Cell-free circulating ALU repeats in serum have a prognostic value for colorectal cancer patients.

BACKGROUND: Carcinoembryonic antigen (CEA) is the only established serum biomarker for colorectal cancer (CRC). To facilitate therapy decisions and improve the overall survival of CRC patients, prognostic biomarkers are required. OBJECTIVE: We studied the prognostic value of five different cell free circulating DNA (fcDNA) fragments. The potential markers were ALU115, ALU247, LINE1-79, LINE1-300 and ND1-mt. METHODS: The copy numbers of the DNA fragments were measured in the peripheral blood serum of 268 CRC patients using qPCR, the results were compared to common and previously described markers. RESULTS: We found that ALU115 and ALU247 fcDNA levels correlate significantly with several clinicopathological parameters. An increased amount of ALU115 and ALU247 fcDNA fragments coincides with methylation of HPP1 (P< 0.001; P< 0.01), which proved to be a prognostic marker itself in former studies and also with increased CEA level (both P< 0.001). ALU115 and ALU247 can define patients with poor survival in UICC stage IV (ALU115: HR = 2.9; 95% Cl 1.8-4.8, P< 0.001; ALU247: HR = 2.2; 95% Cl 1.3-3.6; P= 0.001). Combining ALU115 and HPP1, the prognostic value in UICC stage IV is highly significant (P< 0.001). CONCLUSIONS: This study shows that an increased level of ALU fcDNA is an independent prognostic biomarker for advanced colorectal cancer disease.

Development and External Validation of the Munich Sorafenib Evaluation Score for Hepatocellular Carcinoma.

INTRODUCTION: In recent years, increasing options for systemic HCC treatment have become available. The development of therapy-specific prognostic scores has been encouraged. Tailoring therapy to individual patients requires prognostic scores for treatment success in addition to the Barcelona-Clinic-Liver-Cancer (BCLC) classification. We have developed and validated a prognostic score for patients treated with sorafenib. METHODS: Prognostic factors identified in a multivariate analysis of 108 sorafenib patients were used to construct the Munich Sorafenib Evaluation (M-SE) score. M-SE and 9 established HCC prognostic systems were ranked according to concordance-index and AIC. External M-SE validation was performed in an independent HCC sorafenib cohort (n = 101) derived from the prospective multicenter randomized controlled SORAMIC trial. RESULTS: Ascites (p < 0.0001; HR 2.923), tumor burden ≥50% of the liver (p = 0.0033; HR 1.946), and GOT (p < 0.0001; HR 1.716) were identified as independent prognostic parameters. All three M-SE stages were characterized by significantly different survival times (p < 0.0001). M-SE stage-A patients had a median OS of 18.7 months (95% CI: 15.6-21.8); patients in stage B and C showed a significantly shorter survival of 5.7 (2.7-8.7) and 2.0 months (1.6-2.4), respectively. M-SE (c-index 0.70; AIC 621) outperformed all other prognostic systems. External validation in a prospective cohort confirmed its superior prognostic performance. CONCLUSION: The M-SE score allows classification of sorafenib patients in three distinct prognostic stages. Provided that M-SE successfully passes prospective validation, it can help to predict the outcome of patients evaluated for sorafenib treatment.

Performance testing of four automated coagulation analyzers in a university hospital setting with focus on global coagulation assays.

INTRODUCTION: Several automated coagulation analyzers are available for laboratory use. In a university hospital central laboratory, we compared four different instruments. The results for global coagulation assays are presented here. METHODS: ACL TOP 750 CTS (Instrumentation Laboratory), Atellica COAG 360 (COAG 360), BCS XP (both Siemens Healthineers), and cobas t 711 (Roche Diagnostics) were compared. For inter-instrument comparison, five basic coagulation parameters were analyzed in 476 patient plasma samples. Additional assessments included precision testing using commercial control samples and plasma pools, analysis time for a defined set of samples, sample capacity testing, minimum required sample volumes, and detection quality for hemolytic, icteric, or lipemic (HIL) interferences. RESULTS: Good concordance between different instruments was evident from Bland-Altman plots and comparison of data from each instrument with the overall median (τ≥0.8). Shortest analysis times were found for BCS XP and COAG 360, COAG 360 revealed highest sample capacity. Observed required sample volumes were broadly in line with manufacturer specifications and varied according to instrument configurations. HIL detection differed between instruments and was best with ACL TOP 750 CTS. CONCLUSION: The four analyzers showed similarly high levels of concordance, although some variations were apparent. The most significant differences between the instruments were found in analysis times and sample capacity. Analyzer capabilities must be considered when selecting laboratory equipment and defining algorithms for clinical practice.

A Simple Prognostic Scoring System for Hepatocellular Carcinoma Treated with Selective Internal Radiation Therapy.

INTRODUCTION: Selective internal radiation therapy (SIRT) is a local treatment option for patients with hepatocellular carcinoma (HCC). Its exact role next to other HCC therapies has yet to be defined. In order to identify patients most suitable for SIRT, a SIRT-specific prognostic score should be developed. METHODS: A cohort of 72 SIRT patients treated at the University Hospital of Munich was retrospectively analyzed. The prognostic performance of 12 HCC staging systems and prognostic scores was assessed. Cox-regression analysis was used to identify independent prognostic factors, which formed the basis of the Munich-SIRT score (M-SIRT). All scores were ranked by calculating the c-Index and Akaike information criterion (AIC). External validation was performed in a cohort of 128 SIRT patients treated at the University Hospital of Pamplona, Spain. RESULTS: median overall survival was 13 months (95% confidence interval 9.9-21.9). AFP (p = 0.005; hazard ratio [HR] 2.38), albumin (p < 0.001; HR 5.87), and alkaline phosphatase (p < 0.001; HR 8.38) were identified as independent prognostic factors. M-SIRT comprises 3 prognostic groups with a median survival of 38.9, 14.6, and 7.7 months, respectively (I vs. II: p = 0.003, II vs. III: p < 0.001). AIC (318) and concordance index (0.711) ranked M-SIRT superior to the established HCC staging systems, and the score successfully passed external validation in an independent SIRT cohort (I vs. II: p = 0.03; II vs. III: p = 0.007). CONCLUSION: Therapy-specific prognostic scores can facilitate treatment decisions and prognostication for HCC patients. Considering its performance in 200 SIRT patients, M-SIRT is a promising prognostic tool for HCC patients evaluated for SIRT.

The Munich-Transarterial Chemoembolisation Score Holds Superior Prognostic Capacities Compared to TACE-Tailored Modifications of 9 Established Staging Systems for Hepatocellular Carcinoma.

BACKGROUND/AIMS: The recently proposed Munich-transarterial chemoembolisation-score (M-TACE) was tailored to suit hepatocellular Carcinoma (HCC) patients evaluated for TACE. M-TACE outperformed the established HCC-staging-systems and successfully passed external validation. Modifications of staging-systems through the rearrangement of stages or by adding prognostic factors are methods of improving prognostic power. M-TACEs performance compared to scores modified this way should be tested. METHODS: Seven well-known HCC staging-systems (including Cancer of the Liver Italian Program-score [CLIP] and Barcelona Clinic liver cancer [BCLC]) and 2 TACE-specific scores (Selection for Transarterial Chemoembolisation Treatment [STATE] and Hepatoma Arterial embolisation Prognostic [HAP]) were rearranged in a cohort of 186 TACE-patients through score-point-analysis and subsequent linking of non-significant adjacent score-points. Additionally, a new score was constructed by combining the top established staging-system in TACE patients (CLIP-TACE) and the prognostic parameter with the highest hazard ratio for death in the TACE-cohort [C-reactive protein (CRP)]. Additionally, the TACE-tailored-scores were applied to an external TACE-cohort (n = 71). -Results: Rearrangement resulted in optimal stratification and monotonicity. CLIP-TACE demonstrated the best prognostic capability of all rearranged scores (c-index 0.668, AIC 1294) and the addition of CRP yielded further prognostic improvement (c-index 0.680, AIC 1289). However, superiority over M-TACE could not be achieved by any of the new scores in the internal and external cohort. CONCLUSION: M-TACE outperforms TACE-tailored modifications of all relevant HCC-staging-systems. Prospective validation of M-TACE to promote its role as the preferred staging-system for TACE-patients is therefore justified.

Correlation Between Baseline Osteoprotegerin Serum Levels and Prognosis of Advanced-Stage Colorectal Cancer Patients.

BACKGROUND/AIMS: Osteoprotegerin (OPG) is a soluble receptor of the pro-apoptotic cytokine TRAIL which is thought to contribute to tumour development by inhibiting apoptosis or affecting other aspects of tumour biology, including cell proliferation and immune response. Although immunohistochemical studies suggest that OPG correlates with survival in metastatic colorectal cancer (mCRC), only scarce data are available on serum OPG in CRC patients. METHODS: In this pilot study, we assessed the prognostic significance of serum OPG and CEA (Carcinoembryonic antigen) in 81 patients with UICC (Union for International Cancer Control) stage-IV mCRC. OPG was additionally assessed by immunohistochemistry in primary tissue samples from 33 patients of the same cohort. RESULTS: Baseline serum OPG correlated with CEA (r=0.36, p=0.0011), but independently predicted survival of mCRC patients. Life expectancy was poorer in patients with OPG levels above the median concentration of 51ng/ml (median overall survival [95% confidence interval] 1.8 years [1.3-3.0] vs. 1.0 [0.7-1.2] p=0.013). Patients with high levels of both OPG and CEA had an even poorer life expectancy vs. low-OPG/low-CEA patients (0.9 years [0.6-1.5] vs. 3 years [1.2-4.4], p=0.015), indicating that CEA and OPG have additive prognostic significance. Immunohistochemical analysis of OPG failed to show a correlation between OPG staining and survival (p=0.055) or OPG concentration from matched serum samples. CONCLUSIONS: This pilot study provides evidence of independent prognostic significance of serum OPG in patients with advanced mCRC and warrants its further prospective validation.

Transarterial chemoembolization for hepatocellular carcinoma: development and external validation of the Munich-TACE score.

BACKGROUND: Allocation of patients with hepatocellular carcinoma (HCC) to the adequate therapy is determined by both tumor burden and liver function. The Barcelona Clinic Liver Cancer (BCLC) staging system and therapeutic algorithm recommends transarterial chemoembolization (TACE) based on the best evidence available to patients with intermediate-stage HCC (BCLC-B). However, many centers also treat subgroups of patients outside these recommendations and with more advanced disease by TACE. The purpose of this study was to identify prognostic factors in a TACE cohort, including BCLC-B patients, as well as patients treated outside of BCLC-B, to test the prognostic capabilities of published staging systems and to optimize prognostication for TACE patients. PATIENTS AND METHODS: A cohort of 186 first-line TACE patients was analyzed. Independent prognostic factors were identified and used to construct the Munich-TACE score (M-TACE). M-TACE was tested against established staging systems (including BCLC and two recently published TACE-specific scores) and a ranking using concordance index and Akaike Information Criterion was performed. Finally, an external validation in an independent TACE cohort (n=71) was conducted. RESULTS: Bilirubin, Quick/international normalized ratio, C-reactive protein, creatinine, α-feto protein, and tumor extension were identified as independent prognostic factors and used to construct M-TACE. M-TACE identifies three distinct subgroups (P<0.0001) with median survival times of 35.2, 16.9, and 8.6 months, respectively. Compared with established staging systems, M-TACE showed the best prognostic capabilities in both cohorts of patients (cohort 1: c-index, 0.71; Akaike Information Criterion: 1276; cohort 2: c-index, 0.754). CONCLUSION: We identified independent risk factors for patients treated with TACE. The newly constructed M-TACE score is superior to established staging systems and might prove helpful to identify patients who are most suitable for TACE.

Methylated free-circulating HPP1 DNA is an early response marker in patients with metastatic colorectal cancer.

Detection of methylated free-circulating DNA (mfcDNA) for hyperplastic polyposis 1 (HPP1) in blood is correlated with a poor prognosis for patients with metastatic colorectal cancers (mCRC). Here, we analyzed the plasma levels of HPP1 mfcDNA in mCRC patients treated with a combination therapy containing a fluoropyrimidine, oxaliplatin and bevacizumab to test whether HPP1 mfcDNA is a suitable prognostic and response biomarker. From 467 patients of the prospective clinical study AIO-KRK-0207, mfcDNA was isolated from plasma samples at different time points and bisulfite-treated mfcDNA was quantified using methylation specific PCR. About 337 of 467 patients had detectable levels for HPP1 mfcDNA before start of treatment. The detection was significantly correlated with poorer overall survival (OS) (HR = 1.86; 95%CI 1.37-2.53). About 2-3 weeks after the first administration of combination chemotherapy, HPP1 mfcDNA was reduced to non-detectable levels in 167 of 337 patients. These patients showed a better OS compared with patients with continued detection of HPP1 mfcDNA (HR HPP1(sample 1: pos/ sample 2: neg) vs. HPP1(neg/neg) = 1.41; 95%CI 1.00-2.01, HPP1(neg,pos/pos) vs. HPP1(neg/neg) = 2.60; 95%CI 1.86-3.64). Receiver operating characteristic analysis demonstrated that HPP1 mfcDNA discriminates well between patients who do (not) respond to therapy according to the radiological staging after 12 or 24 weeks (AUC = 0.77 or 0.71, respectively). Detection of HPP1 mfcDNA can be used as a prognostic marker and an early marker for response (as early as 3-4 weeks after start of treatment compared with radiological staging after 12 or 24 weeks) to identify patients who will likely benefit from a combination chemotherapy with bevacizumab.

Variability of piperacillin concentrations in relation to tazobactam concentrations in critically ill patients.

Therapeutic drug monitoring for critically ill patients receiving piperacillin/tazobactam is described as a useful tool. However, the minimum inhibitory concentration of piperacillin depends on a sufficiently high concentration of tazobactam in case of ß-lactamase-producing strains. Therefore, the relationship between piperacillin and tazobactam concentrations was assessed in a heterogeneous group of critically ill patients. Sixty patients with severe infections receiving 4.5 g of piperacillin/tazobactam 2-3 times daily by intermittent infusion were included in this prospective observational study (NCT01793012). Over 4 days, multiple serum samples were obtained to determine the total piperacillin and tazobactam concentrations. The target ranges were defined as trough levels >16 mg/L (>22.5 mg/L) and >4 mg/L (>5.7 mg/L) for the calculated unbound concentrations (measured total concentrations) of piperacillin and tazobactam, respectively. Despite a high correlation coefficient (r = 0.93) comparing piperacillin and tazobactam trough levels, the piperacillin/tazobactam quotients varied between ca. 1 and 10. From linear regression analysis of piperacillin versus tazobactam values, it follows that a piperacillin trough level of 22.5 mg/L might be associated with tazobactam trough levels ranging from 1.5 mg/L to 10.1 mg/L. A 70 mg/L threshold for total piperacillin trough levels would be necessary to ensure that tazobactam concentrations are >5.7 mg/L. Because of the observed variability of piperacillin/tazobactam quotients, defining the total piperacillin target range ≥70 mg/L might be useful to ensure that tazobactam concentrations do not fall below 5.7 mg/L. Further studies are necessary to confirm that the used therapeutic ranges are associated with optimal outcomes in critically ill patients.

Tumor markers in the early detection of tumor recurrence in breast cancer patients: CA 125, CYFRA 21-1, HER2 shed antigen, LDH and CRP in combination with CEA and CA 15-3.

BACKGROUND: Kinetics of CA 15-3 and CEA have a high specificity in the early detection of metastatic breast cancer (MBC). However, this high specificity is associated with a lack of sensitivity. To decrease the number of false negative patients, the additional diagnostic potential of an extended panel of biomarkers was evaluated. METHODS: This analysis was performed as part of a large follow-up study (1998-2010) evaluating 813 patients with a median follow-up of 63months. After primary therapy, all patients underwent tumor marker monitoring for CEA and CA 15-3 at 6-week intervals. A reproducible previously defined increase (≥100%) based on the individual baseline value of each patient was considered as a strong indicator of MBC. For the present analysis, we retrospectively evaluated 1011 blood samples from 95 patients. Forty-seven of these had metastatic disease for the first time at the time of this evaluation, while the remaining 48 patients showed no evidence of disease. The sera of these patients were additionally assessed for the following parameters: cancer antigen (CA) 125, cytokeratin-19 soluble fragment (CYFRA 21-1), HER2 shed antigen, lactate-dehydrogenase (LDH) and C-reactive protein (CRP). RESULTS: 26 of 47 patients with MBC showed a reproducible tumor marker increase of at least CEA and/or CA 15-3 (55.3%, true-positive). The remaining 21 patients with MBC showed no increase in CEA or CA 15-3 (44.7%, false negative, FN). By combining all markers mentioned above, 41 of 47 patients with MBC showed a reproducible marker increase with a sensitivity of 87.2% and specificity of 100%. CONCLUSION: This retrospective analysis indicates that a panel of biomarkers can increase the sensitivity of the CA 15-3/CEA combination without loss of specificity. The combined use is therefore helpful for early detection of MBC.

Piperacillin concentration in relation to therapeutic range in critically ill patients--a prospective observational study.

BACKGROUND: Piperacillin levels after standard dosing have been shown frequently to be subtherapeutic, especially when renal clearance was augmented. Here, we aimed to determine if piperacillin was in its therapeutic range in a typically heterogeneous intensive care unit patient group, and also to describe target attainment dependent on daily dosage, creatinine clearance, and renal replacement therapy (RRT). METHODS: Sixty patients with severe infections were included in this monocentric prospective observational study. Patients received 4.5 g of piperacillin-tazobactam two to three times daily by intermittent infusion depending on renal function according to clinical guidelines. Over 4 days, multiple serum samples (median per patient, 29; in total, 1627) were obtained to determine total piperacillin concentrations using ultra-high-performance liquid chromatography/tandem mass spectrometry. RESULTS: A high heterogeneity of patient characteristics was observed (e.g., on day 1: creatinine clearance 2-233 mL/min and ten patients on RRT). Piperacillin trough levels showed inter-individual variation from 123 to >1785-fold on different study days. Each day, approximately 50% and 60% of the patients had piperacillin levels below the target ranges 1 and 2, respectively [defined for the calculated unbound piperacillin fraction according to the literature as 100% time above MIC (100%fT > MIC) (target range 1) and ≥ 50%fT > 4 × MIC (target range 2); MIC = 16 mg/L]. Whereas only the minority of patients who received piperacillin-tazobactam three times daily (TID) reached target 1 (38% on day 1), most patients who received piperacillin-tazobactam only twice daily (BID) because of severely impaired renal function reached this target (100% on day 1). Patients with RRT had significant higher percentages of fT > MIC. Zero percent, 55% and 100% of patients without RRT who received antibiotics TID reached target 1 when creatinine clearance was > 65 mL/min, 30-65 mL/min and < 30 mL/min, respectively. In patients with causative strains only sensitive to piperacillin-tazobactam of all antibiotics given to the patient, piperacillin levels negatively correlated with CRP concentrations of day 4 (p < 0.05). CONCLUSIONS: A dosage of 4.5 g piperacillin-tazobactam TID seems to be frequently insufficient in critically ill patients, and also in patients where renal function is mildly to moderately impaired. For these patients, prescription of 4.5 g piperacillin-tazobactam four times daily could be considered. TRIAL REGISTRATION: Clinicaltrials.gov NCT01793012. Registered 24 January 2013.

Diagnostic efficacy of CA 15-3 and CEA in the early detection of metastatic breast cancer-A retrospective analysis of kinetics on 743 breast cancer patients.

OBJECTIVE: We investigated the diagnostic capacity of CEA and CA 15-3 kinetics for the early detection of metastatic disease in comparison to fixed cut off values. METHODS: In a retrospective analysis, a total of 743 patients with early breast cancer and available baseline values of CEA and CA 15-3 were included. A reproducible increase of 100% of single or combined markers was considered as a strong indicator of metastatic disease. RESULTS: 187 patients developed metastatic disease and 556 remained disease-free. On the basis of tumor marker kinetics, we reached a specificity of >98% for both biomarkers and a sensitivity of 40.6% for CEA alone, 55.6% for CA 15-3 alone and 66.3% for the combination of both markers. Using fixed cut-off values (CEA: 4ng/mL, CA 15-3: 30U/mL) we ended up with a specificity of 86.3% and a sensitivity of 70.6% for the combination of CEA and CA 15-3. Using higher cut-off values (CEA: 6ng/mL, CA 15-3: 60U/mL) we reached a specificity of 96.9% and a sensitivity of 49.7% for the combination. CONCLUSION: We conclude that the interpretation of these markers in follow-up using individual baseline values and kinetics leads to a significant superior profile of specificity and sensitivity.

Preoperative serum markers for individual patient prognosis in stage I-III colon cancer.

Carcinoembryonic antigen (CEA) remains the only recommended biomarker for follow-up care of colorectal cancer (CRC), but besides CEA, several other serological parameters have been proposed as prognostic markers for CRC. The present retrospective analysis investigates a comprehensive set of serum markers with regard to cancer-specific survival (CSS) and disease-free survival (DFS). A total of 472 patients with colon cancer underwent surgery for curative intent between January 1988 and June 2007. Preoperative serum was analyzed for the following parameters: albumin, alkaline phosphatase (aP), beta-human chorionic gonadotropin (ßhCG), bilirubin, cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), CA 72-4, CEA, C-reactive protein (CRP), cytokeratin-19 soluble fragment (CYFRA 21-1), ferritin, gamma-glutamyltransferase (γGT), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), hemoglobin, haptoglobin, interleukin-6, interleukin-8, creatinine, lactate dehydrogenase (LDH), serum amyloid A (SAA), and 25-hydroxyvitamin D. After a median follow-up period of 5.9 years, the overall 3- and 5-year CSS was 91.7 and 84.9 % and DFS rates were 82.7 % (3 years) and 77.6 % (5 years). Multivariate analyses confirmed preoperative CEA as an independent prognostic factor with regard to CSS and DFS. CA 19-9 and γGT also provided prognostic value for CSS and DFS, respectively. Younger age was negatively associated with DFS. According to UICC stage, CEA provided significant prognostic value with regard to CSS and DFS, while CA 19-9 was only prognostic for CSS. Combined analysis is able to identify patients with favorable prognosis. In addition to tumor baseline parameters, preoperative CEA could be confirmed as prognostic marker in colon cancer. CA 19-9 and γGT also provide additional prognostic value with regard to survival and recurrence in stage III and stage I disease, respectively. The combined use of CEA together with CA 19-9 and γGT improve risk-adapted post-op surveillance.

Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000.

BACKGROUND: Various types of automated hematology analyzers are used in clinical laboratories. Here, we performed a side-by-side comparison of five current top of the range routine hematology analyzers in the setting of a university hospital central laboratory. METHODS: Complete blood counts (CBC), differentials, reticulocyte and nucleated red blood cell (NRBC) counts of 349 patient samples, randomly taken out of routine diagnostics, were analyzed with Cell-Dyn Sapphire (Abbott), DxH 800 (Beckman Coulter), Advia 2120i (Siemens), XE-5000 and XN-2000 (Sysmex). Inter-instrument comparison of CBCs including reticulocyte and NRBC counts and investigation of flagging quality in relation to microscopy were performed with the complete set of samples. Inter-instrument comparison of five-part differential was performed using samples without atypical cells in blood smear (n=292). Automated five-part differentials and NRBCs were additionally compared with microscopy. RESULTS: The five analyzers showed a good concordance for basic blood count parameters. Correlations between instruments were less well for reticulocyte counts, NRBCs, and differentials. The poorest concordance for NRBCs with microscopy was observed for Advia 2120i (Kendall's τb=0.37). The highest flagging sensitivity for blasts was observed for XN-2000 (97% compared to 65%-76% for other analyzers), whereas overall specificity was comparable between different instruments. CONCLUSIONS: To the best of our knowledge, this is the most comprehensive side-by-side comparison of five current top of the range routine hematology analyzers. Variable analyzer quality and parameter specific limitations must be considered in defining laboratory algorithms in clinical practice.

Serum HER2 in combination with CA 15-3 as a parameter for prognosis in patients with early breast cancer.

AIM: HER2 in breast cancer tissue is a marker of high prognostic and predictive relevance. Soluble HER2, the extracellular domain of the HER2/neu receptor (HER2 ECD), which is shed into the blood, has been suggested to be a helpful tumor marker. We investigated the relationship between the concentrations of HER2 ECD, CEA and CA 15-3, the association of these markers with clinicopathological features and the impact of HER2 ECD alone and in combination with known prognostic factors on disease free survival (DFS) and cancer specific survival (CSS) in untreated early breast cancer patients. PATIENTS AND METHODS: HER2 ECD (ADVIA, Bayer), CEA (AxSYM, Abbott) and CA 15-3 (Elecsys, Roche) were measured at time of primary diagnosis in the pre-therapeutic (pre-operative) sera of 241 breast cancer patients and were correlated with clinicopathological parameters and outcome. RESULTS: Higher HER2 ECD levels were significantly correlated with postmenopausal status (p=0.016) and tissue HER2-overexpression (p<0.0001). Higher serum levels of CA 15-3 were associated with larger tumor size (p=0.019), positive lymph nodes (p=0.019), UICC stage III (p<0.01), positive tissue HER2-overexpression (p<0.05) and negative hormone receptor status (p=0.016). In multivariate analysis, serum HER2 ECD levels, CA 15-3 levels, large tumor size and negative hormonal status were independent prognostic factors in DFS. Patients with both high levels of HER2 ECD (>15 ng/mL) and high serum levels of CA 15-3 (>24 U/mL) had the poorest prognosis with a DFS after 3 years of 50.0%. Patients without elevated serum levels had a better outcome with a DFS of 91.2%. CONCLUSIONS: In our retrospective analysis, HER2 ECD and CA 15-3 were independent and better prognostic tools than HER2 in tissue. Prospective validation is necessary to confirm their usefulness in clinical practice.

Comparison of whole blood RNA preservation tubes and novel generation RNA extraction kits for analysis of mRNA and MiRNA profiles.

BACKGROUND: Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA. METHODS: 3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed. RESULTS: RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed. CONCLUSION: We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.

Evaluation of preoperative serum markers for individual patient prognosis in stage I-III rectal cancer.

Several independent serum biomarkers have been proposed as prognostic and/or predictive markers for colorectal cancer (CRC). To this date, carcinoembryonic antigen (CEA) remains the only recommended serological CRC biomarker. The present retrospective analysis investigates the prognostic value of several serum markers. A total of 256 patients with rectal cancer underwent surgery for curative intent in a university cancer center between January 1988 and June 2007. Preoperative serum was retrospectively analyzed for albumin, alkaline phosphatase (aP), beta-human chorionic gonadotropin, bilirubin, CA 125, cancer antigen 19-9, cancer antigen 72-4 (CA 72-4), CEA, CRP, CYFRA 21-1, ferritin, gamma-glutamyl transpeptidase, glutamate oxaloacetate transanunase, glutamate pyruvate transaminase, hemoglobin, haptoglobin, interleukin-6, interleukin-8, creatinine, lactate-dehydrogenase, serum amyloid A (SAA), and 25-hydroxyvitamin D. Cancer-specific survival (CSS) and disease-free survival (DFS) were estimated. Median follow-up time was 8.4 years. Overall 3- and 5-year CSS was 88.6 and 78.9 %, respectively. DFS rates were 72.8 % (3 years) and 67.5 % (5 years). Univariate analysis of CSS indicated aP, CA 72-4, CEA, and SAA as prognostic factors, while aP, CEA, and SAA were also prognostic with regard to DFS. Multivariate analysis confirmed SAA together with T and N stage as prognostic factors. According to UICC stage, CEA and SAA add prognostic value in stages II and III with regard to DFS and CSS, respectively. The combined use of CEA and SAA is able to identify patients with favorable and poor prognosis. In addition to tumor baseline parameters, routine analysis of SAA together with CEA provided markedly improved prognostic value on CSS and DFS in resected rectal cancer.

Variability of linezolid concentrations after standard dosing in critically ill patients: a prospective observational study.

INTRODUCTION: Severe infections in intensive care patients show high morbidity and mortality rates. Linezolid is an antimicrobial drug frequently used in critically ill patients. Recent data indicates that there might be high variability of linezolid serum concentrations in intensive care patients receiving standard doses. This study was aimed to evaluate whether standard dosing of linezolid leads to therapeutic serum concentrations in critically ill patients. METHODS: In this prospective observational study, 30 critically ill adult patients with suspected infections received standard dosing of 600 mg linezolid intravenously twice a day. Over 4 days, multiple serum samples were obtained from each patient, in order to determine the linezolid concentrations by liquid chromatography tandem mass spectrometry. RESULTS: A high variability of serum linezolid concentrations was observed (range of area under the linezolid concentration time curve over 24 hours (AUC24) 50.1 to 453.9 mg/L, median 143.3 mg*h/L; range of trough concentrations (Cmin) < 0.13 to 14.49 mg/L, median 2.06 mg/L). Furthermore, potentially subtherapeutic linezolid concentrations over 24 hours and at single time points (defined according to the literature as AUC24 < 200 mg*h/L and Cmin < 2 mg/L) were observed for 63% and 50% of the patients, respectively. Finally, potentially toxic levels (defined as AUC24 > 400 mg*h/L and Cmin > 10 mg/L) were observed for 7 of the patients. CONCLUSIONS: A high variability of linezolid serum concentrations with a substantial percentage of potentially subtherapeutic levels was observed in intensive care patients. The findings suggest that therapeutic drug monitoring of linezolid might be helpful for adequate dosing of linezolid in critically ill patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT01793012. Registered 24 January 2013.

Comparison of three tests for faecal calprotectin in children and young adults: a retrospective monocentric study.

OBJECTIVE: Faecal calprotectin is used as a sensitive marker for gastrointestinal mucosal inflammation. We compared the performance of three different assays in a large cohort of symptomatic paediatric patients. DESIGN: Retrospective monocentric study. SETTING: Inpatients and outpatients of a tertiary referral centre for paediatric gastroenterology. PARTICIPANTS: 304 symptomatic patients (163 males, aged 2-20 years) with active inflammatory bowel disease (IBD/A, n=130), IBD in clinical remission (IBD/R, n=62), other intestinal diseases (n=45) and controls without identified intestinal disease (n=67). INTERVENTIONS: Calprotectin was measured in homogenised faecal samples with three tests (A: EliA Calprotectin, Phadia AB, Sweden; B: PhiCal, Calpro AS, Norway; C: EK-Cal, Bühlmann Laboratories, Switzerland). OUTCOMES: Concordance between tests was calculated using Kendall's τ coefficient. RESULTS: IBD/A and controls were correctly classified as 97.7%/82.1% (A), 97.7%/85.1% (B) and 98.4%/62.7% (C; not significant). Test C tended to have higher calprotectin values with a lower specificity compared to tests A and B. The concordance between two tests was 0.835 for tests A and B, 0.782 for tests A and C and 0.765 for tests B and C. CONCLUSIONS: All three tests are very sensitive for detecting mucosal inflammation, but major differences exist between specificity and absolute values. It is highly advisable to use the test of the same manufacturer for follow-up and to monitor for disease activity.

Circulating cell-free methylated DNA and lactate dehydrogenase release in colorectal cancer.

BACKGROUND: Hypermethylation of DNA is an epigenetic alteration commonly found in colorectal cancer (CRC) and can also be detected in blood samples of cancer patients. Methylation of the genes helicase-like transcription factor (HLTF) and hyperplastic polyposis 1 (HPP1) have been proposed as prognostic, and neurogenin 1 (NEUROG1) as diagnostic biomarker. However the underlying mechanisms leading to the release of these genes are unclear. This study aimed at examining the possible correlation of the presence of methylated genes NEUROG1, HLTF and HPP1 in serum with tissue breakdown as a possible mechanism using serum lactate dehydrogenase (LDH) as a surrogate marker. Additionally the prognostic impact of these markers was examined. METHODS: Pretherapeutic serum samples from 259 patients from all cancer stages were analyzed. Presence of hypermethylation of the genes HLTF, HPP1, and NEUROG1 was examined using methylation-specific quantitative PCR (MethyLight). LDH was determined using an UV kinetic test. RESULTS: Hypermethylation of HLTF and HPP1 was detected significantly more often in patients with elevated LDH levels (32% vs. 12% [p = 0.0005], and 68% vs. 11% [p < 0.0001], respectively). Also, higher LDH values correlated with a higher percentage of a fully methylated reference in a linear fashion (Spearman correlation coefficient 0.18 for HLTF [p = 0.004]; 0.49 [p < .0001] for HPP1). No correlation between methylation of NEUROG1 and LDH was found in this study. Concerning the clinical characteristics, high levels of LDH as well as methylation of HLTF and HPP1 were significantly associated with larger and more advanced stages of CRC. Accordingly, these three markers were correlated with significantly shorter survival in the overall population. Moreover, all three identified patients with a worse prognosis in the subgroup of stage IV patients. CONCLUSIONS: We were able to provide evidence that methylation of HLTF and especially HPP1 detected in serum is strongly correlated with cell death in CRC using LDH as surrogate marker. Additionally, we found that prognostic information is given by both HLTF and HPP1 as well as LDH. In sum, determining the methylation of HLTF and HPP1 in serum might be useful in order to identify patients with more aggressive tumors.