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[Diffuse melanosis and melanuria in malignant melanoma].

A new case of diffuse melanosis with melanuria is reported. Light and electron microscopic technics were employed. Several hypothesis were proposed to explain the pathogenesis of this particular pigmentation: Dermal perivascular histiocitic cells phagocyte organelles originated in lysed circulating melanoma cells. Phagocytosis of the altered circulating melanoma cells by the endothelium. Macrophages containing ingested melanin migrate from the capillary lumen into the dermis. We consider that in this particular pigmentation participate two mechanism: a) The perivascular dermal macrophage phagocyte the pigment originated in the lysis of the circulating melanoma cells. b) Macrophages loaded with pigment granules cross the capillary wall and remain around the blood vessels.

Adult↗

[Prostate-specific membrane antigen. A new sensitive molecular indicator in metastasizing prostatic cancer].

Prostate cancer is the most common malignant disease in men in western societies. Extracapsular spread of carcinoma is found in approximately half of the patients that are treated by radical prostatectomy. Recently, a new prostate-specific membrane glycoprotein was cloned and sequenced. A highly sensitive and specific nested reverse transcriptase polymerase chain reaction has been developed to detect early occult haematogeneous micrometastatic prostate cells. We analysed venous samples from 17 patients with metastatic prostate cancer using a modified reaction assay. This showed presence of micrometastatic prostate cells in 14 patients. Molecular detection of circulating prostatic epithelial cells could improve clinical staging and treatment of early prostate cancer.

Aged↗

Morphologically intact melanoma cells may be detected in peripheral blood of melanoma patients.

The detection of circulating melanoma cells has been the subject of numerous investigations in recent years. We developed a cellular approach to identifying circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. The examination covered 205 blood samples from 155 melanoma patients and 30 samples from healthy persons and nonmelanoma patients. After density gradient centrifugation, the interphase was incubated with the 9.2.27 antibody. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-anti-alkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per milliliter of whole blood could be detected reliably. Circulating melanoma cells were not found in 30 controls, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and disseminated disease, circulating 9.2.27-positive cells could be detected in 3 of 29 patients (10%) and 13 of 85 patients (15%) examined, respectively. We conclude that immunomagnetic cell sorting is a promising method with high sensitivity and specificity. The method is not suitable for early detection of metastases but is a valuable tool for further investigating the biological characteristics of circulating melanoma cells.

Adolescent↗

Detection of telomerase-positive circulating epithelial cells in ovarian cancer patients.

The aim of this study is the detection of circulating tumor cells in peripheral blood of ovarian cancer patients. We applied immunomagnetic beads coated with an epithelial or leukocyte-specific antibody to isolate epithelial cells from peripheral blood and we measured their telomerase activity. Both enrichment methods showed high sensitivity and specificity to isolate ovarian tumor cells from peripheral blood. Our data also suggested that disseminated ovarian tumor cells isolated from ascites consistently express telomerase enzyme. We have tested epithelial cell enriched blood samples from 20 stage III patients, 8 stage IV patients and 30 healthy volunteers for telomerase activity. Telomerase activity was detectable in the blood samples of all stage IV patients (100%) and seven stage m patients (35%), but none of the healthy donors. Our results showed that CA-125 level, an established diagnostic marker for ovarian cancer, is significantly higher in telomerase-positive patients than telomerase-negative patients. In summary, our data demonstrated that this non-invasive blood test is sensitive and specific for detecting disseminated ovarian epithelial cells and telomerase is a potential marker for the detection of circulating ovarian tumor cells.

Adult↗

[Molecular genetic principles of progression of malignant diseases].

During the past decade, the molecular mechanisms in the process of tumor progression, including metastasis and angiogenesis, have become better understood. Cancer metastasis consists of multiple, complex interacting steps. Each of these steps is crucial and limiting, since a failure to complete any one prevents the tumor cell from producing a metastasis. Detachment from the solid tumor by loosening the intercellular junctions and proteolysis of the extracellular matrix enables tumor cells to enter blood- and lymph vessels. The intravasation into the circulation is supported by the secretion of angiogenic factors, which induce degradation of the basal membrane in blood vessels. Adhesion to endothelial cells, extravasation from the circulation, and induction of angiogenesis are further essential steps for completing the metastatic process. Furthermore, it is well known that once a tumor cell has entered circulation, it will survive only by evasion of the immune system. The systematic identification of tumor antigens opens up new possibilities for immunotherapeutic approaches.

Animals↗

Circulating human hepatic lipase mRNA in patients with hepatocellular carcinoma and healthy controls.

OBJECTIVE: To evaluate circulating human hepatic lipase mRNA as an indicator of circulating hepatoma cells by reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with hepatocellular carcinoma (HCC). DESIGN: Prospective study SETTING: University hospital, Germany. SUBJECTS: 15 patients with hepatocellular carcinoma and 8 healthy volunteers. INTERVENTIONS: Peripheral venous blood was obtained and total RNA was extracted from the lymphocytic layer by caesium chloride gradient centrifugation. The mRNA was reverse transcripted, and hepatic lipase cDNA was amplified by hepatic lipase specific primers with PCR. The integrity of isolated RNA was confirmed by RT-PCR with beta-actin specific primers. Amplificates were visualised by agarose gel electrophoresis and ethidium bromide staining. MAIN OUTCOME MEASURES: Detection of hepatic-lipase-specific RT-PCR products in peripheral blood. RESULTS: Circulating hepatic lipase-specific PCR products were detected in all patients with HCC and in all healthy controls. CONCLUSION: Detection of circulating human hepatic lipase-mRNA by RT-PCR does not indicate metastasising HCC.

Biomarkers, Tumor↗

Genomics and circulating tumor cells: promising tools for choosing and monitoring adjuvant therapy in patients with early breast cancer?

PURPOSE OF REVIEW: Gene expression profiling using a class discovery approach has consistently shown that breast cancer emerges as a group of different disease entities: the basal-like, the human epidermal growth factor receptor 2 type, the normal breast-like, and at least two different hormone receptor positive or luminal types. Initial reports have also suggested that certain expression patterns are associated with relapse. Current data on adjuvant therapy are only beginning to consider this biologic heterogeneity (estrogen receptor and human epidermal growth factor receptor 2 status). Similarly, the search for isolated tumor cells in blood or bone marrow has been regarded as another approach for defining prognosis and tailoring adjuvant therapies. The purpose of this review is to highlight the recent data emerging from both approaches as a means of assessing prognosis and tailoring therapy in patients with early breast cancer. RECENT FINDINGS: The initial reports on prognosis assessment of breast cancer using the expression profile have been corroborated, but the differences between the actual genes selected for the different prognostic signatures remain difficult to explain. Others have introduced added signatures, such as those associated with proliferation or with wound healing, or selected subgroups on the basis of, for example, estrogen receptor level prior to class comparison. The data on the quantification of expression of a limited number of selected genes are promising for both prognosis and prediction. The segregation of histological grade 2 tumors into genetically defined grade 1 or 3 tumors and its associated prognostic significance is a critical observation. The standardization of methods for measuring isolated tumor cells in blood or bone marrow has resulted in validated data both on prognosis in early breast cancer and in the advanced setting. Methylation of circulating DNA might become another method for selecting patients for extended adjuvant regimens. SUMMARY: The new molecular knowledge must be incorporated into prospective clinical studies in patients with early breast cancer. This is the challenge for the years to come.

Biomarkers, Tumor↗

Positive prostate-specific antigen circulating cells detected by reverse transcriptase-polymerase chain reaction does not imply the presence of prostatic micrometastases.

OBJECTIVES: Detection of circulating tumor cells may improve the preoperative local staging of prostate cancers. The aim of this study was to perform enhanced reverse transcriptase-polymerase chain reaction (RT-PCR) of prostate-specific antigen (PSA) mRNA to define the predictive value of PSA-positive circulating cells in a large series of patients. METHODS: The study included 46 patients with Stage T1 to T2 prostate cancer, 94 with benign prostatic hyperplasia (BPH), and 51 (including 9 women) with nonprostatic disease. PSA-positive cells from peripheral blood samples were detected by Southern blot analysis of the RT-PCR products. Original oligonucleotide primers were defined to exclusively detect the three PSA mRNA splices. RESULTS: Circulating PSA-positive cells were observed in 8 (8.5%) of 94 patients with BPH, 10 (22%) of 46 with Stage T1 to T2 prostate cancer, and 9 (17.6%) of 51 with nonprostatic disease. The detection rate of PSA-positive circulating cells was significantly increased in patients with prostate cancer versus patients with BPH (P = 0.03). Among clinically localized prostate cancers with a Gleason score less than 8, a correlation was observed between PSA-positive circulating cells and Stage pT3 cancer (P = 0.038), capsular penetration (P = 0.04), and a positive margin (P = 0.038). The specificity of the assay for Stage pT3 cancer detection was 84.6%, with a positive predictive value of 60%. CONCLUSIONS: Although RT-PCR assay may have a role in preoperative local staging, this study demonstrated the absence of tissue and tumor specificity of PSA-positive circulating cells, accounting for the weak positive predictive value of this technique.

Aged↗

Cadherin-6 gene expression in conventional renal cell carcinoma: a useful marker to detect circulating tumor cells.

BACKGROUND: Dissemination of cancer cells into the circulation is an essential step in the development of a metastasis. Detection of circulating cancer cells may improve the monitoring methods for cancer patients. However, the detection of circulating renal cancer cells is mainly hampered by the lack of markers available for renal cell carcinoma (RCC). In this study, we evaluated cadherin-6 mRNA as a new molecular marker for the detection of circulating renal cancer cells. MATERIALS AND METHODS: Forty-six blood samples of conventional RCCs were included. A standard protocol of RT-PCR, assisted by computer densitometric analysis to establish a cut-off, was performed to examine cadherin-6 mRNA expression by using specific primers. A renal cancer cell line, SKRC-59 and forty tumor biopsies from conventional RCCs were used as positive controls. Twenty-five blood samples from non-RCC patients were also analyzed. RESULTS: Cadherin-6 mRNA could be detected in 38140 (95%) conventional RCC specimens. Cadherin-6 mRNA was positive in 21/46 (45.7%) blood samples of RCC patients, while no positivity was found in non-RCC blood samples. Among the localized RCCs, 14/35 (40.0%) blood samples were positive while 7/11 (63.6%) were positive among the blood samples from metastatic RCCs. CONCLUSION: Our data indicate that cadherin-6 gene is frequently expressed in conventional RCCs. Cadherin-6 is a useful molecular marker to detect the circulating cancer cells disseminated from conventional RCC.

Biomarkers, Tumor↗

Clonal circulating cells are common in plasma cell proliferative disorders: a comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma.

The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45-CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).

Alleles↗

Micrometastasis in colorectal cancer.

Colorectal cancer is one of the most common malignancies in the word and can be usually cured if diagnosed at an early stage. In the European area the estimated number of new cases of cancer in 1995 were approximately 334.000 and 189.000 died of cancer in that year. Despite recent progress in early detection and surgical therapy, the mortality remained unchanged over the past decades. The major reason for this disturbing discrepancy is that occult dissemination of viable cancer cells can occur at any stage of tumorigenesis. Occult dissemination of the tumor cells in patients with operable cancer may be considered a determinant of subsequent metastasis formation. Several groups have therefore designed immunocytochemical and molecular assays to identify such minimal amounts of residual tumor cells that have successfully invaded secondary organs. The question whether circulating tumor cells represent metastatic dissemination or are merely cancer cells without metastatic potential that have detached from the primary tumor, has been debated for over half a century.

Colorectal Neoplasms↗

Osteosarcoma: improved survival with anticoagulation and amputation.

A study of warfarin anticoagulation as an adjunct to amputation of osteosarcomas was undertaken after finding dramatic results in experimental systems. Anticoagulation was started 7 days preoperatively, continued during the operation, and for up to six months postoperatively. Three of 21 (14%) non-anticoagulated control patients are alive at 5-11 years. Five of 9 (56%) of the anticoagulated patients remain alive 5-8 years. The presumed mechanism of increased survival is an inhibition of fibrin deposition around circulating tumor cells, thereby preventing their adherence to capillary endothelium to initiate metastasis formation.

Adolescent↗

Carcinocythemia (carcinoma cell leukemia). An acute leukemia-like picture due to metastatic carcinoma cells.

Observation of a unique population of cells on a Wright-stained blood smear of a patient with metastatic breast carcinoma prompted a study to determine their origin. The primary carcinoma contained a marker, the presence of "signet cells." These were demonstrated in direct peripheral smears and buffy coat preparation of peripheral blood and confirmed histochemically by showing positive periodic acid-Schiff, alpha-napthol and beta-glucuronidase reactions. "Carcinocythemia" is suggested as a name for this unusual process observed over a six month period. Studies of the patient's immunocompetence, of circulating cell surface immunoglobulins and karyotype analysis were made. Postmortem examination revealed retroperitoneal fibrosis, splenic atrophy and extensive metastatic carcinoma but no evidence of leukemia. The cells will be contrasted to those seen in a second patient who appeared to have acute myelocytic leukemia complicating extensive cancer involving the bone marrow. The observations suggest that a leukemia-like blood picture due to circulating cancer cells may occur during the course of metastatic breast carcinoma.

Acute Disease↗

Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction.

BACKGROUND: Application of the reverse transcriptase polymerase chain reaction (RT-PCR) to identification of circulating tumour cells in colorectal cancer. AIMS: To assess whether circulating malignant cells in patients with colorectal liver metastasis could be identified by RT-PCR recognition of mRNA coding for the tumour marker carcinoembryonic antigen (CEA). PATIENTS: A total of 31 with colorectal liver metastases and 22 no-cancer controls. METHODS: Specific cDNA primers for CEA transcripts were used to apply RT-PCR to tissue biopsy specimens, colon carcinoma cell lines, and peripheral blood samples from patients with colorectal liver metastases. A strongly CEA-expressive HT115 colorectal carcinoma cell line was used to spike blood samples from no-cancer control subjects. RESULTS: The limit for detection of CEA cDNA by Southern blotting using HT115 cells was 50 cells per 14 ml of spiked blood. There was a significant difference (p = 0.007) in RT-PCR positive expression between patients with liver metastasis (26/31) compared with controls (5/22). There was no significant relation between the prevalence of CEA cDNA amplification and serum CEA level or metastasis volume in patients with liver metastasis. CONCLUSIONS: This is the first study to suggest that identification of circulating colorectal cancer cells using RT-PCR for detection of CEA cDNA is feasible.

Biomarkers, Tumor↗

[Micro-metastases and non-small cell lung cancer].

INTRODUCTION: Non-small cell lung cancer has a poor prognosis, including those with operable, apparently localised, disease. Preoperative staging investigations and histo-pathological analysis are poor at detecting small clusters of tumour cells, particularly in lymph nodes. STATE OF THE ART: New methods based on immunohistochemistry, or molecular biology, have been developed to detect these so-called micro-metastases. We present a ten-year review of the literature published on this topic. PERSPECTIVE: These publications primarily reported on the detection of micro-metastases within mediastinal lymph nodes removed at operation in order to identify patients at risk of recurrence, for whom adjuvant therapy might be offered. CONCLUSIONS: Lymph node micro-metastases have been demonstrated to be an independent prognostic factor for survival, especially in stage I patients. On the other hand, the presence of bone marrow micro-metastases did not appear to be of significant prognostic value in non-small-cell lung cancer. Finally, the clinical relevance of circulating tumour cells is still debatable, although recent published studies show interesting results.

Bone Marrow Neoplasms↗