The significance of soluble CD138 in diagnosis of monoclonal gammopathies.

Report is summary of the results of study designed to ascertain the significance of soluble CD138 (sCD138) assessment in patients with different monoclonal gammopathies. Previous studies have shown that sCD138 is shed from the surface of myeloma cells into serum and that this marker is a new independent prognostic parameter in multiple myeloma. In presented study was evaluated serum sCD138 level in 14 patients with monoclonal gammopathy of undetermined significance (MGUS) and in 17 patients with multiple myeloma (MM), all MM patients were treated by high-dose chemotherapy regimen with subsequent autologous transplantation of peripheral blood stem cells. To determine the sCD138 level we used a rapid and simple ELISA procedure. The mean serum sCD138 level of patients with MGUS was 32 ng/ml (range: 5-128). Soluble CD138 levels were elevated in the sera of 10 out of 17 (59%) multiple myeloma patients, the mean baseline sCD138 concentration was 1542 ng/ml (range: 10-17300). In spite of small number of patients the difference between MGUS and MM group was highly statistically significant (p<0.001). Multiple myeloma patients with high level of sCD138 at diagnosis (cut-off value: 500 ng/ml) had worse prognosis despite of good response to chemotherapy in some of them (p=0.029). It seems that determination of sCD138 can be recommended as a helpful and reliable marker for differential diagnosis as well as prognosis of monoclonal gammopathies.

Phenotype of peripheral blood leukocytes and survival of patients with metastatic colorectal cancer.

Immune dysfunction is prevalent in metastatic cancer. Few patients with colorectal cancer metastases are cured, and among the strategies aimed at improving the therapeutic results in patients with metastatic colorectal cancer, immunotherapy is being increasingly investigated. We evaluated retrospectively the prognostic significance of peripheral blood leukocytes in 59 patients with metastatic colorectal cancer. The relative numbers of CD3+, CD3+CD4+, CD3+CD8+, NK (CD3-CD16+CD56+), CD3+DR+, CD3+CD25+, CD3+CD69+, CD19+, CD19+CD23+, CD8+CD28+, CD8-CD28+, CD8+CD57+, CD14+DR+ and CD14+CD16+ leukocytes were analyzed by two-color flow cytometry. A three-step approach was adopted to identify predictors of prognosis using regression analysis. Based on the results of univariate survival analysis, the absolute number of white blood cells, NK/CD3+CD69+ and NK/white cell count ratios were significant indicators of prognosis. In the multivariate regression analysis a model was obtained using a single parameter, the NK/CD3+CD69+ ratio, predicting the survival with 10-15% power of regression. The present results indicate that the NK/CD3+CD69+ ratio in peripheral blood may be an independent variable in a regression model predicting the overall survival of patients with colorectal cancer metastases to be tested in prospective studies.

[Importance of selected laboratory indicators in the differential diagnosis and monitoring of multiple myeloma]. / Význam vybraných laboratorních ukazatelu pro diferenciální diagnostiku a sledování aktivity mnohocetného myelomu.

Multiple myeloma is one of the most common haematologic malignancies. Currently there are numerous studies looking for new prognostic markers in multiple myeloma. The most important of them are the markers related to proliferative activity of neoplastic cells or to size of tumor mass. The subject of this paper are the results obtained from investigation of some such laboratory markers in a group of patients with monoclonal gammopathies diagnosed at our department in the last 3 years. We analyzed blood and bone marrow samples from 51 patients with new diagnosed monoclonal gammopathies, 14 of them were patients with monoclonal gammopathy of undetermined significance and 37 patients had multiple myeloma. 17 patients with multiple myeloma were treated by high-dose chemotherapy regimen. We assessed significance of selected laboratory markers for differential diagnosis of monoclonal gammopathies and for monitoring of activity of multiple myeloma. Among the investigated parameters, we verified the significance of cell cycle analysis of bone marrow plasmatic population and of the determination of the number of circulating myeloma cells in differential diagnosis of monoclonal gammopathies. In our opinion, the determination of soluble CD138, beta 2-microglobulin and neopterin serum levels can be also recommended as helpful markers for a solution of this problem. Except of beta 2-microglobulin serum level we did not find statistically significant correlation with activity of multiple myeloma in any of the investigated parameters.

CD4+ T-lymphocytopenia and systemic immune activation in patients with primary and secondary liver tumours.

Using flow cytometry, we evaluated peripheral blood leucocyte subsets in 84 patients with primary and secondary liver cancer. The patients had significantly lower absolute (659+/-386 vs. 906+/-360 cells per microl, p=0.004) numbers of CD3+ CD4+, relative (9+/-5 vs. 12+/-4%, p=0.02) and absolute (154+/-115 vs. 221+/-83 cells per microl, p=0.02) numbers of CD8+ CD28+, absolute numbers of CD3+ and relative and absolute numbers of CD19+. Relative and absolute numbers of CD3+ DR+, CD3+ CD69+ and CD14+ CD16+ cells were significantly elevated in patients compared to controls. The phenotype was similar in 54 patients exposed to chemotherapy compared to 30 untreated patients. Urinary neopterin, a marker of systemic immune activation, was significantly higher in patients with liver tumours compared to controls. A negative correlation was observed between urinary neopterin and the absolute numbers of CD3+ CD4+ (Spearman rank correlation coefficient, rs = -0.54, p<0.0025) and CD19+ (rs = -0.49, p<0.01) in untreated patients. We conclude that, independently of prior chemotherapy, patients with liver present with markedly decreased numbers of CD3+ CD4+ lymphocytes as well as with other abnormalities of peripheral blood leukocyte phenotype. Similar to patients with human immunodeficiency virus infection, the decrease in CD3+ CD4+ lymphocytes is associated with systemic immune activation.

The phenotype of ascitic fluid lymphocytes in patients with ovarian carcinoma and other primaries.

BACKGROUND: Ascitic tumor-infiltrating lymphocytes (TIL) are a potential source of effectors for adoptive immunotherapy. PATIENTS AND METHODS: The TIL phenotype was examined by two-color flow cytometry in malignancy-related ascites of 49 patients with different primaries. Interleukin-10 (IL-10) and neopterin were determined in ascitic fluid by enzyme-linked immunoassay. RESULTS: Malignant melanoma patients had significantly higher CD3(+), CD3(+)CD8(+) and CD3(+)CD95(+), and lower CD3(+)CD4(+) lymphocyte numbers than patients with other primaries. Ovarian cancer patients had higher CD3(+)CD45RO(+), CD8(+)CD28(+), CD19(+)CD86(+), CD19(+) and CD19(+)CD86(+) lymphocyte numbers, and lower NK cell numbers than patients with gastrointestinal and pancreatic primaries. Pretreated patients had significantly lower concentrations of IL-10, lower CD8(+)CD28(+), CD3(+)CD45RA(+), and higher CD3(+)CD80(+) numbers than chemotherapy-naïve patients. Patients with hepatic metastases had lower CD3(+), CD3(+)CD4(+) and CD3(+)CD45RO(+), and higher CD3(+)CD25(+) and NK cell numbers than patients without liver metastases. A substantial number of cells exhibited dendritic cell phenotype. Significant correlations were observed between neopterin and IL-10 concentrations, and numbers of CD8(+)CD28(+) and CD3(+)CD80(+) lymphocytes. CONCLUSIONS: Some parameters of TIL phenotype differ depending on primary, previous treatment, or the presence of liver metastases. A negative correlation was observed between IL-10 and neopterin, and an opposing effect of local concentrations of IL-10 and neopterin on the numbers of CD8(+)CD28(+) and CD3(+)CD80(+) was noted.

The peripheral blood leukocyte phenotype in patients with breast cancer: effect of doxorubicin/paclitaxel combination chemotherapy.

Presence of functional immune system is critical for any attempt aimed at improving survival of breast cancer patients by strategies based on immune system manipulation. We evaluated by flow cytometry the phenotype of peripheral blood leukocyte of 43 breast cancer patients. In 11 patients, the phenotype was evaluated before and during the chemotherapy by combination of doxorubicin and paclitaxel (AT). Compared with controls breast cancer patients had significantly higher relative and absolute numbers of CD3 HLA-DR+, CD3+CD69+ and CD14+CD16+, and significantly lower percentages of CD3 and CD8+CD28+ cells. After one cycle of AT, the absolute numbers of CD3 , CD3+CD4+, CD3+CD8+ and CD8+CD28+ cells increased significantly. Present data show a presence of T-cell activation in breast cancer patients. Administration of AT may lead to an increase in functional T-cells in peripheral blood, indicating a potential for combining chemotherapy with immunotherapy in the treatment of breast cancer patients.

Intraarterial chemotherapy of malignant melanoma metastatic to the liver.

BACKGROUND/AIMS: The prognosis of malignant melanoma metastatic to the liver is poor. The aim of the present report was to analyze retrospectively the effectiveness of regional chemotherapy and biologic therapy in patients with hepatic metastases of malignant melanoma. METHODOLOGY: Seven patients with hepatic metastases of malignant melanoma were treated by intraarterial administration of the combination of cisplatin, vinblastine and dacarbazine, or melphalan, with or without interleukin-2, interferon-alpha and interferon-gamma. RESULTS: The median survival of 4 patients with metastatic involvement initially limited to the liver is 19+ months in contrast to a median survival of 5 months in patients with concurrent extrahepatic disease. Intraarterial administration of cytokines led to an initial decrease in circulating lymphocyte numbers, with subsequent return to pretreatment levels, and to an increase in urinary neopterin, a marker of immune activation. CONCLUSIONS: Regional intraarterial administration of chemotherapy with or without cytokines may be effective for controlling hepatic metastases of malignant melanoma in patients with disease limited to the liver, but little benefit is evident in patients who present with concurrent extrahepatic disease.

Regional administration of irinotecan in combination with 5-fluorouracil and leucovorin in patients with colorectal cancer liver metastases--a pilot experience.

BACKGROUND/AIMS: Regional chemotherapy represents an effective approach for the control of isolated liver metastases of colorectal cancer. Fluoropyrimidines have been the basis of systemic and regional chemotherapy of this disease for several decades, but recent studies have demonstrated that the addition of irinotecan (CPT-11) ameliorates the results of systemic treatment. METHODOLOGY: Fifteen patients with isolated liver metastases of colorectal carcinoma were treated with regional administration of CPT-11 in combination with 5-fluorouracil/folinic acid (5-FU/FA). CPT-11 (100-200 mg, mean: 93 +/- 25, range: 51-125 mg/m2) was administered weekly in combination with 5-FU (1000-2000 mg, 857 +/- 182, range: 538-1301 mg/m2) and FA (100-350 mg). Peripheral blood leukocyte phenotype was examined during the treatment in selected patients. RESULTS: One patient achieved complete response, 4 patients had partial response, 7 patients had stable disease, 1 patient progressed and 2 patients were not evaluable. The response rate was 38% in evaluable patients, and a more than 50% decrease in serum carcinoembryonic antigen levels was observed in 12 out of 14 patients. The treatment was generally well tolerated. All patients, except one, are currently alive at mean follow-up of 11 +/- 6 (median: 10, range: 5-24) months. The therapy is still being continued in 8 patients. CONCLUSIONS: Weekly CPT-11/5-FU/FA is highly effective in the treatment of patients with metastatic colorectal cancer limited to the liver, even after failure of previous 5-FU/FA. The present regimen should be tested as a first line treatment in phase III trial.

[Determination of intracellular molecules in cells in hematopoietic malignancy using flow cytometry]. / Stanovení intracelulárních molekul bunek krevních malignit prutokovou cytometrií.

BACKGROUND: Detection of membrane molecules by immunophenotypisation is a routine counterpart of the laboratory tests which are needed for the complex diagnostic procedures of hematopoetic malignancies at present. The immunochemical detection of intracellular molecules is essential in such cases, where the expression of surface molecules on malignant cells is poor, or where the results of other diagnostic analyses are unequivocal (cases with ectopic expression of other lineage-specific molecules and cases of biphenotypic leukemias). METHODS AND RESULTS: We followed the procedure using 4% solution of paraformaldehyd in phosphate buffered saline for fixation of cells. Lysing solution G (Becton-Dickinson) was used for permeabilisation of cells to detect nuclear and cytoplasmatic molecules. The permeabilised cell suspension was than washed with 0.5% solution of Tween 20 in phosphate buffered saline. This simple and rapid procedure is readily available for the routine detection of intracellular molecules. This approach is important for the diagnosis of hematopoetic malignancies.

[Detection of multiple myeloma cells using multicolor immunofluorescence and flow cytometry]. / Prukaz bunek mnohocetného myelomu vícebarevnou imunofluorescencí s vyhodnocením prutokovou cytometrií.

The advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by the immunofluorescence using flow cytometry. We have examined the co-expression of CD19, CD38, CD45, CD54, CD56, and CD138 molecules in cells of peripheral blood and bone marrow aspirates in patients with multiple myeloma by 3-color flow cytometry. For the detection and characterization of multiple myeloma cells, combinations of following antibodies were used: anti-CD19 FITC, anti-CD38 FITC, anti-CD38 PE, anti-CD54 FITC, anti-CD56 PE-Cy5, anti-CD45 PE, anti-CD45 PE-Cy5 (Immunotech) and anti CD138 PE (Serotec). The samples were analyzed using EPICS XL (Coulter) flow cytometer, and the analysis was based on at least 10,000 events. Samples from 17 patients were analyzed. The percentage of multiple myeloma cells ranged between 0.3% and 54.2% in bone marrow aspirates and between 0.0 and 11.8% in periferal blood. The expression of CD138, CD38, CD54 and CD56 molecules was found in 100%, 100%, 85% and 68% of examined cases, respectively. In our opinion, multiple myeloma cells are best characterized by following combinations of antibodies: CD38 FITC/CD138 PE/CD45 PE-Cy5, CD54 FITC/CD138 PE/CD56 PE-Cy5 or CD54 FITC/CD38 PE/CD 56 PE-Cy5. The identification of a malignant clone is the first and the most important step in the characterization of the disease, determination of its prognosis and the detection of residual disease after treatment. Three-color flow cytometry represents a method which can meet these goals.

Decreased peripheral blood gamma delta T cells in patients with bronchial asthma.

Many cell populations are thought to be involved in the etiopathogenesis of bronchial asthma. We examined by flow cytometry the relative and absolute number of CD3+, CD4+, CD8+, alpha beta TcR+ or gamma delta TcR+ T cells, CD19+ B cells; and CD56+ natural killer (NK) cells in the peripheral blood of 26 adult patients with difficult-to-control asthma (DCA) and 22 patients with minimally symptomatic asthma (MSA). Statistically higher relative and absolute numbers of NK cells (18.39 +/- 10.67% and 0.38 +/- 0.17 x 10(9)/l) in comparison with healthy controls (11.77 +/- 8.06% and 0.25 +/- 0.19 x 10(9)/l) and significantly decreased relative and absolute numbers of gamma delta T cells (3.02 +/- 2.16% and 0.06 +/- 0.04 x 10(9)/l) in comparison with controls (5.65 +/- 2.90% and 0.13 +/- 0.08 x 10(9)/l) in the DCA patient group were found. After pooling of data from both MSA and DCA patients and dividing the patients according to the presence of allergy, the relative and absolute numbers of gamma delta T cells were found to be diminished in both the allergy (3.77 +/- 2.98 and 0.07 +/- 0.05 x 10(9)/l) and nonallergy (3.06 +/- 1.78% and 0.06 +/- 0.03 x 10(9)/l) groups in comparison with healthy controls. The reason for the low number of gamma delta T cells in the peripheral blood of patients suffering from bronchial asthma is under investigation.

Some serum activity markers of airways inflammation in difficult-to-control asthma patients.

UNLABELLED: The main aim of the present study was a search for a characteristic serum marker of inflammatory activity in the airways of asthmatics with difficult-to-control disease. Therefore, serum levels of interleukin-4 (IL-4), serum low-affinity Fc Epsilon Receptor II (sFcER II), Interferon-gamma (INF-gamma) Immunoglobulin-E (IgE), Interleukin-2 (IL-2), serum Interleukin Receptor 2 (sIL-2R) and Intercellular Adhesion Molecule-1 (sICAM-1) were measured in 2 groups of asthmatics: 1-26 patients with difficult-to-control asthma (DTCA), 2-22 asthmatics, minimally symptomatic (MSA). RESULTS: No significant difference in either measured parameters between the DTCA and MSA group in peripheral blood samples was found. CONCLUSION: The above mentioned serum markers of T- and B-cell activation as well as the serum ICAM-1 level are not sensitive enough to determine the type, activity and severity of the inflammatory process in the asthmatic airways.