin on the pulmonary lodgement of circulating tumour cells in normal and traumatized rats.

As shown in earlier studies, the lodgement of circulating tumour cells in the lungs is reduced by thrombocytopenia in both normal and traumatized rats. Other experiments have shown that thrombocytopenia and antiserotonin treatment with ketanserin, which has a selective effect on 5-HT2 receptors, decrease the hepatic lodgement of intraportally injected tumour cells. The present studies show that treatment with ketanserin also reduces the lodgement of i.v.-injected tumour cells in the lungs in both normal and traumatized rats.

Animals↗

Serotherapy of a patient with a monoclonal antibody directed against a human lymphoma-associated antigen.

A preliminary serotherapeutic trial was undertaken with a monoclonal antibody designated antibody 89 (Ab 89) directed against a lymphoma-associated antigen. In vitro studies demonstrated that Ab 89 could mediate complement-dependent lysis and macrophage adherence but not antibody-dependent cell-mediated cytotoxicity. To evaluate toxicity and therapeutic efficacy, two courses of Ab 89 were administered to a patient with an Ab 89-reactive tumor. Transient decreases in the number of circulating tumor cells and the appearance of circulating dead cells were noted with the infusion of Ab 89. Following administration of 150 mg or more of Ab 89, small amounts of antibody could be demonstrated on circulating tumor cells at a time when no free antibody was found in the serum. The inability to deliver a significant amount of Ab 89 to tumor cells in vivo is thought to be secondary to a circulating tumor antigen. Following each infusion, the amount of this blocking antigen decreased but could not be entirely cleared from the serum. This study provides preliminary evidence for the lack of clinical toxicity of a monoclonal antibody and identifies circulating blocking antigens as a significant obstacle to serotherapy.

Antibodies, Neoplasm↗

Molecular detection of disseminated tumor cells in the peripheral blood in patients with gastrointestinal cancer.

PURPOSE: In this study, 62 preoperative peripheral blood samples from patients with gastrointestinal carcinomas, 12 healthy volunteers, and ten patients with inflammatory gastrointestinal diseases were analyzed for the determination of CEA, CK19, and CK20 mRNA expression in peripheral blood, and its clinical significance was evaluated. METHODS: Nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) was used to analyze CEA, CK19, and CK20 mRNA expression in peripheral blood. Fresh tumor tissues from patients with colorectal cancer (n=15) were used as a positive control. RESULTS: Among 62 blood samples from patients with gastrointestinal cancer, the expression of CEA, CK19, and CK20 mRNA was 51.6% (32/62), 35.5% (22/62), and 48.4% (30/62), respectively. 74.2% (46/62) were positive for at least one marker. Fifteen tumor tissues were all positive for CEA, CK19, and CK20 mRNA. Only one blood sample from patients with no gastrointestinal carcinoma was positive for CEA mRNA. CONCLUSION: Our results indicate that the molecular detection of circulating tumor cells in peripheral blood in patients with gastrointestinal carcinoma was significantly correlated with its malignant biological properties, and may be helpful in the selection of clinical treatment and judgment of prognosis.

Adult↗

Circulating tumor cells in patients with solid malignancy treated by high-intensity focused ultrasound.

The theoretical possibility that exposure of a solid malignancy to high-intensity focused ultrasound (US), or HIFU, could lead to an increased rate of metastasis still remains. Using reverse transcriptase polymerase chain reaction (RT-PCR), the potential risk of hematogenous dissemination was assessed in HIFU-treated patients with solid malignancy. RT-PCR can demonstrate the presence or absence of specific RNA fragments. On the day before HIFU ablation, 5-mL peripheral blood samples were collected, and again 5 to 7 days after HIFU, from 26 enrolled patients (hepatocellular carcinoma, HCC: 10; osteosarcoma: 16). Total RNA was isolated and RT-PCR was performed to analyze the mRNA expression of (alpha-fetoprotein (AFP) and bone-specific alkaline phosphatase (BALP) genes. Positive AFP mRNA expression was preoperatively detected in 8 of 10 patients with HCC. In the postoperative specimens, positive expression was also detected in 8 of 10 patients. In 2 patients, circulating tumor cells were found preoperatively, but not postoperatively. Conversely, 2 patients with no circulating tumor cells preoperatively were found to have circulating tumor cells after HIFU. Of 16 osteosarcoma patients, 12 patients had circulating tumor cells and 4 had none. After HIFU treatment, 2 of the 12 patients had converted from presence to absence of circulating cells and the remaining 4 patients remained negative. It is concluded that patients undergoing complete HIFU ablation may demonstrate conversion from presence to absence of circulating tumor-specific marker mRNA, and that HIFU would not enhance the potential risk of metastasis in patients with malignant diseases.

Alkaline Phosphatase↗

Detection of micrometastasis of gastric carcinoma in peripheral blood circulation.

AIM: To detect the micrometastasis of gastric carcinoma in peripheral blood circulation using immunomagnetic beads sorting technique and RT-PCR technique, and to discuss its significance and the difference between the two methods. METHODS: Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood, immunomagnetic beads sorting technique and RT-PCR technique were used to detect the disseminated carcinoma cells. HE, immunocytochemical and immunofluorescence staining were also used to identify the characteristics of the cells separated with immunomagnetic beads sorting technique. RESULTS: Cells expressing cytokeratin were separated and enriched from the peripheral blood specimens of patients suffering from gastric carcinoma or chronic gastritis. After HE staining, two kinds of cells with little cytoplasm were found. Majority of these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal. Immunohistochemical staining indicated that there were CD34 and CD45 expression on the cell membrane of this kind of cells and these cells also showed expressed human telomerase reverse transcriptase by immunofluorescence staining, but the expression of carcinoembryonic antigen was absent. So, these cells might hematopoiesis precursors. Another kind of cells had larger and abnormal nuclei with thicker nuclear membranes. Massed chromatins and poly-nucleoli were found in the nuclei. These cells expressed human telomerase reverse transcriptase and carcinoembryonic antigen, but CD34 and CD45 were not found on the cell membrane. So, these cells were considered as gastric carcinoma cells escaping from the original focuses and existing in the peripheral blood circulation. Carcinoma cells were found in 25 of 60(41.7%) specimens of peripheral blood from patients with gastric carcinoma, while there were no such cells separated from the blood specimens of chronic gastritis patients. The difference of positive rates of disseminated carcinoma cells between two groups was markedly significant (P<0.005). The expressions of CK20 mRNA in peripheral blood specimens were examinated with RT-PCR. CK20 mRNA was detected from 32 of 60(53.3%) peripheral blood specimens in the group of gastric carcinoma patients, while none of the specimens from patients suffering from chronic gastritis had CK20 mRNA. Significant difference was also found between two groups (P<0.005). Statistic analyses also showed that there was a significant difference between the positive rates of two methods in detecting the disseminated carcinoma cells from the peripheral blood circulation of gastric carcinoma patients (P<0.05). CONCLUSION: The results demonstrated that there were disseminated carcinoma cells in the peripheral blood circulation of some patients with gastric carcinoma. Disseminated carcinoma cells can be detected from the peripheral blood samples with immunomagnetic beads sorting technique and RT-PCR technique. The positive rate of RT-PCR technique is higher than that of immunomagnetic beads sorting technique in detecting micrometastasis.

Carcinoma↗

Differences among myeloproliferative disorders in the behavior of their restricted progenitor cells in culture.

We have studied the behavior in culture of circulating restricted hemopoietic progenitor cells from patients with idiopathic myelofibrosis (IMF), polycythemia vera (PV), and essential thrombocytopenia (ET). We have found differences in circulating granulocyte-macrophage, erythroid, and megakaryocytic progenitors that appear to be specific for these chronic myeloproliferative disorders. In IMF, most affected were granulocyte-macrophage progenitor cells (CFU-C), which circulated in increased numbers and were heterogeneous in their sensitivity to the regulatory factor(s) present in phytohemagglutinin (PHA) stimulated T-lymphocyte conditioned medium (CM). Most CFU-C were either highly sensitive to, or independent from, stimulatory factors, while others showed normal sensitivity. In some IMF patients, circulating megakaryocytic progenitors (CFU-M) were present that were capable of giving rise to colonies in the absence of added CM or erythropoietin (EPO). In PV, we confirmed the presence of circulating erythroid progenitor cells that give rise to colonies in culture without the addition of EPO. The number of circulating CFU-C was normal and they responded normally to CM. In ET, failure to detect 7-day circulating restricted progenitor cells was a common observation; the level of other circulating restricted progenitors was in the low normal range. Thus, despite certain common features, including a primary lesion at the level of the pluripotential hemopoietic stem cell, the myeloproliferative disorders differ with respect to the behavior in culture of their circulating restricted progenitor cells. These results have led us to postulate a second regulatory lesion in the pluripotential stem cell that differs in these disorders and is expressed at the level of the respective restricted progenitor cells.

Aged↗

Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients.

Detection and quantitation of circulating cancer cells in peripheral blood may improve cancer staging and monitoring. This study explored the feasibility of using circulating cancer cell detection in peripheral blood for the rapid assessment of chemotherapeutic response. Cytokeratin 19 mRNA was amplified by nested reverse transcriptase-PCR in the peripheral blood of 29 healthy volunteers, 33 pneumonia patients, and 86 lung cancer patients. Circulating cancer cells in the peripheral blood were semiquantitatively determined by taking the ratio of cytokeratin 19 band intensity from the second round of nested PCR to the glyceraldehyde-3-phosphate dehydrogenase band intensity from the first round of PCR amplification. The detection limit of the method was 1 cancer cell in 107 peripheral blood mononuclear cells. The positive detection rate was 40% for lung adenocarcinoma patients of all stages, 41% for squamous carcinoma patients of all stages, and 27% for small cell lung cancer patients. Only one control sample from a pneumonia patient showed a positive result (1.6%). The quantitative method reliably and sensitively estimated cancer cell numbers in the peripheral blood of lung cancer patients. Serial measurement of the relative number of circulating cancer cells correlated with the tumor burden and treatment response of patients. This method may help rapidly assess the efficacy of anticancer treatment, redefine cancer staging, and facilitate the design of better therapeutic strategies for the treatment of cancer patients.

Adult↗

Detection of occult breast cancer cells by amplification of CK19 mRNA by reverse transcriptase-polymerase chain reaction: role of surgical manipulation.

BACKGROUND: To assess if surgical manipulation increases peripheral blood cancer cells dissemination in early stage (I and II) breast cancer patients. PATIENTS AND METHODS: We analyzed 64 patients using RT-PCR for cytokeratin-19 as a marker for peripheral blood breast cancer cell dissemination. Peripheral blood was obtained at 4 different time-points (24 hours before and after surgery, one week and one month later). RESULTS: RT-PCR was positive in 14 (24%) out of 59 evaluable patients. Circulating cells were detected in 4 out of 14 patients before surgery (7%) while in the remaining 10, the positivity was observed after surgery (17%). The percentage of patients with occult breast cancer cells increased significantly after surgery (p = 0.01). CONCLUSION: 1) 7% of early breast cancer patients had circulating tumor cells before surgery. 2) After surgery tumor cells were detected in 17% of patients. 3) Surgery significantly increased the presence of occult breast cancer cells. 4) The clinical significance of occult breast cancer cells should be tested within a larger clinical trial trying to assess their role as an independent prognostic factor.

Adult↗

Development of a membrane array-based multimarker assay for detection of circulating cancer cells in patients with non-small cell lung cancer.

Combination of multiple mRNA markers has been largely investigated for detection of circulating cancer cells. However, current PCR-based methods are relatively expensive and time consuming. The aim of this study was to develop a membrane array-based multimarker assay for detection of circulating cancer cells in nonsmall cell lung cancer (NSCLC) patients. At first, we selected 22 candidate genes by means of suppression subtractive hybridization and Northern blot analysis. The diagnostic value of each candidate gene was then preliminarily evaluated in 50 pairs of blood samples by membrane array method. Accordingly, 17 genes with area under the ROC curve (AUC) > or = 0.8 were selected as target genes to reconstruct the diagnostic membrane array, which was then used to test peripheral blood samples from 100 NSCLC patients and 147 control subjects. ROC curve analysis demonstrated that the optimal threshold number of overexpressed markers on membrane array for discrimination between NSCLC patients and control subjects was 12. As a result, the diagnostic membrane array could detect circulating cancer cells in 90 (90%) of 100 NSCLC patients and in 14 (9.5%) of 147 control subjects (including 6 of 100 normal persons, 3 of 20 breast cancer patients, 3 of 15 colorectal cancer patients and 2 of 12 gastric cancer patients). Moreover, the detection rate was significantly correlated with NSCLC patients' metastatic status and overall stage (p = 0.028 and 0.014, respectively). These results suggested that our blood-based membrane array assay for molecular detection of circulating lung cancer cells has great potential for clinical applications.

Adult↗

Altered melanoma cell surface glycosylation mediates organ specific adhesion and metastasis via lectin receptors on the lung vascular endothelium.

Adhesive interactions between the molecules on cancer cells and the target organ are one of the key determinants of the organ specific metastasis. In this communication we show that b1,6 branched N-oligosaccharides which are expressed in a metastasis-dependent manner on B16-melanoma metastatic cell lines, participate in the adhesion process. We demonstrate that high metastatic cells show significantly increased translocation of one of the major carriers of these oligosaccharides, lysosome associated membrane protein (LAMP1), to the cell surface. LAMP1 on high metastatic cells, carry very high levels of these oligosaccharides, which are further substituted with poly N-acetyl lactosamine (polylacNAc), resulting in the expression of high density of very high affinity ligands for galectin-3 on the cell surface. We show that galectin-3 is expressed in highest amount in the lungs as compared to other representative organs. Blocking galectin-3 by pre-incubating the frozen sections of the lungs with 100 mM lactose, substantially inhibited the adhesion of high metastatic cells, while pre-incubation with sucrose had no effect. Finally, by in situ labeling and immunoprecipitation experiment, we demonstrated that the lung vascular endothelial cells express galectin-3 constitutively on their surface. Galectin-3 on the organ endothelium could thus serve as the first anchor for the circulating cancer cells, expressing high density of very high affinity ligands on their surface, and facilitate organ specific metastasis.

Animals↗

Detection of circulating prostatic cells during radical prostatectomy.

The detection of micrometastasis of prostate cancer could help to decide more appropriate therapeutic strategies in an individual patient. We have developed a flow cytometric method for detecting cytokeratin-positive cells in the peripheral blood before, during and after radical prostatectomy in patients with prostatic carcinoma. By means of this technique we were able to detect a higher number of cytokeratin-positive cells in the intraoperative blood sample than in the pre- and postoperative blood sample in 15 patients with prostate cancer (P < 0.05). Our results show an increase in the number of cytokeratin-positive cells with increasing tumor stage and grade, as well a good correlation of prostate-specific antigen (PSA) value with the number of cytokeratin-positive cells (r > 0.6). Our results underline the importance of no-touch techniques at prostatectomy to minimize release of tumor cells into the circulation during surgery. In the light of our results we consider that the indication for cell savers during radical prostatectomy should be reevaluated. The possibility of detecting single metastatic cells in peripheral blood will enable better individual patient management, and open up new modalities for diagnosing early prostate cancer and enhancing patient monitoring in relapse and tumor progression.

Aged↗

Quantitation of tumorigenic disseminating and arrested cancer cells.

The numbers of potentially tumorigenic cancer cells released into the circulation and secondarily arrested in the lungs of mice bearing B16F10 melanomas or Lewis lung carcinomas were systematically quantified throughout i.m. tumour growth using a bioassay procedure capable of detecting as few as 10 to 100 tumorigenic cells in the circulation or lungs. Viable disseminating cancer cells were detectable within 4 days of i.m. tumour growth and reached 10(6) per 0.5 ml of blood in carcinoma-bearers and 2 X 10(4) per 0.5 ml in melanoma-bearers; 98% of mice with circulating cancer cells had potentially tumorigenic cells in their lungs, even in the absence of overt metastases. The numbers of cancer cells present in the circulation and lungs were related to the growth rate of the i.m. lesion, more cells being released from faster-growing tumours. The numbers of tumorigenic carcinoma cells were compared with the total numbers of cells released into the circulation as quantitated by direct counting procedures, and it was found that the vast majority of these circulating cells were potentially tumorigenic. These studies provide quantitative information about cancer cell input into the metastatic process. Also, the bioassay procedure provides a useful experimental model for the development of regimens for therapy of metastases since it is a sensitive method of monitoring not only the size of disseminated populations of cancer cells but also their clinically relevant property namely, their tumorigenic potential.

Animals↗

Molecular detection of micrometastases and circulating tumor cells in solid tumors.

The detection of circulating tumor cells and micrometastases may have important prognostic and therapeutic implications. Because their numbers can be very small, these tumor cells are not easily detected using conventional methods. In the last decade, molecular techniques have been widely used for the detection of occult tumor cells. The objective of this report is the application of these molecular tools to solid tumors. A systematic review of all related English-language articles published in the last 32 years was performed. The molecular detection of occult tumor cells can be accomplished by PCR amplification of tumor-specific abnormalities present in the DNA or mRNA of malignant cells. The other main PCR strategy for the detection of CTC and micrometastases involves amplification of tissue-specific mRNA. This latter method was often applied to solid tumors, whereas the former was occasionally used. PCR was shown to be superior to conventional techniques in detecting occult tumor cells, allowing the identification of 1 malignant cell mixed with 1 to 10 million normal cells. In some reports, PCR is shown to be a strong predictor of outcome. The molecular detection of circulating tumor cells and micrometastases in solid tumors can be accomplished using highly sensitive PCR assays. The central question of whether PCR reliably predicts relapse and survival remains unanswered for many types of solid tumor. If PCR-based assays are found to be a reliable tool, they will likely have a major impact on the management of these malignancies.

False Negative Reactions↗

Mobilization of tumour cells during biopsy in an infant with Ewing sarcoma.

UNLABELLED: Ewing sarcoma and the closely related peripheral primitive neuroectodermal tumour, recently referred to as Ewing tumour (ET), are characterised by unique gene rearrangements on chromosome 22. The breakpoints have been cloned and shown to fuse the Ewing sarcoma gene to one of two closely related ETS proto-oncogens, FLI-1 or ERG, which reside on chromosomes 11 and 21, respectively. The rearrangement results in the expression of specific hybrid transcripts which can be detected with high sensitivity by the reverse transcriptase polymerase chain reaction technique (RT-PCR) in primary tumours, blood and bone marrow. We report on a 7-month-old boy with a pelvic Ewing sarcoma in whom circulating tumour cells were identified in the peripheral blood during open tumour biopsy by RT-PCR. However, before and 6 days after surgery no tumour cells could be detected in the peripheral blood. CONCLUSION: The application of RT-PCR to monitor shedding of tumour cells during surgical intervention will help to evaluate if open biopsy potentially contributes to metastatic tumour cell spread.

Base Sequence↗

Factor VIIIR:Ag plasma levels in patients with cervical and ovarian carcinoma.

The Authors measured Factor VIII Related Antigen (FVIIIR:Ag), Factor VIII Coagulant Activity (FVIII:C) and fibrinogen plasma levels in 21 patients with cervical carcinoma and in 14 ones with ovarian carcinoma. In the women with cervical cancer, FVIIIR:Ag was significantly higher than controls only in advanced stages. No significant variation of fibrinogen according to stage and no correlation between FVIIIR:Ag and fibrinogen plasma levels were observed. In the patients with ovarian cancer FVIIIR:Ag was significantly increased in stage III-IV and not significantly in stage I. In these subjects too there was a lack of correlation between FVIIIR:Ag and fibrinogen plasma levels. These results seem to indicate that FVIIIR:Ag increase in advanced stages is due to its major release in circulation resulting from invasion of vascular endothelium and not to an aspecific reactivity. These data could suggest a role of FVIIIR:Ag as an aspecific marker of vascular involvement by cervical and ovarian neoplasms.

Adult↗

Carcinocythemia (carcinoma cell leukemia). Report of two cases with English literature review.

Cancer cells of nonhematologic origin are rarely seen on routinely prepared peripheral blood smears. This report describes two patients with disseminated carcinoma, in near terminal state, one with malignant melanoma and another with transitional cell carcinoma of the ureteropelvic junction who developed carcinocythemia (CCA). A review of the English literature revealed only eight other cases in which CCA was documented. Four patients had a small cell bronchogenic ("oat cell") carcinoma, two had disseminated breast carcinoma and one each had advanced adenocarcinoma of ileum and abdominal rhabdomyosarcoma. The circulating malignant cells usually accounted for less than 10% of the nucleated blood cells, but in one patient comprised up to 80% of the leukocyte count. The mean time between detection of CCA and death was 5 weeks (median, 2 weeks) reflecting the fact that CCA is generally a terminal event.

Aged↗

Detection of tumour cells in peripheral blood and bone marrow from Ewing tumour patients by RT-PCR.

The Ewing family of tumours (ET) is characterised at the cytogenetic level by unique chromosome 22 rearrangements. The breakpoints have been cloned and were shown to fuse the EWS gene on chromosome 22 to one of two closely related ETS proto-oncogenes, FLI-I or ERG, which reside on chromosomes II and 21, respectively. The rearrangement results in the expression of chimaeric transcripts, which can be identified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). We applied this method for the monitoring of ET cells circulating in the peripheral blood or infiltrating the bone marrow. The presence of tumour cells could be detected with a sensitivity of I in I x 10(6) nucleated cells. When samples were kept at 4 degrees C, tumour cells could still be identified after 48 hr of storage. Positive RT-PCR signals originated from intact ET cells rather than from free RNA released by ruptured tumour cells. We analysed peripheral blood, bone marrow samples and peripheral blood stem cell collections from 16 ET patients. At diagnosis, bone marrow specimens collected from 6 patients and I peripheral blood specimen tested positive for EWS chimaeric RNA. During therapy tumour cells were detected in bone marrow aspirations obtained from 2 patients. Our results show that ET cells infiltrating the bone marrow or circulating in peripheral blood can be identified by RT-PCR. The clinical implications for the presence of ET cells in samples detected by RT-PCR at diagnosis and during therapy requires further evaluation.

Antineoplastic Combined Chemotherapy Protocols↗