Arto Orpana

Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors

Detection of recurrent somatic rearrangements routinely allows monitoring of residual disease burden in leukemias, but is not used for most solid tumors. However, next‐generation sequencing now allows rapid identification of patient‐specific rearrangements in solid tumors. We mapped genomic rearrangements in three cancers and showed that PCR assays for rearrangements could detect a single copy of the tumor genome in plasma without false positives. Disease status, drug responsiveness, and incipient relapse could be serially assessed. In future, this strategy could be readily established in diagnostic laboratories, with major impact on monitoring of disease status and personalizing treatment of solid tumors.

Procalcitonin, soluble interleukin-2 receptor, and soluble E-selectin in predicting the severity of acute pancreatitis.

ObjectiveTo investigate whether marker(s) of systemic inflammation detect, at an early stage of acute pancreatitis, patients who may ultimately develop severe disease. DesignProspective study. SettingUniversity hospital emergency unit. PatientsThirty patients with mild acute pancreatitis (SEV0 group) and 27 with severe acute pancreatitis. Of the latter, 11 did not develop organ failure (SEV1 group), whereas the other 16 patients developed acute respiratory failure and 9 of them also developed renal failure (SEV2 group). InterventionsBlood samples were collected at admission to the hospital (T0), and at 12 hrs (T12) and 24 hrs (T24) after admission. Measurements and Main Results The plasma concentrations of procalcitonin (PCT), soluble E-selectin (sE-selectin), soluble interleukin-2 receptor (sIL-2R), and the serum concentration of C-reactive protein (CRP) were monitored. PCT levels at T0 were significantly higher in the SEV1 group (median 0.4 ng/mL, range 0.2–2.3) and the SEV2 group (0.8 ng/mL, 0.2–73.5) than in the SEV0 group (0.3 ng/mL, 0.1–3, p < .05 and p < .001, respectively). At T12, PCT level in the SEV2 group was significantly higher than that in the SEV1 group (2.2 ng/mL, 0.2–86.6 vs. 0.4 ng/mL, 0.3–2.8, p = .05), as it also was at T24 (2.2 ng/mL, 0.4–73.3 vs. 0.5 ng/mL, 0.3–4, p < .01). Among SEV2 patients, PCT concentration correlated negatively with the time elapsed between admission and the diagnosis of organ failure. At T12, sIL-2R levels of the SEV1 group (1011 U/mL, range 334–2211) and the SEV2 group (1495 U/ml, range 514–4526) both differed significantly from the SEV0 group (636 U/ml, range 356–1678, p < .05 and p < .001, respectively) as they also did at T24. Although CRP level in the SEV1 group at T12 did not differ from the SEV0 group, the difference between SEV2 (272 &mgr;g/mL, range 46–462) and SEV0 was significant (53 &mgr;g/mL, range 5–243, p < 0.01). sE-selectin levels did not differ between groups. ConclusionsAt admission to hospital, concentrations of PCT, but not those of CRP, sE-selectin, or sIL-2R, are higher in patients with severe acute pancreatitis than in patients with mild pancreatitis. PCT test had sensitivity of 94% and specificity of 73% for development of organ failure. PCT may be useful to identify the patients who benefit from novel therapies aimed at modifying the course of systemic inflammation.

A prospective study of inflammation markers in patients at risk of indirect acute lung injury.

Systemic inflammation triggered by insults like sepsis and acute pancreatitis may play a role in development of indirect acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Because little is known about the course of systemic inflammation on the days preceding diagnosis of ARDS, we prospectively monitored immune inflammatory status in 52 patients at risk and we assessed the presence of ALI and ARDS on day 7 after admission to the intensive care unit. On admission, serum interleukin (IL) 8, IL-6, and soluble IL-2 receptor concentrations were significantly higher in patients with subsequent ALI (n = 18) than in patients without ALI (n = 30). During a 4-day follow-up, IL-8 and IL-6 levels of ALI patients remained high and those of non-ALI patients decreased. None of the markers discriminated ARDS patients (n = 9) from non-ARDS ALI patients (n = 9). Among 11 patients with acute pancreatitis, ALI patients had significantly higher IL-8, IL-6, and phagocyte CD11b expression levels than did non-ALI patients, whereas among 14 patients with massive transfusion, respective findings in ALI and non-ALI patients were comparable. Results give credence to the view that systemic inflammation plays a role in development of ALI triggered by pancreatitis, but not in that by massive transfusion. This finding, if confirmed in studies with sufficient statistical power, suggests that the patients with massive transfusion do not necessarily benefit from novel biotherapies aimed at altering the course of systemic inflammation.

The calcium-dependent nitric oxide production of human vascular endothelial cells in preeclampsia ☆ ☆☆ ★ ★★

OBJECTIVE Nitric oxide is an important vasodilator, and in this study we studied whether the calcium-dependent nitric oxide production capacity of human umbilical vein endothelial cells was affected by preeclampsia. STUDY DESIGN Human umbilical vein endothelial cells were isolated from 11 preeclamptic and 10 normotensive pregnancies. The maximal calcium ionophore A23187-stimulated nitric oxide production capacity was measured as accumulation of nitrate and nitrite into the culture medium, and it was related to the number of viable endothelial cells by measurement of their mitochondrial dehydrogenase activity. RESULTS The cell number-related nitric oxide production capacity was similar in preeclamptic and normotensive pregnancies. The total nitric oxide production of cells from preeclamptic pregnancies was significantly lower (p <0.001). This difference, however, was mainly caused by larger amount of viable endothelial cells recovered from normotensive pregnancies. CONCLUSION The maximal calcium-dependent nitric oxide production capacity of individual human umbilical vein endothelial cells is not affected by preeclampsia.

Human vascular endothelial cells produce tumor necrosis factor-α in response to proinflammatory cytokine stimulation

OBJECTIVE To determine whether human vascular endothelial cells produce tumor necrosis factor-alpha (TNF-alpha) after stimulation with proinflammatory cytokines and bacterial lipopolysaccharides (LPS). DESIGN Prospective, in vitro repeated-measurements analysis of cellular responses. SETTING Research laboratory in an academic medical center. SUBJECTS Human umbilical vein endothelial cells (HUVECs). INTERVENTIONS HUVECs were incubated with interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), and LPS, or their different combinations for 2 to 48 hrs. MEASUREMENTS AND MAIN RESULTS TNF-alpha was measured by time-resolved immunofluorometric assay. Unstimulated HUVECs did not produce detectable amounts of TNF-alpha, but IFN-gamma, IL-1beta, and LPS when added together induced TNF-alpha production of HUVECs in a time-dependent manner. Immunofluorescent staining confirmed that the TNF-alpha was produced by endothelial cells. IFN-gamma, IL-1beta, or LPS alone did not induce TNF-alpha production, whereas IFN-gamma and IL-1beta in combination were able to induce TNF-alpha production to some extent, and the production could be further increased with LPS. TNF-alpha messenger RNA expression was detected with reverse transcriptase-coupled polymerase chain reaction in stimulated, but not in unstimulated, HUVECs. CONCLUSIONS HUVECs are capable of producing TNF-alpha after proinflammatory cytokine stimulation and may therefore contribute to the increased amount of TNF-alpha found in pathologic states such as septic shock.

Relative levels of SCCA2 and scca1 mRNA in primary tumors predicts recurrent disease in squamous cell cancer of the head and neck

Squamous cell carcinoma antigen (SCCA) is widely used as a serum marker in cancers of the uterine cervix, the head and neck, lung and esophagus. Two isoforms of SCCA, deriving from 2 highly homologous serine proteinase inhibitor genes, are co‐expressed in normal and malignant squamous epithelium, but it is mainly the acidic isoform SCCA2 that is present in the circulation of cancer patients. We studied the relative levels of SCCA2 and SCCA1 mRNA in frozen sections of squamous cell carcinomas of the head and neck (SCCHN) in relation to disease recurrence, using a new reverse transcription‐polymerase chain reaction‐based technique for accurate quantitation of relative mRNA levels. Primary tumors from 30 SCCHN patients, recurrent tumors from 11 patients and normal epithelium from 16 controls were examined. In patients responding to initial therapy (n = 26), an elevated SCCA2/SCCA1 mRNA ratio in the primary tumor predicted recurrence independent of clinical stage (p = 0.011). The relative risk of developing a recurrence was 7.2 (CI 1.2–13.3) in patients with elevated vs. normal SCCA2/SCCA1 mRNA ratios. We demonstrate that subtle differences in expression levels of the SCCA genes are reflected in the course of the SCCHN disease and may provide a target for molecular grading of SCCHN tumors. If this finding can be confirmed in a larger study the SCCA2/SCCA1 mRNA ratio in primary tumors could be useful for individual selection of treatment strategy for patients with head and neck cancer.

Effect of physiological concentrations of estradiol on PGI2 and NO in endothelial cells

OBJECTIVES To elucidate the mechanisms by which estrogens protect against occlusive vascular disorders, we studied the effect of 17 beta-estradiol on the production of prostacyclin (PGI2) and nitric oxide (NO) in primary cultures of human umbilical vein endothelial cells (HUVECs). METHODS To study the effect of 17 beta-estradiol on PGI2 production, HUVECs were incubated in the absence and presence of 17 beta-estradiol (0.01-10 nmol/l) encapsulated within beta-cyclodextrin for 12 h in serum-free medium. To study the effect of 17 beta-estradiol (100 nmol/l) on maximal calcium-dependent NO production, we used different approaches. First, HUVECs were incubated with 2 mumol/l calcium ionophore A23187 with or without 17 beta-estradiol (100 nmol/l) for 24 h in serum-free medium. Second, HUVECs were preincubated with or without 17 beta-estradiol (100 nmol/l) for 12 h in medium supplemented with 2% fetal calf serum, and thereafter incubated in serum-free medium with 2 mumol/l of A23187 and with 100 nmol/l of 17 beta-estradiol (cells which contained 17 beta-estradiol during the preincubation period as well as cells which did not) or without it (only cells which did not contain 17 beta-estradiol during the preincubation period) for 6 h or 24 h. RESULTS 17 beta-Estradiol (0.1 nmol/l) increased the concentration of 6-keto-prostaglandin F1 alpha, a stable metabolite of PGI2 in the incubation medium, by 16%, and no further increase occurred with higher 17 beta-estradiol concentrations. The stimulation was prevented by tamoxifen. 17 beta-Estradiol did not affect NO production in any of our experiments measured as accumulation of nitrate and nitrite in the experimental medium. CONCLUSIONS The stimulatory effect on PGI2 production of physiological concentrations of 17 beta-estradiol, shown now for the first time, may provide one explanation for the ability of 17 beta-estradiol to protect against occlusive vascular disorders.

Transhepatic neutrophil and monocyte activation during clinical liver transplantation

BACKGROUND During experimental liver transplantation, neutrophil sequestration results in increased oxygen free radical production and correlates inversely with graft viability. Neutrophil activation in clinical liver transplantation is poorly understood. METHODS We assessed leukocyte sequestration and transhepatic differences of neutrophil and monocyte CD11b expression, neutrophil free radical production, and plasma concentrations of interleukin 6 and interleukin 8 in nine patients during liver transplantation. RESULTS Significant hepatic neutrophil sequestration occurred during initial graft rewarming with portal blood, after inferior vena cava declamping, and after hepatic artery declamping (all P<0.05). A positive transhepatic difference (i.e., outcoming - ingoing) in CD11b expression of neutrophils was observed after portal vein declamping (51+/-32 relative fluorescence unit [RFU]) and in CD11b expression of monocytes during initial graft rewarming (67+/-86 RFU, both P<0.05). A transcoronary increase in both unstimulated (74+/-80 RFU) and N-formyl-methionyl-leucylphenylalanine-stimulated (112+/-168 RFU) neutrophil free radical production took place after hepatic artery declamping (both P<0.05). A negative transcoronary difference of interleukin 6 occurred during initial graft rewarming (-192+/-176 pg/ml) and a positive difference of interleukin 8 occurred after hepatic artery declamping (17+/-23 pg/ml, both P<0.05). CONCLUSIONS Hepatic sequestration and transhepatic activation of neutrophils, and hepatic production of interleukin 8 occur during clinical liver transplantation. A splanchnic influx of interleukin 6 occurs to the graft, possibly modulating neutrophil-mediated graft reperfusion injury.

Accurate determination of relative messenger RNA levels by RT-PCR

The increasing focus on functional genomics spurs new techniques for accurately quantitating differences in mRNA levels.

Systemic Inflammation in Hemorrhagic Fever with Renal Syndrome Correlates with Hypotension and Thrombocytopenia but Not with Renal Injury

Systemic inflammation is common in patients with nephropathia epidemica (NE), a European form of hemorrhagic fever. Markers of inflammation were studied in a patient with NE with respiratory insufficiency (patient 1), 18 other patients with NE, and 13 patients with a viral infectious disease other than NE. Neutrophil and monocyte CD11b expression levels, determined by flow cytometry; soluble interleukin (IL)-2 receptor (sIL-2R), IL-6, and IL-8 concentrations, determined by means of Immulite; and soluble E-selectin, determined by ELISA, were higher in patients with NE than in healthy subjects. The findings were not specific for NE and did not correlate with serum creatinine levels, but the findings correlated inversely with mean arterial pressure (sIL-2R and monocyte CD11b expression) and minimum platelet count (sIL-2R, IL-6, neutrophil, and monocyte CD11b expression). Monocyte CD11b expression in patient 1 was extremely high, suggesting that monocytes may contribute to development of lung injury. Severity of inflammation in patients with NE is related to hypotension and platelet consumption but not to renal injury.