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Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction.

We have prospectively analyzed blood samples of 122 patients with breast disease for the presence of circulating expressing MUC1 cells before and after treatment. Among them, 28 patients had histologically confirmed benign breast disease (group 1), 34 patients had operable breast cancer (group 2), and 60 patients had advanced breast cancer (group 3). Circulating epithelial cells were isolated with BerEP4-coated immunomagnetic beads. Total RNA was extracted and reverse transcribed before analysis by real-time PCR of a MUC1-specific cDNA sequence. The sensitivity of the reverse transcription-PCR tested with blood spiked with MCF7 cells was one cell in 5 ml of blood. The immunomagnetic separation step was mandatory to obtain the maximum specificity. Control samples from healthy donors never displayed cycle threshold (Ct) values for MUC1 lower than 38. Circulating cells (Ct, <38) were detected in 3 of 28 (11%) cases in group 1, in 8 of 34 (24%) cases in group 2, and in 27 of 60 cases (45%) in group 3. A semiquantitative estimate of blood-borne cells could be derived from the Ct value when below 32 (the lowest was 28) or by the number of positive aliquots of the same blood sample. Thus, immunomagnetic separation, followed by MUC1-specific RT-PCR, allows the semiquantitative detection of circulating mammary cells. A significant correlation between the presence of MUC1-positive cells and the group of breast tumors was observed. The clinical significance of blood-borne cells in breast cancer, especially at the operable stage, may be investigated by following these patients.

Adult↗

Differential experimental micrometastasis to lung, liver, and bone with lacZ-tagged CWR22R prostate carcinoma cells.

LacZ-tagged human prostate carcinoma CWR22Rv1 cells metastasize spontaneously to lung, liver, and bone from subcutaneous primary tumors in athymic nude mice; these organs are 'natural' targets of metastasis for the human disease. To evaluate the mechanism(s) of metastasis to these organs, an experimental metastasis model was used by taking advantage of the ultrasensitive detection of lacZ. Within I h after tail vein injection, micrometastases were forming in lung, liver, bone, kidney, and brain with very different quantitative levels. The kinetics of loss of unstable micrometastases and retention of stable ones were also very different in these organs. After injecting suspensions of single cells, both whole-organ and serial-section staining for lacZ revealed considerable heterogeneity in cell number of individual lung micrometastases while micrometastases in liver contained only I or 2 cells. The size of individual bone micrometastases also suggested only 1 or 2 cells. Tumor cells could also be detected in the small blood vessels of the lung within minutes after injection. These studies indicate that lacZ-tagged CWR22Rv1 cells after tissue culturing contain subsets of cells capable of establishing transient micrometastases in lung, liver, and bone after direct injection into the animal's circulation. Moreover, the quantitative and qualitative properties of the micrometastases in the three organs differ significantly, suggesting different mechanisms for stabilization and fates of micrometastases in these organs.

Adenocarcinoma↗

Enhanced recognition of hydroxyl radical modified plasmid DNA by circulating cancer antibodies.

Reactive oxygen species have been implicated in various human diseases which are also responsible for the elimination of invading pathogens. In disease state and inflammatory responses, the excess of these radicals damage cellular macromolecules. DNA is susceptible to attacks by OH-induced damage. Oxidative DNA damage is an important factor in mutagenesis and carcinogenesis. In the present study, purified plasmid Bluescript DNA was modified by hydroxyl radical. Modifications incurred in DNA were characterized by physico-chemical techniques. Sera from patients of cancer were studied for their binding to native and hydroxyl radical modified plasmid DNA. Direct binding ELISA and competition binding results indicated that autoantibodies in cancer showed higher recognition to ROS-plasmid DNA as compared to the native form. Retarded mobility of the immune complex formation between IgG isolated from cancer sera using native and ROS-plasmid DNA as antigens reiterated preferential recognition of modified plasmid DNA by cancer autoantibodies. Therefore, it can be concluded that circulating autoantibodies in cancer sera bind preferentially to ROS-plasmid DNA as compared to native polymer. The data presented in the present communication suggest a role of ROS in the etiology of cancer.

Antibodies, Neoplasm↗

Cardiac effects of noncardiac neoplasms.

Clinically significant cardiovascular abnormalities may occur as secondary manifestations of noncardiac neoplasms. The principal cardiac effects of noncardiac tumors include the direct results of metastases to the heart or lungs, the indirect effects of circulating tumor products (causing nonbacterial thrombotic endocarditis, myeloma-associated amyloidosis, pheochromocytoma-associated cardiac hypertrophy and myofibrillar degeneration, and carcinoid heart disease), and the undesired cardiotoxicities of chemotherapy and radiotherapy.

Amyloidosis↗

Cell adhesion molecules: a critical examination of their role in metastasis.

It is proposed that the arrest of circulating cancer cells in the microvasculature is mediated by mechanical interlocking of the cancer cell and vessel surfaces, as distinct from the formation of adhesive bonds utilizing cell adhesion molecules. It is also proposed that the major role of cell adhesion molecules in metastasis occurs after cancer cell arrest, when linkage to the extracellular matrix, initially the subendothelium, and consequent signal transduction results in cancer cell proliferation which is the ultimate determinant of metastasis.

Animals↗

Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers.

Recently several noninvasive methods have been employed to detect circulating tumor cells (CTCs) in cancer patients. In this study, we have developed a highly sensitive, high-throughput colorimetric membrane-array method that was designed to detect a panel of mRNA markers including human telomerase reverse transcriptase (hTERT), cytrokeratin-19 (CK-19), carcinoembryonic antigen (CEA) and mucin 1 (MUC1) mRNA for the presence of CTCs in the peripheral blood of patients with gastric cancer (GC). Digoxigenin-labeled cDNA targets synthesized following total RNA isolation from peripheral blood samples of 64 GC patients and 80 healthy individuals were subjected to membrane-array hybridization. The results showed that membrane array could positively detect 5 cancer cells/ml of peripheral blood in GC cell-dilution experiments. The sensitivity, specificity and diagnostic accuracy for hTERT, CK-19, CEA and MUC1 mRNA ranged from 78.1% to 82.8%, 76.3% to 85% and 81.3% to 83.3%, respectively. Both CEA and MUC1 mRNA expression was correlated significantly with all malignant biological properties of GC, such as macroscopic type, depth of tumor invasion, lymph-node metastasis, TNM stage and coexisting distant metastasis (all p < 0.05). Using these 4 markers in combination, the sensitivity, specificity and diagnostic accuracy of membrane array were raised to 89.1%, 91.3% and 90.3%, respectively. The expression of all 4 mRNA markers was an independent predictor for postoperative recurrence/metastasis. GC patients with the expression of all the 4 mRNA markers showed a poorer survival rate than those without the expression of any 1 mRNA marker (p = 0.0223). These findings demonstrated that our membrane-array method could detect CTCs in the circulation of GC patients with considerably high sensitivity and specificity. The identification of CTCs in the peripheral blood may be useful in the auxiliary cancer diagnostics or postoperative surveillance of GC patients for recurrence/metastasis.

Adult↗

Rapid apoptosis in the pulmonary vasculature distinguishes non-metastatic from metastatic melanoma cells.

The presence of metastases indicates an ominous prognosis in patients with malignancies, yet the factors that distinguish metastatic from non-metastatic tumors remain poorly understood. Here we pursued the hypothesis that apoptosis in vivo would distinguish metastatic cells from non-metastatic cells and developed a novel method for observation of apoptosis induction in living cells. One hour after the infusion of metastatic or non-metastatic human melanoma or transformed rat embryo fibroblasts, arrest of tumor cells in the pulmonary vasculature was equivalent. In order to demonstrate the induction of apoptosis in living cells, we observed the translocation of cytoplasmic BAD-GFP fusion proteins to the mitochondria during apoptosis. Microscopic observation of the tumor cells transfected with BAD-GFP in isolated lung preparations after intravenous injection into nu/nu mice revealed translocation of BAD-GFP in many more of the arrested, non-metastatic melanoma or transformed rat embryo cells over 4-24 h than of the metastatic cells. TUNEL staining confirmed enhanced apoptosis by non-metastatic tumor cells after injection in vivo. Metastatic melanoma cells or metastatic embryo fibroblasts were better able to negotiate the barrier of survival in the circulation after pulmonary arrest than non-metastatic cells confirming the hypothesis that susceptibility to apoptosis after arrest in the pulmonary vasculature distinguishes metastatic from non-metastatic cells and introducing a new assay for in vivo induction of apoptosis.

Animals↗

[Circulating cancer cells in the peripheral blood].

Occasional observations on this subject were seen until 1995 when Engell reported the large study on circulating cancer cells in the peripheral blood. Engell's report generated great enthusiasm in our country also. But, these cytologic method have a low sensitivity. Consequently, studies on the circulating tumor cells have been abandoned. However, since 1987, RT-PCR and other molecular biologic method were introduced for the identification of circulating cancer cells and micrometastases in bone marrow as well as in lymphnode. Serial analysis of a large number of patients by well-qualified laboratories have been performed. Several authors have shown a significant correlation between detection of cancer cells and relapse in some forms of malignancies. These research may be beneficial in the staging of cancer, in monitoring the effectiveness of cancer treatment, etc. Detection of cancer cells in the peripheral blood likely to have a profound impact on the practice of clinical oncology.

Cytological Techniques↗

Evaluating neoadjuvant therapy effectiveness on systemic disease: use of a prostatic-specific membrane reverse transcription polymerase chain reaction.

OBJECTIVE: An on-going study at the Memorial Sloan-Kettering Cancer Center assessed the effectiveness of androgen deprivation therapy (ADT) prior to surgical removal of the prostate. In this report, we evaluate the effectiveness of ADT on systemic disease by monitoring the presence or absence of circulating prostatic epithelial cells using a reverse transcription polymerase chain reaction (RT-PCR) assay for prostatic-specific membrane antigen (PSM). METHODS: PSM RT-PCR was performed on a total of 38 prostate cancer patients. There were 12 pT2 patients in the ADT group and 10 patients in the control pT2 group and 5 pT3 patients in the ADT group and 11 pT3 patients in the control group. RESULTS: For pT2 patients, 2 of the 12 patients (17%) were positive for circulating prostatic cells during androgen deprivation therapy but before radical retroprostatectomy (RRP). Within a 6-month period after RRP, 3 of 12 patients (25%) were positive. For the period between the 7th and 12th month after RRP, 6 of 12 patients (50%) were positive. For the period 12-36 months after RRP, 2 of the 12 patients (17%) remained positive for circulating prostatic cells. In contrast, the pT2 control group had higher positive rates in comparable periods: 4 of 10 patients (40%) were positive prior to surgery; 6 of 10 patients (60%) were positive during the 6 months following surgery. For the period between the 7th and 12th month following surgery, 4 of 7 patients (57%) were positive for PSM. Finally, 3 of 6 patients (50%) were positive for the period longer than 12 months. Regarding patients who have extraprostatic disease (stage pT3), the ADT group had a lower rate of circulating PSM positive cells. Before RRP and during androgen deprivation therapy, 1 out of 5 patients (20%) in the ADT group were positive as compared to 4 out of 11 patients for the control group. Within a 6-month period after RRP, the ADT group had 4 out of 9 (44%) patients positive for PSM as compared to 9 of 11 (82%) for the control group. For the period between the 7th and 12th months postsurgery, 1 of 5 patients (20%) of the ADT group were positive as compared to 4 of 7 (57%) of the control patients. CONCLUSIONS: These results indicate that patients with pT2 and pT3 lesions who receive neoadjuvant ADT are less likely to have circulating tumor cells detected compared to a control group both prior to and after surgery. In addition, irrespective of ADT or control group, there were increases in the detection of circulating tumor cells in the period after RRP, and this rise gradually decreased, suggesting that surgical manipulation may cause hematogenous dissemination of tumor cells and that ADT reduces such dissemination of tumor cells. Overall, these results indicate that the use of neoadjuvant ADT decreases the number of circulating prostatic cells. These data represent the initial results of an on-going study. As additional patients are added to the studies, attempts to correlate PSM positivity and serum PSA values postoperatively, recurrence, and margin positivity will be made.

Androgen Antagonists↗

Quantitative analysis of circulating tumor cells in peripheral blood of osteosarcoma patients using osteoblast-specific messenger RNA markers: a pilot study.

Metastasis is a major cause of mortality and morbidity in osteosarcoma (OS) patients. To monitor tumor dissemination, we assessed the circulating tumor burden in OS patients by semiquantitative reverse transcription-PCR using osteocalcin, osteonectin, osteopontin, and type I collagen (COLL) mRNAs as molecular markers. We distinguished levels of the mRNAs in peripheral blood between OS patients and healthy subjects using an OS-derived cell line (Saos-2) as a reference standard. We prospectively analyzed 40 peripheral blood samples from 11 OS patients at diagnosis and 29 healthy subjects. In all 29 (100%) healthy subjects, we detected osteocalcin, osteonectin, and osteopontin mRNAs that were most likely attributed to illegitimate transcription in normal hematopoietic cells. In contrast, we found low COLL mRNA levels in only 35% (10 of 29) of healthy subjects, but significantly higher COLL mRNA levels in 91% (10 of 11) of OS patients (P < 0.0001). The reverse transcription-PCR assay for COLL mRNA was sensitive down to the detection of 10 Saos-2 cells among 10(6) normal peripheral blood nucleated cells. The upper limit of COLL mRNA determined among the healthy subjects was found exceeded by six OS patients. The substantially elevated COLL mRNA levels in peripheral blood seemed to originate from circulating malignant cells in these six OS patients, all of whom subsequently developed clinical metastases within 12 months of diagnosis (P = 0.002). Conversely, no metastases were detected in the remaining OS patients with normal COLL mRNA levels. Quantification of COLL mRNA may prove valuable for diagnosing OS micrometastasis and assessing prognosis.

Adolescent↗

The detection of renal carcinoma cells in the peripheral blood with an enhanced reverse transcriptase-polymerase chain reaction assay for MN/CA9.

BACKGROUND: Using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, the authors previously determined the expression of MN/CA9 mRNA in renal cell carcinoma (RCC) and its absence in benign renal tissue. In the current study, the utility of an enhanced RT-PCR assay in the detection of renal carcinoma cells in the peripheral blood was assessed. METHODS: An enhanced MN/CA9 RT-PCR assay was applied to peripheral blood samples from a total of 96 patients. Forty-two patients had renal tumors, including 5 with benign renal lesions, 28 with localized RCC, and 9 with metastatic RCC. Fifty-four control patients without renal tumors were similarly tested. Pathologic staging for patients with localized cancer was T1N0M0 for 5, T2N0M0 for 9, and T3N0M0 for 14 patients. RESULTS: Cells expressing MN/CA9 were detected in 1 of 54 controls (1.8%) and in 18 of 37 cancer patients (49%). Thirteen of twenty eight patients (46%) with localized RCC and 5 of 9 (56%) with metastatic disease tested positive with the assay. No patient with a benign renal tumor exhibited MN/CA9 expression. All blood test results for patients with clear cell RCC were noted to be positive. No correlation was noted between MN/CA9 results and age, gender, or tumor grade. The differences in MN/CA9 results according to T classification were not statistically significant. CONCLUSIONS: The enhanced RT-PCR assay for MN/CA9 is a highly specific technique for detecting circulating renal carcinoma cells in the peripheral blood, and it may prove useful in the diagnosis and monitoring of RCC.

Adult↗

Polymerase chain reaction-based detection of circulating melanoma cells as an effective marker of tumor progression. Melanoma Cooperative Group.

PURPOSE: Reverse transcriptase (RT) polymerase chain reaction (PCR) with multiple markers has been demonstrated to be highly sensitive in detecting circulating cells from patients with malignant melanoma (MM). We evaluated the clinical significance of the presence in peripheral blood of specific PCR-positive mRNA markers as an expression of circulating melanoma cells. PATIENTS AND METHODS: Total cellular RNA was obtained from the peripheral blood of 235 patients with either localized (n = 154) or metastatic (n = 81) melanoma. We performed RT-PCR using tyrosinase, p97, MUC18, and MelanA/MART1 as gene markers. The PCR products were analyzed by gel electrophoresis and Southern blot hybridization. In addition, 20 healthy subjects and 21 patients with nonmelanoma cancer were used as negative controls. RESULTS: Although detected at various levels among assessable patients, each mRNA marker was significantly correlated with disease stage. A significant correlation with disease stage was demonstrated for patients who were positive to all four markers (P < .0001) or to at least three markers (P < .001). Univariate analysis showed a significant correlation between risk of recurrence (evaluated in stage I, II, and III patients) and increasing number of PCR-positive markers (P = .0002). Logistic regression multivariate analysis indicated that each single marker (except tyrosinase) and, more especially, the presence of four PCR-positive markers remained statistically independent prognostic factors for tumor progression. CONCLUSION: Our data establish the existence of a significant correlation among clinical stages, tumor progression, and presence of circulating melanoma-associated antigens in peripheral blood of MM patients. Preliminary assessment of a subset of patients with a higher risk of recurrence needs longer follow-up and further studies to define the role of RT-PCR in monitoring MM patients.

Adult↗

Amplification of MelanA messenger RNA in addition to tyrosinase increases sensitivity of melanoma cell detection in peripheral blood and is associated with the clinical stage and prognosis of malignant melanoma.

An improved protocol for reverse transcription-polymerase chain reaction (RT-PCR), amplifying tyrosinase and MelanA/MART-1 mRNA from peripheral blood, was used to test 340 blood samples from 225 patients with malignant melanoma for the presence of circulating tumour cells. Positive results for tyrosinase or MelanA were obtained in 19% of patients in stage I (n = 74), 31% in stage II (n = 45), 29% in stage III (n = 48) and 52% in stage IV (n = 58). Amplification of MelanA in addition to tyrosinase resulted in a 30% enhanced sensitivity of melanoma cell detection compared with amplification of tyrosinase alone. The sensitivity was further enhanced by analysis of at least two blood samples per patient and performing at least two PCR analyses per sample. During a median follow-up of 4 months, patients with a positive PCR showed a 2. 4-fold increased risk for relapse compared with PCR-negative patients. These data indicate that the detection of circulating melanoma cells in peripheral blood using our optimized protocol for RT-PCR correlated with the clinical stage of disease and is therefore likely to be a prognostic marker for recurrence. MelanA is a sensitive additional marker to tyrosinase in detecting micrometastases using RT-PCR.

Adolescent↗

[Molecular detection of circulating cancer cells in patients with renal cell carcinoma].

We have developed a highly sensitive technique to detect circulating renal cell carcinoma (RCC) cells in the blood using the reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the MN/CA9 gene. RT-PCR analysis of RCC specimens resulted in the clear detection of MN/CA9 mRNA signal in 93%. In contrast, no expression of MN/CA9 was observed in normal kidney specimens. Highly sensitive RT-PCR analysis of blood samples from RCC patients revealed the presence of circulating MN-positive cancer cells in the blood. Fifty samples obtained from the patients with RCC and 31 samples from healthy donors were investigated. The sensitivity and specificity of this RT-PCR analysis were 72% and 78%, respectively. These findings suggest that the MN antigen may be a potential diagnostic biomarker for early detection of RCC.

Adult↗

Screening and identification of differentially expressed transcripts in circulating cells of prostate cancer patients using suppression subtractive hybridization.

BACKGROUND: Tumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model. RESULTS: We established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients. CONCLUSION: The comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.

Biomarkers, Tumor↗

Mechanisms of metastasis.

The role of dermatologists in the diagnosis and treatment of skin cancer continues to increase. Consequently, they will more frequently be involved in the diagnosis, treatment, and management of patients with metastatic or potentially metastatic tumors. Squamous cell carcinomas and malignant melanomas are frequently seen in dermatologic practices and have the capability to metastasize. Metastases are the result of a complex process that is characterized by a sequence of steps, each of which requires acquisition by the malignant cell of key biologic properties. The metastatic sequence can be conceptualized as detachment from the primary tumor followed by invasion, intravasation into a vessel, circulation, stasis within a vessel, extravasation, invasion of the recipient tissue bed, and ultimately proliferation. The basic steps of the metastatic sequence are described as well as how these steps and other tumor cell adaptations can affect the clinical patterns of metastasis. Finally, practical applications of the understanding of these principles of metastasis are discussed.

Cell Aggregation↗

Molecular prediction of progression in patients with conservatively managed prostate cancer.

OBJECTIVES: To report our results with the use of reverse transcriptase-polymerase chain reaction (RT-PCR) as a potential predictor of prostate cancer (CaP) progression in patients managed with watchful waiting. There has been much recent debate about the safety of treating older patients with localized CaP with watchful waiting. The RT-PCR is an assay that can detect small numbers of prostate cells in circulating blood. METHODS: Blood samples were taken from male and female control patients and from patients with advanced, hormone-treated and untreated localized (watchful-waiting) CaP. Sensitive nested RT-PCR assays were carried out on these samples using primers for both prostate-specific antigen (PSA) and prostate-specific membrane antigen mRNA. RESULTS: Fifty-one blood samples were taken from patients managed with watchful waiting. Fourteen of these had positive RT-PCR results. These patients had a significantly higher PSA velocity than did the patients with negative RT-PCR results. Circulating prostate cells were detected in 18 of 24 patients with advanced CaP, 2 of 34 patients with stable, hormone-treated CaP, and in none of the negative controls. The assay was able to detect 20 LNCaP cells reliably when added to a 5-mL volunteer blood sample. CONCLUSIONS: A significant minority (27%) of patients with untreated localized CaP had detectable circulating prostate cells, and these patients tended to have a progressively rising serum PSA level. Despite low-grade disease and sometimes low serum PSA values, these patients may be at risk of early metastatic progression. RT-PCR, in conjunction with existing prognostic tests, may be of use in predicting which "watchful-waiting" patients are at risk of early progression.

Aged↗

Detection of circulating prostate-specific antigen-positive cells in patients with prostate cancer by flow cytometry and reverse transcription polymerase chain reaction.

The presence of prostate-specific antigen (PSA)-positive cells has previously been demonstrated in the peripheral blood of prostate cancer patients by flow cytometry (FC), but the identity of these cells has not been established. In this study, the reverse transcriptase polymerase chain reaction (RT-PCR) was compared with analytical FC in an attempt to detect and characterise these cells. Peripheral blood was obtained from 12 patients with newly diagnosed and untreated prostate cancer and five controls. Nine of the 12 patients with prostate cancer (75%) had circulating PSA-positive cells as shown by FC. Only one of those patients (11.1%) was found to express PSA mRNA by RT-PCR. The absence of PSA mRNA in the majority of samples showing PSA-positive cells suggests that they do not represent haematogenous micrometastases. PSA-positive cells in the blood could represent monocytes that express PSA, either following binding/phagocytosis of free serum PSA or phagocytosis of tumour cells.

Base Sequence↗