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Introduction: Thymidine kinase 1 (TK1) is a metabolic enzyme involved in DNA synthesis. Most standard treatment protocols for lung cancer (LC) include cytotoxic agents, which are potential modulators of TK1. We aimed to assess the prognostic significance of serum TK1 activity and its role in monitoring chemotherapy in LC patients., Methods: TK1 activity was measured using the DiviTum (Biovica) assay in sera from 233 patients with non-small-cell lung cancer (NSCLC), 91 with small-cell lung cancer (SCLC), and 90 with benign lung disease., Results: TK1 activity was significantly associated with age, performance status, and stage in NSCLC and with stage and weight loss in SCLC. In multivariate analysis, pretreatment TK1 activity, adjusted for performance status, stage, and weight loss, independently affected survival in NSCLC (relative risk =1.45, p = 0.031) and SCLC (relative risk = 2.49, p = 0.001). In NSCLC patients, adjusted elevated TK1 activity (>100 Du/L) at pretreatment was a significant predictor of treatment failure (odds ratio = 2.55, p = 0.01). A small (less than twofold) increase in TK1 activity after the first and second cycle of chemotherapy was significantly associated with treatment failure and poor overall survival., Conclusions: Elevated pretreatment serum TK1 activity was an independent, adverse prognostic factor, based on survival, in the two main histological types of LC. A small (less than twofold) increase in TK1 activity after the first and second cycle of chemotherapy was associated with treatment failure and poor overall survival.

Previous studies indicated that BRCA haploinsufficiency was associated with activation of the EGF receptor (EGFR) signaling pathway and increased proliferative activity in mammary epithelial cells of healthy women. We hypothesized that these processes might be reflected in the expression of serologic soluble EGFR (sEGFR) and thymidine kinase 1 (TK1) activity, which signal the initial and final steps of the proliferative pathway, respectively. We found that healthy carriers of BRCA1/2 mutations (n = 80) showed a significantly higher TK1 activity than age-matched controls (P = 0.0003), and TK1 activity was similar in women with BRCA1 and BRCA2 mutations (P = 0.74). The sEGFR concentration was significantly higher in women with BRCA1 than in controls and BRCA2 mutation (P = 0.013 and 0.002, respectively). During follow-up, four of 80 BRCA1/2 mutation carriers developed breast cancer. These women showed a significantly higher TK1 activity and somewhat higher sEGFR concentrations than the other 76 BRCA1/2 carriers (P = 0.04 and 0.09, respectively). All tumors were negative for ovarian hormone receptors, but showed a high EGFR expression. This study was limited by the short-term follow-up (mean, 27 months; range, 5-45), which resulted in a small sample size. Women with BRCA1 and BRCA2 mutations that had undergone risk-reducing bilateral salpingo-oophorectomy (BSO) showed significantly lower sEGFR compared with those without surgery (P = 0.007 and 0.038, respectively). Larger, prospective studies are warranted to investigate whether TK1 and sEGFR measurements may be useful for identifying healthy BRCA1/2 carriers with high risk of developing breast cancer; moreover, sEGFR measurements may serve as effective tools for assessing risk before and after BSO., (©2013 AACR.)

Background: We compared two recently developed immunoassays for serum thymidine kinase 1 (TK1) activity: one manual assay (DiviTum, Biovica(®)) and one fully automated assay (Liaison, Diasorin(®))., Methods: The study included 368 women: 149 healthy blood donors (control), 59 patients with benign breast disease (BBD) and 160 patients with primary breast cancer (BC)., Results: A regression analysis of the Liaison (y) and DiviTum (x) assays for all three groups yielded the equation y=3.93+0.03x (r=0.85, n=368). The r-value in BC was higher than in control and BBD (0.90 vs. 0.81 and 0.64). The correlation between the two assays for TK1 values above the cut-off was higher compared to that below (0.88 and 0.59). Breakdown of the BBD group into subgroups with proliferative and non-proliferative lesions was effective only with the measurement of TK1 with DiviTum assay (p=0.03). The TK1 activity determined preoperatively in BC patients with DiviTum and Liaison assays was significantly associated with T-stage (for both p=0.01), presence of vascular invasion (p=0.002 and p=0.02), lack of estrogen receptor (ER) (p=0.001 and p=0.01) and progesterone receptor (PR) (p=0.01 and p=0.03) expression. Only TK1 analyzed with the DiviTum assay was associated with tumor grade and molecular subtype of BC (p=0.02 and p=0.003). Multivariate Cox proportional hazards analyses demonstrated that T-stage, PR status and TK1 activity measured by both methods (DiviTum, RR=3.0, p=0.02 and Liaison, RR=3.1, p=0.01) were independent predictors of disease recurrence., Conclusions: In spite of differences observed between TK1 activity measured by the DiviTum and Liaison assays, both of them may be used for recurrence prediction in preoperative evaluation of BC patients.

Aim: To investigate the prognostic significance of cancer antigen 15-3 (CA15-3) in primary breast cancer (BC)., Patients and Methods: This prospective study included 368 women: 62 patients with benign breast disease (BBD), 159 patients with invasive BC and 88 healthy blood donors (control). The median follow-up was 76 months (range, 43-99 months). Serum CA15-3 was measured with LIAISON® CA15-3® chemilluminescence immunoassay., Results: Significantly high levels of CA15-3 were found in patients with BC compared to controls (p=0.029), but not to the BBD group (p=0.16). Preoperative CA15-3 in patients with BC was significantly associated with tumor size (p=0.003), TNM stage (p=0.005), vascular invasion (p=0.018) and tumor necrosis (p<0.05). Increased CA15-3 (>30 U/ml) concentrations were more often found in patients with larger tumors (p<0.05), advanced stage (p=0.004) and node-positive disease (p=0.007). Patients with normal levels of CA15-3 had better recurrence-free survival (RFS) than those with elevated levels (p<0.001). After adjustment for T-stage, grade, tumor necrosis, estrogen receptor (ER) and progesterone receptor (PR) status, CA15-3 remained an important preoperative characteristic with independent impact on RFS (hazard ratio=4.4, 95% confidence interval=1.5-13.1, p=0.007). The independent prognostic contribution of CA15-3, considered on a continuous scale was significant among subgroups of the BC patients with ER/PR-positive (p=0.002), node-positive (p=0.028), node-negative (p=0.003), T1-stage node-negative (p=0.017), luminal-A (p=0.003), luminal-B (p=0.028) and human epidermal growth factor receptor-2 (HER2)/non-luminal disease (p=0.045)., Conclusion: Preoperative measurement of CA15-3 allowed identifying high-risk of recurrence for patients with primary BC and might be combined with existing prognostic factors in planning adjuvant treatment.

Objectives: Pyruvate kinase type M2 (TuM2-PK) and thymidine kinase 1 (TK1) are the key enzymes involved in tumor cells metabolism and proliferation. We explored the association of their preoperative circulating levels with disease recurrence in patients with renal cell carcinoma (RCC)., Methods: We measured the plasma levels of TuM2-PK levels and serum TK1 activity preoperatively in patients with RCC, using a quantitative ELISA, and correlated the results with clinicopathological parameters., Results: Significantly higher levels of TuM2-PK and TK1 were found in 116 patients with RCC compared with 20 healthy participants (P < .001 and P = .03), but not compared with 27 patients with benign kidney tumors (P = .13 and P = .72). There was a significant association between the level of TuM2-PK and of TK1 activity with T stage (P = .01 and P = .04). Of 2 markers only TuM2-PK was significantly associated with tumor grade (P = .001). The presence of extensive tumor necrosis (> 50%) was associated with high TuM2-PK (P = .001) and low TK1 (P = .03). The 5-year recurrence-free survival for patients with elevated TuM2-PK or TK1 was significantly lower compared with those for patients with normal marker levels (55% vs 94%, P < .001 and 21% vs 90%, P = .002). Multivariate Cox Proportional Hazard analysis demonstrated that TuM2-PK and TK1, adjusted for stage, grade and tumor necrosis were retained as independent predictors of disease recurrence (HR = 7.3, P = .04 and HR = 3.8, P = .03)., Conclusions: The measurements of 2 circulating biomarkers, TuM2-PK and TK1, in RCC patients before nephrectomy can be useful for predicting recurrence and stratifying the patients into risk groups for possible adjuvant treatment., (Copyright 2010 Elsevier Inc. All rights reserved.)

As the release and amount of circulating biomarkers show considerable variations between individuals, single value determinations are often difficult to be interpreted on their diagnostic or prognostic significance on the individual level. However, changes of the biomarker levels in a specific person during the disease course are quite informative for the estimation of the efficacy of therapy or the early detection of recurrent disease because they consider only intraindividual variations. If methods for marker determination are maintained, preanalytical and analytical standard prerequistits are respected, thresholds for each marker have to be defined which exceeds the normal, intraindividual biological variation. Then continuous biomarker increases may be indicative for disease activity in terms of inefficient therapy response or tumor recurrence while decreasing values often are associated with activity reduction of cancer disease. Here, we review the current knowledge on biomarker kinetics in patients with non-small cell lung cancer (NSCLC) and discuss the conditions and pitfalls of their relevance for the estimation efficacy of therapy and the early detection of recurrent disease. Further, we suggest a scenario to reveal the power of the defined biomarker use in future and to include those markers into the individual management of NSCLC patients.

Aims: Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis and an important proliferation marker. We explored the association of preoperative serum TK1 activity with clinicopathological parameters and prognosis in terms of recurrence-free survival (RFS) in breast cancer (BC) patients., Patients and Methods: TK1 activity in serum of 120 healthy women and 161 BC patients was measured by quantitative ELISA., Results: Serum TK1 activity in BC patients was significantly higher than in healthy women (P < 0.0001). In BC patients elevated TK1 activity was significantly associated with advanced T stage (P = 0.015), higher grade (P = 0.013), presence of tumor necrosis (P = 0.006), vascular invasion (P = 0.002), and lack of estrogen receptor (ER) and progesterone receptor (PR) expression (P = 0.0004 and P = 0.003). Higher TK1 activity was found in patients with BRCA1/2 mutations compared to those without the mutation (P = 0.004). Multivariate Cox proportional hazards analyses demonstrated that TK1, adjusted for stage, grade, necrosis, ER and PR negativity was retained as an independent predictor of disease recurrence (Hazard Ratio = 3.9, 95%CI 1.3-11.6, P = 0.013)., Conclusion: Elevated serum TK1 is an important risk factor indicating a high proliferation potential of tumors at the time of excision. In multivariate analysis TK1 activity was found to be an independent prognostic factor for RFS.

Aim: To investigate the diagnostic and prognostic significance of pro-gastrin-releasing peptide (ProGRP) in non-small cell (NSCLC) and small cell lung cancer (SCLC) and compare this marker with other known serum markers in lung cancer., Patients and Methods: Serum levels of ProGRP, neuron-specific enolase (NSE), CYFRA 21-1 and carcinoembryonic antigen (CEA) were measured in 37 patients with benign pulmonary disease (BPD), 88 with advanced NSCLC and 37 with SCLC., Results: The ProGRP assay showed a better clinical performance than that of NSE in discriminating between SCLC and BPD or NSCLC, especially at specificity higher than 90%. ProGRP and NSE sensitivity in SCLC at 95% specificity versus the BPD group was 78.4% and 48.6%, (p=0.001) and at 97.7% specificity versus NSCLC, 75.7% and 37.8%, respectively (p=0.001). A significant association of low ProGRP levels with high-grade NSCLC tumors was found (p=0.002). A univariate analysis showed a significant association of ProGRP with survival both in NSCLC and SCLC (p=0.03 and p=0.04, respectively). In multivariate analysis, performance status (PS) and CYFRA 21-1 in NSCLC, and PS, CYFRA 21-1 and serum lactic dehydrogenase in SCLC were found as significant variables with an independent impact on survival., Conclusion: ProGRP is a useful marker in SCLC, with diagnostic performance better than that of NSE and demonstrating association with survival in NSCLC and SCLC limited to univariate analysis.

Aim: This study aimed to evaluate the accuracy of urine cytology, bladder ultrasound (US), urine cytokeratin 19 fragment assay (CYFRA 21-1) and the combination of these noninvasive modalities in the detection of recurrent bladder cancer., Patients and Methods: In a total of 154 patients that were followed with cystoscopy after endoscopic resection of non-muscle-invasive bladder cancer, we performed and analyzed results of 311 observations that included cytology, CYFRA 21-1, US. The urine concentration of CYFRA 21-1 was measured by an immunoradiometric assay., Results: Cystoscopy and biopsy revealed recurrent bladder tumors in 21 patients. Most of the tumors (77%) were less than 10 mm in their largest diameter. Urine cytology, US and urine CYFRA 21-1 resulted in overall sensitivity of 19.1, 52.4 and 71.4% and specificity of 96.6, 99.7 and 68.6%, respectively. Each of these methods used alone yielded false-negative results in patients harboring tumors at high risk of progression. The combination of all three methods had sensitivity and specificity of 90.5 and 67.2%, respectively. All three tests were negative in 197 of 311 observations (63.3%), missing only 2 low-risk tumors., Conclusion: The combined use of US, urinary CYFRA 21-1 and cytology appears to be an effective, noninvasive approach for the detection of recurrent bladder tumors.

Heparanase is an endo-beta-glucuronidase capable of cleaving heparan sulfate (HS), an activity implicated in tumor metastasis. Heparanase expression is upregulated in primary human tumors, correlating with reduced post operative survival and elevated microvessel density. An ELISA method was used to quantify heparanase in urine from 282 individuals. Urine was collected from healthy volunteers (n = 41), patients diagnosed with noncancerous pathologic disorders (n = 90), and bladder cancer patients (n = 92). Fifty-nine bladder carcinoma patients after transurethral resection (TUR) with no evidence of disease (NED) were also included. Heparanase levels were significantly elevated in urine from bladder cancer patients compared with healthy controls (P < .001) and with noncancerous urinary disorders (P < .05). Heparanase elevation strongly correlated with tumor grade (P < .001) and stage (P = .027). An optimal cutoff value of 154 pg/ml was determined. Of 199 individuals enrolled (59 patients after TUR and 24 patients with recurring disease were excluded), 65 had heparanase levels above 154 pg/ml. Only 3 of 65 (4.6%) were healthy individuals. In contrast, 52.3% (34 of 65) of individuals with heparanase levels above 154 pg/ml were bladder cancer patients. The results indicate that urine heparanase levels are elevated during bladder cancer progression, suggesting that the ELISA method may be applied for bladder cancer diagnosis.

Heparanase is a mammalian endo-beta-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis, and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50 kDa active heparanase heterodimer vs. the latent 65 kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml and is suitable for quantification of heparanase in tissue extracts and urine.

Background: Chromogranin A (CGA), Pro-gastrin-releasing peptide (ProGRP) and neuron-specific enolase (NSE) are known as immunohistochemical tissue markers closely associated with neuroendocrine differentiation in non-small-cell lung carcinoma (NSCLC). The aim of the present study was to assess the value of serum levels of these markers in predicting response to chemotherapy and survival of patients with unresectable NSCLC., Methods: The study included 67 patients with advanced NSCLC treated with chemotherapy. Before treatment, serum levels of CGA, ProGRP and NSE were measured with commercial kits., Results: No association was found between serum NSE and age, gender, histology, performance status or extent of the disease. Distribution of serum CGA differed significantly according to gender and histology, with higher levels being found in men (p = 0.01) and in squamous cell carcinoma (p = 0.01). Serum ProGRP levels correlated with disease extent, being higher in patients with metastatic disease (M1) than in those with locoregional disease (M0; p = 0.02). The association of NSE, CGA and ProGRP levels with response to chemotherapy was not significant. While NSE had no impact on survival, the median survival was shorter for patients with elevated serum CGA and longer for patients with high ProGRP levels. Association with survival was significant when the Classification and Regression Tree (CART)-derived or median cutoff points were explored. On inclusion in multivariate Cox models, both CGA and ProGRP retained significance with high levels showing an opposite effect on survival [CART-derived cutoff points: CGA, relative risk (RR) -4.0; p < 0.001, and ProGRP, RR -0.4; p = 0.006, and median cutoff points: CGA, RR -1.8; p = 0.04, and ProGRP, RR -0.5; p = 0.03]. The combined use of CGA, ProGRP and NSE allowed for definition of two sets of patients with significantly different median survival times (25.2 vs. 8.8 months, p = 0.0001)., Conclusions: In the circulation, CGA and Pro-GRP appear to bear important information related to the prognosis for NSCLC patients before chemotherapy. While a high CGA before treatment was found as an unfavorable prognostic determinant, a high ProGRP conferred a survival advantage. The combined use of serum CGA, ProGRP and NSE may supply additional information to prognosis., (Copyright 2006 S. Karger AG, Basel.)

The prognostic value of various demographic, clinical and laboratory characteristics was investigated in 54 patients with metastatic breast cancer during first-line paclitaxel chemotherapy. As a single-agent treatment, paclitaxel (175 mg/m2) was given by 3-hour infusion every three weeks. The overall response rate was 30%. The follow-up ranged from 3 to 65 months (median 17 months). The most important pretreatment prognostic factors for survival were found to be hemoglobin (Relative Risk-2.26; p = 0.02) and serum lactic dehydrogenase (RR-1.81; p = 0.04) levels. The survival showed a strong association to the type of response. The median survival for responders was 5-fold greater than for patients with progressive disease (30.2 months and 5.7 months, respectively). Following the first-line paclitaxel treatment the estimates of tumor response became the major predictor of survival (RR-12.3; p < 0.0001).

Background: The urinary concentration of soluble cytokeratin 19 fragments, measured by the CYFRA 21-1 assay, may be used for the noninvasive, early detection of bladder carcinoma., Methods: This prospective study examined urine samples from 325 patients. The authors included 152 patients who presented with hematuria or irritative voiding symptoms (Group 1), 107 patients who were under surveillance after undergoing transurethral resection of bladder carcinoma (Group 2), 46 patients with urinary tract pathology other than bladder carcinoma (Group 3), and 20 healthy participants (Group 4). The urine concentration of CYFRA 21-1 was measured by an immunoradiometric assay. The patients in Groups 1 and 2 underwent cytoscopy and urine cytopathology. Biopsies were obtained if a tumor was seen on cytoscopy or if there was a suspicion of carcinoma in situ (CIS)., Results: The optimal cut-off concentration for the detection of primary bladder tumors, 4.9 microg/L, resulted in a sensitivity of 79.3% and a specificity of 88.6%. The optimal threshold for the detection of recurrent bladder tumors (excluding patients who had been treated with intravesical bacillus Calmette-Guerin [BCG]), 4.04 microg/L, resulted in a sensitivity of 76.2% and a specificity of 84.2%. There was no significant advantage for centrifugation of the urine samples or for determination of the creatinine concentration in the urine samples. The CYFRA 21-1 assay of urine samples provided a three-fold greater sensitivity compared with the sensitivity of cytology for detecting Grade 1 transitional cell tumors. CYFRA 21-1 detected 91.9% of Grade 3 tumors, 100% of CIS, and 92.8% of invasive bladder tumors (T2 or higher classification). The CYFRA 21-1 assay detected all tumors that had positive cytology with the exception of only one tumor. Conversely, the assay identified 71% of primary tumors and 65% of recurrent tumors that were missed by cytopathology. Urinary stones, infection, and previous intravesical BCG immunotherapy caused many false positive results., Conclusions: The urinary CYFRA 21-1 assay is a useful test for the noninvasive detection of bladder carcinoma and for surveillance of patients who were not treated previously with BCG. It may be used in combination with urine cytology and bladder ultrasound. Multi-institutional trials are required to compare the accuracy as well as the cost of this combination of tests with cystoscopy., (Copyright 2002 American Cancer Society.)

The prognostic value of the tumor markers CYFRA 21-1, tissue polypeptide specific antigen (TPS) and carcinoembryonic antigen (CEA) was investigated in three histologic subtypes of non-small cell lung cancer. Pretreatment serum marker levels were measured in 38 patients with adenocarcinoma (AC), in 43 patients with squamous cell carcinoma (SQC) and in 35 patients with large cell carcinoma (LCC). Univariate analysis in AC showed significant lower survival of patients with elevated levels of TPS, CYFRA 21-1 and CEA. In LCC, elevated levels of TPS and CEA were found to predict lower survival, while in SQC--only TPS was a predictor. A multivariate analysis of survival identified CEA (Relative Risk-5.5; p = 0.004), CYFRA 21-1 (RR-3.4; p = 0.008) and TPS (RR-3.0; p = 0.02) as independent prognostic factors in AC. In SQC, only TPS (RR-2.3; p = 0.03) was such a factor whereas in LC--none of the markers studied retained statistical significance. Thereafter, the combinations of the two strongest prognostic factors in the AC group--CEA and CYFRA 21-1 were explored to divide this group into subsets with different prognosis. In cases where both markers were positive, the relative risk of death was 10.5 times higher as compared to cases where both markers were negative (p = 0.002).

Gaucher's disease (GD) is characterized by hepatosplenomegaly, bone marrow infiltration, osteonecrosis, which may all be associated with the presence of pathological macrophages that contain undegraded glycosphingolipids. Levels of serum cytokines, which are soluble products of mononuclear phagocytes (MNP), were evaluated in 24 GD patients. Levels of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and soluble interleukin-2 receptor (sIL-2R) in GD patients were significantly higher than in normal controls. We attempted to correlate cytokine levels with disease severity. Type I GD patients with more severe clinical manifestations had significantly higher levels of IL-1beta, IL-1Ra and IL-6, relative to type I patients with milder disease. Three patients homozygous for the 1448C mutation with neuropathic type III disease, had significantly higher levels of sIL-2R than type I patients or controls. We speculate that cytokine over-expression may relate to the pathophysiology of some of the clinical manifestations of GD. Thus, the elevated IL-1beta, TNF-alpha and IL-6 levels may induce the bone manifestations, the neutrophil chemotaxis and the increased incidence of hyper-gammaglobulinemia present in GD patients.

Hairy cell leukemia (HCL) is a well recognized indolent B-cell lymphoproliferative disorder. HCL cell proliferation is regulated by growth factors and cytokines, of which tumor necrosis factor-alpha (TNF-alpha) may be one of the most important. The mechanism of TNF-alpha-induced HCL cell growth is mediated via 2 receptors, which are present in both cellular and soluble forms. In this study we determined the serum levels of TNF-alpha and their soluble receptors - sTNF-R60 and sTNF-R80 - in 23 HCL patients and correlated them with clinical parameters before and after therapy. Patients were classified according to their clinical status as either "active" at diagnosis or during relapse and "non-active" (responding to therapy with partial and complete remission). Most patients were treated with 2-CDA, following which serum levels of TNF-alpha, sTNF-R60 and sTNF-R80 were significantly decreased, particularly in CR. Significant differences in paired observation values were noted for TNF-alpha and sTNF-R80, indicating a good correlation with the clinical status of disease, but this was not the case for sTNF-R60 (coefficients of correlation between levels of TNF-alpha and sTNF-R80 and of TNF-alpha and sTNF-R60 were r = 0.85 and r= 0.64, respectively). These results suggest that decreases in both TNF-alpha and sTNF-R80 are indicative of response to treatment, while increased levels accompany active disease. Accordingly, we conclude that serum levels of the TNF family, as for the sIL-2R and IL-1 family, may also be used as sensitive markers for monitoring HCL status. Levels may be indicative of the clinical efficacy of therapy and can be used as an indicator of the presence of residual disease.

A clinical randomized study was performed on advanced breast cancer patients who were treated by interferons (IFN) beta and gamma in combination with hormonotherapy (Megace or Tamoxifen). Cytokine levels (IL-1beta, IL-2, IL-6, TNF-alpha, IFN-gamma) and sIL-2R of individual patients before, during (3 months) and after (6 months) therapy were evaluated and correlated to clinical response according to UICC criteria (responder patients-partial or Complete Response versus non-responder patients-Stable/Progression). Decreases in IL-1beta, IL-6 and sIL-2R were associated with clinical response to therapy versus increases in their levels which corresponded to progression of disease. A significant and dramatic increase in IFN-gamma levels was associated with a favourable response to therapy in the IFNs-treated patients, mainly in the group of Tamoxifen. Baseline levels of sIL-2R and of IFN-gamma were prognostic of clinical response and were found to be the most sensitive cytokine parameters for defining the clinical utility of the combination of IFNs and hormonotherapy in breast cancer patients., (Copyright 1998 Academic Press.)

Inflammatory cytokines have been shown to play an important role in the pathogenesis of various inflammatory processes. In throat infections, intracellular inflammatory cytokines have been detected from the sites of inflammation. The present study aimed to evaluate serum cytokine levels of patients with throat infections and correlate them to the inflammatory parameters and type of inflammation. Significantly higher inflammatory cytokine levels (interleukin [IL]-6 > 7 pg/mL, IL-1 > 1 beta pg/mL, tumor necrosis factor alpha > 1 pg/mL) were detected in most of the patients as opposed to healthy controls. Clinical parameters of infection (fever > 38 degrees C, leukocytosis > 11,000 white blood cells per cubic millimeter, polymorphonuclear neutrophils > 75%) were significantly correlated with high levels of inflammatory cytokines: mainly IL-6 and tumor necrosis factor alpha, and to a lesser degree with IL-1 beta. No correlation, however, was found between the type of inflammation and cytokine levels. The present study indicates a role of inflammatory cytokines in the pathogenesis of throat infections and the need for an anti-inflammatory and anticytokine therapeutic approach.

Hairy cell leukemia (HCL) is a well-recognized chronic lymphoproliferative disorder of B cell lineage, which may be regulated by growth factors including cytokines and cytokine antagonists. Previous studies have shown a good correlation between circulating soluble interleukin-2 receptor (sIL-2R) levels and disease activity and response to therapy was always associated with a decrease in sIL-2R levels. The interleukin-1 (IL-1) family of agonists and antagonists may also be involved in the regulation of hematopoietic malignancies. In the present study, we evaluated members of the IL-1 family (IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 soluble receptors Type I and Type II (IL-1sRI and IL-1sRII) in 23 patients with HCL. Patients were classified according to the clinical state of their disease. Most were treated with 2-chloro-2'-deoxyadercosine (2-CDA) and treatment was associated with a significant decrease in the serum levels of sIL-2R, IL-1beta and IL-1sRII in patients achieving a complete or partial response. In contrast to the above, levels of IL-1Ra increased during response to treatment and clinical response to 2-CDA was associated with an increase of 122% in IL-1Ra levels, in parallel with a decrease of 63% in IL-1beta and 47% in IL-1sRII levels. These results suggest that the balance between IL-1beta, IL-2 and their soluble receptors or antagonists may be involved in the pathogenesis and immunoregulation of HCL. Serum levels of these cytokines may therefore be used to monitor therapeutic efficacy of therapy in HCL and to detect any residual disease.

Previously available serum tumor markers had a low clinical value in malignant pleural mesothelioma (MPM). The recently developed tissue polypeptide-specific antigen (TPS) and CYFRA 21-1 assays identify the soluble cytokeratin 18 and 19 fragments, respectively. In MPM these cytokeratins are expressed and may therefore be used as serum tumor markers. In this preliminary study, TPS and CYFRA 21-1 assays were evaluated to determine their potential for management of patients with MPM. Carcinoembryonic antigen (CEA) was evaluated as an additional marker. The study group consisted of 95 patients with benign lung and pleural diseases (BLPD), 14 patients with MPM, 41 patients with adenocarcinoma of lung (AC), and 40 patients with squamous cell carcinoma of lung (SQC). The utilized cutoff points corresponded to a 95% specificity for patients with BLPD. In MPM, TPS showed greater sensitivity (64.3%) than CYFRA 21-1 (50.0%), while CEA showed no sensitivity. In SQC, the marker CYFRA 21-1 had the highest sensitivity (52.5%), whereas in AC the most sensitive marker was CEA (56.1%). Significantly lower levels of CEA were found in MPM compared with BLPD (p < 0.001) or AC and SQC (p < 0.0001). Conversely, TPS levels in MPM were significantly higher than in SQC (p < 0.05). Close correlation of various individual pretreatment marker levels was observed only between TPS and CYFRA 21-1, both in MPM (r = 0.84; p < 0.001) and in non-small cell lung cancer (NSCLC) (r = 0.71; p < 0.001). In serial determinations of the markers during chemotherapy of MPM (n = 10), TPS and CYFRA 21-1 were shown to demonstrate more or less the same pattern of reactivity, although the changes in the TPS levels better reflected the clinical response to therapy. In conclusion, TPS seems to be a more sensitive marker than CYFRA 21-1.

Graft rejection and graft failure represent major obstacles in allogeneic bone marrow transplantation (BMT). Cytokines possibly play a central role in the inflammatory and allospecific components of allograft rejection. Therefore, we evaluated inflammatory cytokine levels following BMT in 12 consecutive patients with graft rejection (GR). Seven of the patients underwent BMT from siblings (6 matched and 1 mismatched), 4 patients received bone marrow from other family members (3 mismatched and 1 matched), and 1 patient underwent HLA-matched unrelated BMT. Nine of 12 had a sex-mismatched BMT and 5/12 had an ABO-mismatched BMT. Nine of 12 underwent T cell-depleted (Campath anti-CDw52 moAb) BMT. Rejection was defined as marrow hypoplasia with a peripheral white blood cell count < 0.5 x 10(9)/L 21 days after BMT, in conjunction with the absence of donor cells by polymerase chain reaction analysis using a sex-mismatched probe and/or a tumor-specific probe (BCR/ABL). Twenty-five patients who underwent uneventful BMT with no GR served as controls. The levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1 (IL-1) were evaluated by a high sensitive RIA or an enzyme immunoassay. The levels of TNF and IL-6 were found to be higher in 10/12 and 7/7 evaluated GR patients, respectively, as compared with controls (P < 0.05). The level of IL-1 was high only in 2/12 patients. TNF elevation occurred in all patients immediately after GR. TNF and IL-6 levels were significantly higher for patients with early rejection (< 35 days after BMT) as compared with patients with late rejection (> 35 days after BMT) (P < 0.049 and P < 0.006, respectively). Eight patients engrafted after the second transplant (2 only transient). All 6 patients with stable engraftment are alive (4 with basic disease), while the 4 patients who did not engraft and the 2 patients with only transient engraftment died. In the 6 patients with no engraftment or only transient engraftment, the elevated TNF levels remained high; in the 6 patients who had stable engraftment after retransplant, TNF levels, but not IL-6 levels, decreased. In conclusion, a majority of the patients with GR displayed high levels of inflammatory cytokines (TNF and IL-6). Dysregulation of inflammatory cytokines may be involved in the pathogenesis of GR.

Some evidence points towards a possible autoimmune role in the aetiology of schizophrenia. Experimental findings provide contradictory results regarding abnormalities in cytokine production in this disorder. In the present study we tested the production of cytokines in CSF and serum in 16 schizophrenic patients and 10 healthy controls (tumor necrosis factor alpha - TNF alpha; interleukins IL-1 beta, IL-2, IL-6, soluble IL-2 receptor). Cytokine levels were evaluated by radioactively-labeled antibodies (IL-1 beta, IL-2, IL-6), by enzyme-linked immunoassay (TNF) and by a sandwich enzyme immunoassay (soluble IL-2 receptor). No significant differences were found in either CSF fluid or serum levels of TNF and IL-2 or IL-6. Interleukin-1 beta was significantly decreased in patients' CSF and serum as compared to controls. Soluble interleukin-2 receptor levels were decreased in CSF of patients, but highly increased in their serum in comparison with controls. Changes in various cytokine levels in CSF fluid and serum of schizophrenic patients probably reflect interrelated process of growth, degeneration or neuroimmunological abnormalities, which may all play a role in the pathophysiology of schizophrenia. The present study supports evidence for change in immune activation, probably of peripheral origin, in schizophrenic patients.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder of the B cell lineage. In the search for specific markers with prognostic significance we evaluated the clinical value of IL-1 beta and sIL-2R levels in HCL patients. HCL patients (25) were classified according to their clinical status as "active", "non active" disease or partial or complete remission and response treatment. We found a good correlation between IL-1 beta or IL-2R levels and disease activity: improved clinical status or response to different therapies were associated with decreasing IL-1 beta or sIL-2R levels. In contrast, lack of response to therapy or disease progression was reflected in increases in both IL-1 beta and sIL-2R levels. In some patients increases of both cytokines preceded clinical symptoms. In conclusion our results show that IL-1 beta and s-IL-2R levels may be used as reliable markers for monitoring HCL activity, assessing response to treatment and predicting early progression of disease.

By sequential selection for a resistance to a short-term heating at 45 degrees C, two independent clones, CHSR5 and CHSR6, have been isolated from ovarian cells of the Chinese hamster (CHO-K1). A subline CHO-A40, capable of proliferation at 40 degrees C, has been obtained as mass culture by culturing CHO-K1 cells for 2 months at 40 degrees C. The lines obtained have no cross-resistance to the temperature used in their selection: the CHSR5 and CHSR6 cells are not capable of reproduction at 40 degrees C, while the CHO cells do not survive over a 60 min heating at 45 degrees C. This bears evidence that mechanisms, underlying the cell resistance under different thermal regimens used, may be different. The heat resistant sublines maintain their heat resistance for 6 months of cultivation at 37 degrees C. The acquisition of heat resistance by cells correlates with their high primary resistance, and with their resistance to colchicine and actinomycin D.

The ability of the mouse neuroblastoma cell line NTR to proliferate at 40 degrees C correlates with the position of the temperature optimum of protein kinases A and C activities in the region of higher temperatures compared to those for cells of the original line N18AI, and with higher thermostability of protein kinase A after its heating at various elevated temperatures. The found changes in protein kinases A and C in the cells of NTR line mean that the selection of variants, capable of growing at elevated temperatures, is accompanied with conformational protein changes.

Under study was the dynamics of functional properties of leukocytes of healthy people and patients after UV-irradiation of their blood, mixing the UV-irradiated and non-irradiated blood. It was shown that UV-irradiation and mixing the irradiated and non-irradiated blood facilitated the liberation of bactericidal cation proteins of leukocytes, higher expression of membrane receptors, growth factors, activation of reparation of DNA after its injury by gamma-rays. It proves the role of immediate activation of leukocytes of the circulating blood in trigger mechanisms of effects of autotransfusion of UV-irradiated blood.

The recombinant plasmid DNA YEp secl-v-sis was constructed. This plasmid was able to code for the synthesis and secretion into the cultural medium of the protein-product of oncogene v-sis. Transcription, copy number and stability of the plasmid DNA were studied under the conditions of galactose induction. The v-sis protein was determined by gel electrophoresis and immunoblotting methods and assayed for cell-proliferation activity in the mammalian cell culture.

One-step selection of murine neuroblastoma (N18TG2 cell line) for the resistance to injuring action of higher temperature resulted in obtaining cell line NTR1 stably capable of proliferating at 40 degrees C. The thermoresistance is shown to be coupled with the changes in some phenotypic features of NTR1 cells: the multiplication rate, saturation density of cells, morphological features, inducibility of long neurite-like processes and adhesion. A possible significance of plasma membrane changes in genetically stable thermoresistance of NTR1 neuroblastoma cells is discussed.

The influence of cycloheximide on currents through sodium and potassium channels in dialysed neuroblastoma cells of clone N18A-1 has been studied under voltage clamp conditions. When cells are incubated with cycloheximide, the conductance of sodium decreases, whereas that of potassium changes only slightly, if at all. The cycloheximide concentration, at which sodium conductance is reduced by 50% during 24 hours incubation, was about 0.5 microgram/ml. The half-time of inhibition at the concentration of 15 microgram/ml was about 9 hours, with the time of 50% inhibition of sodium being more complex than single exponential. The density of sodium channels in control and after the maximal inhibition was estimated to be about 25 and 2.2 micron-2, respectively. The sodium-potassium selectivity and the gating mechanism parameters were shown to be unchanged following cycloheximide treatment.

The initiation of both transcription and translation occurs in membrane-associated nucleoid of E. coli without the addition of cytoplasmic fraction. Initiation of translation, however, is stimulated by adjunction of S 165. Our results suggest that the system is limited in synthesizing F-met-tRNA. The capacity of nucleoid-extracted ribosomes to form initiation complexes is only slightly higher than that of supernatant ribosomes. RNA synthesis is partially rifampicin-sensitive, which implies that initiation of new chains takes place in our system.

The induction of beta-galactosidase and alkaline phosphatase by the nucleoid of Escherichia coli was studied. Only the membrane-associated form was active in the presence of S 30. The induction of beta-galactosidase showed an absolute requirement for the inducer and was enhanced by cyclic AMP and cyclic GMP. Further-more, in our hands, the synthetic activity of the membrane-associated nucleoid proved to be far higher than that of the soluble system described by Zubay. Our results suggest that membrane shield the structure which is necessary for the integrity of the initiation step of both transcription and translation.

The effect of cycloheximide (an inhibitor of protein synthesis) on the ionic currents through sodium and potassium channels was investigated in dialysed voltage-clamped N18 A-1 neuroblastoma cells. The cycloheximide concentration needed for half-inhibition of sodium peak conductance was about 0.5 micrograms/ml for 24 h of incubation. Half-inhibition time of the sodium peak conductance in cells incubated with 15.0 micrograms/ml of cycloheximide was about 9 h. Sodium against potassium ion selectivity, the activation and inactivation parameters were shown to be not affected by cycloheximide. Potassium conductance in similarly treated cells exhibited no consistent changes. The main conclusion is that the decay in peak sodium conductance is caused by diminishing the sodium channel density in the membrane (from 25 to 2.2 channels per micron2). The inhibition effect was evidently mediated by block of protein synthesis and was not the result of direct drug-channel interaction. The half-decay time of sodium peak conductance is interpreted as a possible life-time characteristic of sodium channels in the neuroblastoma cell membrane.

General and primary thermoresistance of mouse neuroblastoma clonal cell lines derived from N18A subline was studied: the N18A1 clonal cell line was not treated by heat, the NTR1 was obtained by one-step selection for resistance to the long action of the temperature 40 degrees C, the NHSR1 was obtained by multistep selection for resistance to short-time treatment at 44 degrees C. The NHSR1 clonal line was shown to have higher general and primary thermoresistance by comparison with that of N18A1 cells. The NTR1 cell line, capable of unlimited proliferation at 40 degrees C, did not differ in general resistance but displayed a slower primary resistance compared to that in the N18A1 cells. Cells of all the three clones were found to be capable of temporary increasing in primary thermoresistance, i.e. hardening. A possible contribution of the primary resistance into the general one in cells of all the selected clones has been discussed.

Ionic currents of cells of neuroblastoma clone N18 A-1 was studied under conditions when the internal medium was placed for artificial fluoride or phosphate solutions. The specific membrane leakage resistance was measured to be 8.1 +/- 2.6 kOhm.cm2 and 1.3 +/- 0.3 Kohm.cm2, respectively. The presence of usual sodium and tetraethylammonium sensitive potassium channels is demonstrated. Potassium conductance is shown to amount to 0.25--0.025 of sodium conductance. Dialysis of the cells by phosphate solutions induces a slow outward current, which is not inhibited by tetraethylammonium ions.

Electrophoretic analysis of cell proteins of 3 mouse neuroblastoma strains differing in thermostability has shown that a distinctive feature of one of the strains (stable to the action of temperature at 44 degrees C) is the accumulation under normal conditions (37 degrees C) of heat shock protein with the molecular mass of 70 kD. Such accumulation of the specific protein does not take place in cells of the thermosensitive strain, stable to temperature of 40 degrees C.