Sequential Blood Samples Research Articles

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy

Background:The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors.Methods:Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA).Results:The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047).Conclusion:Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.

Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment in Tanzanian children with acute uncomplicated malaria

BackgroundThis study aimed to explore Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment with artemisinin-based combination therapy in children with clinical malaria in a high transmission area in Africa.MethodsA total of 50 children aged 1-10 years with acute uncomplicated P. falciparum malaria in Bagamoyo District, Tanzania, were enrolled. Participants were hospitalized and received supervised standard treatment with artemether-lumefantrine according to body weight in six doses over 3 days. Blood samples were collected 11 times, i.e. at time of diagnosis (-2 h) and 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 h after initiation of treatment. Parasite population dynamics were assessed using nested polymerase chain reaction (PCR)-genotyping of merozoite surface protein (msp) 1 and 2.ResultsPCR-analyses from nine sequential blood samples collected after initiation of treatment identified 20 and 21 additional genotypes in 15/50 (30%) and 14/50 (28%) children with msp1 and msp2, respectively, non-detectable in the pre-treatment samples (-2 and 0 h combined). Some 15/20 (75%) and 14/21 (67%) of these genotypes were identified within 24 h, whereas 17/20 (85%) and 19/21 (90%) within 48 h for msp1 and msp2, respectively. The genotype profile was diverse, and varied considerably over time both within and between patients, molecular markers and their respective families.ConclusionPCR analyses from multiple blood samples collected during the early treatment phase revealed a complex picture of parasite sub-populations. This underlines the importance of interpreting PCR-outcomes with caution and suggests that the present use of PCR-adjustment from paired blood samples in anti-malarial drug trials may overestimate assessment of drug efficacy in high transmission areas in Africa.The study is registered at http://www.clinicaltrials.gov with identifier NCT00336375.

Detection of minimal residual disease following induction immunochemotherapy predicts progression free survival in mantle cell lymphoma: final results of CALGB 59909

In the present study, the prognostic impact of minimal residual disease during treatment on time to progression and overall survival was analyzed prospectively in patients with mantle cell lymphoma treated on the Cancer and Leukemia Group B 59909 clinical trial. Peripheral blood and bone marrow samples were collected during different phases of the Cancer and Leukemia Group B 59909 study for minimal residual disease analysis. Minimal residual disease status was determined by quantitative polymerase chain reaction of IgH and/or BCL-1/JH gene rearrangement. Correlation of minimal residual disease status with time to progression and overall survival was determined. In multivariable analysis, minimal residual disease, and other risk factors were correlated with time to progression. Thirty-nine patients had evaluable, sequential peripheral blood and bone marrow samples for minimal residual disease analysis. Using peripheral blood monitoring, 18 of 39 (46%) achieved molecular remission following induction therapy. The molecular remission rate increased from 46 to 74% after one course of intensification therapy. Twelve of 21 minimal residual disease positive patients (57%) progressed within three years of follow up compared to 4 of 18 (22%) molecular remission patients (P=0.049). Detection of minimal residual disease following induction therapy predicted disease progression with a hazard ratio of 3.7 (P=0.016). The 3-year probability of time to progression among those who were in molecular remission after induction chemotherapy was 82% compared to 48% in patients with detectable minimal residual disease. The prediction of time to progression by post-induction minimal residual disease was independent of other prognostic factors in multivariable analysis. Detection of minimal residual disease following induction immunochemotherapy was an independent predictor of time to progression following immunochemotherapy and autologous stem cell transplantation for mantle cell lymphoma.

Utility of Urothelial mRNA Markers in Blood for Staging and Monitoring Bladder Cancer

To test the efficiency of 6 mRNA bladder markers in staging urothelial cell carcinoma (UCC) and monitoring UCC dissemination from blood samples. From 2002 to 2009, 347 blood samples were collected from 150 patients with UCC and 29 healthy controls. Sequential blood sampling was performed in patients undergoing cystectomy at surgery and 6, 12, 18, and 24 months postoperatively. The median follow-up was 33 months. The presence of KRT20, FXYD3, C10orf116, UPK2, AGR2, and KRT19 markers in blood was evaluated in all patients and controls by measuring the gene expression using preamplified cDNA and reverse transcriptase quantitative polymerase chain reaction. Gene expression data were correlated with the tumor risk, follow-up, and outcomes data. Expression of C10orf116 and KRT19 genes differed between patients and controls (P<.001). KRT20, C10orf116, and AGR2 differentiated between low- and high-risk nonmuscle-invasive bladder cancer (P=.001, P=.011, and P=.001, respectively). FXYD3 differentiated between patients with high-risk nonmuscle-invasive bladder cancer and those with muscle-invasive bladder cancer (P=.009). In contrast, the 6 markers showed no differences in gene expression between metastatic and patients without metastases who had not undergone cystectomy (P=NS). None of the markers were significantly increased in the metastatic patients at 6, 12, 18, or 24 months after surgery. The gene expression of bladder-specific mRNA markers in blood was different among the various tumor risk groups of patients with UCC. However, this gene expression analysis is not suitable for predicting metastases or monitoring UCC hematogenous dissemination in patients who have undergone cystectomy.

Biomarkers of Anti-Angiogenic Therapy in Metastatic Colorectal Cancer (mCRC): Original Data and Review of the Literature

Tumour angiogenesis via vascular endothelial growth factor (VEGF) is essential for promoting tumour progression and is overexpressed in colorectal cancer. The humanised monoclonal anti-VEGF antibody bevacizumab (Avastin®, Genentech Inc., South San Francisco, CA) has shown activity in metastatic colorectal cancer (mCRC) combined with conventional chemotherapy. The search for biomarkers to predict response to anti-angiogenic therapy in mCRC is of great interest. We investigated several potential predictive anti-angiogenic markers including circulating endothelial progenitor cells (EPC) in patients with mCRC receiving bevacizumab containing treatment within a randomised multicenter phase 2 study of the German AIO GI tumour study group. We collected sequential blood samples and tumour tissues from patients participating in a clinical trial for patients with mCRC. We performed flow cytometry of mononuclear cells isolated from peripheral blood to assess CD 133 + or CD 34 + /KDR + EPC before the first bevacizumab containing chemotherapy and after 21 days. Circulating VEGF blood levels before a bevacizumab containing chemotherapy regimen and after 21 days and VEGF expression in tumour tissue were examined. Patients with mCRC and a partial remission after six months of immuno-chemotherapy containing bevacizumab showed a reduction of CD 34 negative KDR positive cells as early as 3 weeks after start of therapy. In contrast, no remarkable change in the number of CD 34 /KDR positive or CD 34 /CD133 positive cells was seen. Furthermore, there was no correlation between treatment response and VEGF expression within the tumour tissue. The mAb bevacizumab reduced serum-VEGF levels in patients independent of their treatment response to bevacizumab. We examined circulating endothelial progenitor cells (EPC), serum-VEGF levels and the tumour tissue VEGF expression of patients with mCRC under a bevacizumab containing chemotherapy. The patients with a partial remission after six months of immuno-chemotherapy showed a reduction of CD 34 negative KDR positive cells as early as 3 weeks after start of therapy. Neither serum nor tissue markers were of significant predictive value in our pilot study. Furthermore, we review the current data on biomarkers for anti-angiogenic therapy of mCRC.

Inhaled Nitric Oxide Therapy Increases Blood Nitrite, Nitrate, and S-Nitrosohemoglobin Concentrations in Infants with Pulmonary Hypertension

To measure the circulating concentrations of nitric oxide (NO) adducts with NO bioactivity after inhaled NO (iNO) therapy in infants with pulmonary hypertension. In this single center study, 5 sequential blood samples were collected from infants with pulmonary hypertension before, during, and after therapy with iNO (n = 17). Samples were collected from a control group of hospitalized infants without pulmonary hypertension (n = 16) and from healthy adults for comparison (n = 12). After beginning iNO (20 ppm) whole blood nitrite levels increased approximately two-fold within 2 hours (P<.01). Whole blood nitrate levels increased to 4-fold higher than baseline during treatment with 20 ppm iNO (P<.01). S-nitrosohemoglobin increased measurably after beginning iNO (P<.01), whereas iron nitrosyl hemoglobin and total hemoglobin-bound NO-species compounds did not change. Treatment of pulmonary hypertensive infants with iNO results in increases in levels of nitrite, nitrate, and S-nitrosohemoglobin in circulating blood. We speculate that these compounds may be carriers of NO bioactivity throughout the body and account for peripheral effects of iNO in the brain, heart, and other organs.

Urbanization of congenital transmission of Trypanosoma cruzi: prospective polymerase chain reaction study in pregnancy

Chagas disease ranks among the world's most neglected tropical diseases and congenital transmission is increasingly responsible for urbanization of Chagas disease in non-endemic areas. Molecular assays for amplification and profiling of parasite minicircle DNA (kDNA) and identification of discrete typing units (DTUs) were prospectively conducted in bloodstream and placental samples from pregnant women cursing chronic Chagas disease residing in Buenos Aires city. Sensitivity of kDNA-PCR increased from 75.6% to 95.6% when one to three sequential blood samples were analysed. Congenital infection (CI) was diagnosed in 3 neonates born to kDNA-PCR positive mothers, one who had transmitted CI in a previous gestation, pointing to family clustering of congenital transmission. Fourteen of 44 placental samples were kDNA-PCR positive, all from non-CI transmitting women, indicating that placental PCR is not useful for CI diagnosis. Placental PCR positivity was not related to maternal bloodstream PCR positivity and placental parasitic subpopulations not observed in bloodstream were detected by minicircle signatures. PCR targeted to intergenic regions of spliced-leader genes and serological tests using trypomastigote small surface recombinant antigens showed predominance of DTU group TcII/V/VI and only one patient infected with TcI. To our knowledge, this is the first PCR-based follow-up study of bloodstream and placental T. cruzi infections during pregnancy, including identification of DTUs. kDNA-PCR assays in serial blood samples provided high sensitivity for detection of T. cruzi DNA in pregnant women with chronic Chagas disease.

Pharmacokinetics of combined treatment with praziquantel and albendazole in neurocysticercosis

Neurocysticercosis is the most common cause of acquired epilepsy in the world. Antiparasitic treatment of viable brain cysts is of clinical benefit, but current antiparasitic regimes provide incomplete parasiticidal efficacy. Combined use of two antiparasitic drugs may improve clearance of brain parasites. Albendazole (ABZ) has been used together with praziquantel (PZQ) before for geohelminths, echinococcosis and cysticercosis, but their combined use is not yet formally recommended and only scarce, discrepant data exist on their pharmacokinetics when given together. We assessed the pharmacokinetics of their combined use for the treatment of neurocysticercosis. A randomized, double-blind, placebo-controlled phase II evaluation of the pharmacokinetics of ABZ and PZQ in 32 patients with neurocysticercosis was carried out. Patients received their usual concomitant medications including an antiepileptic drug, dexamethasone, and ranitidine. Randomization was stratified by antiepileptic drug (phenytoin or carbamazepine). Subjects had sequential blood samples taken after the first dose of antiparasitic drugs and again after 9 days of treatment, and were followed for 3 months after dosing. Twenty-one men and 11 women, aged 16 to 55 (mean age 28) years were included. Albendazole sulfoxide concentrations were increased in the combination group compared with the ABZ alone group, both in patients taking phenytoin and patients taking carbamazepine. PZQ concentrations were also increased by the end of therapy. There were no significant side effects in this study group. Combined ABZ + PZQ is associated with increased albendazole sulfoxide plasma concentrations. These increased concentrations could independently contribute to increased cysticidal efficacy by themselves or in addition to a possible synergistic effect.

Effect of Organic Anion-Transporting Polypeptide 1B1 (OATP1B1) Polymorphism on the Single- and Multiple-Dose Pharmacokinetics of Enalapril in Healthy Chinese Adult Men

BackgroundEnalapril is an angiotensin-converting enzyme (ACE) inhibitor approved for the treatment of mild to severe hypertension and congestive heart failure. There is evidence that enalapril may be an organic anion-transporting polypeptide 1B1 (OATP1B1) substrate, suggesting that genetic polymorphisms of the OATP1B1 gene may play a role in causing the interindividual pharmacokinetic differences of this drug. ObjectiveThe purpose of this study was to investigate the functional significance of the OATP1B1 genetic polymorphism on the pharmacokinetics of enalapril and its active metabolite enalaprilat in healthy Chinese adult male participants. MethodsThis was a single-center, open-label, single- and multiple-dose study conducted in healthy Chinese male participants. Each participant received a single oral dose of 10 mg enalapril under fasting conditions, followed by enalapril 10 mg/d for 7 days. In the single-dose phase, sequential blood samples were collected from 0 to 24 hours after drug administration. In the multiple-dose phase, samples were obtained before drug administration on days 4, 5, 6, and 7; on day 7, samples were collected from 0 to 72 hours after drug administration. An HPLC-MS/MS method was used to determine plasma concentrations of enalapril and enalaprilat. A polymerase chain reaction technique was used for genotyping of 2 single nucleotide polymorphisms (SNPs) of the OATP1B1 gene: T521C and A388G. The pharmacokinetic parameters of enalapril and enalaprilat were then compared according to genotype groups, using 1-way ANOVA, except for Tmax in which the Mann-Whitney test or Kruskal-Wallis test was used. ResultsThe study included 32 healthy Han Chinese male participants (age range, 18–28 years; weight range, 50.0–80.0 kg; height range,159–182.0 cm). Twenty-six were OATP1B1*15 noncarriers (homozygous for 521TT), the others were *15 carriers with at least one 521 T>C mutant allele. After single and multiple oral doses of 10 mg enalapril, plasma concentrations of enalapril in *15 noncarriers were lower than that in *15 carriers, with significant difference in area under the curve at steady state (AUCss) between *15 noncarriers and *15 carriers (P = 0.048) in the multiple-dose phase. There were no significant differences in enalapril's AUC0–24, Cmax, or the ratio of the AUC0–24h in the single-dose study to the AUCss (Rac) between the *15 carriers and noncarriers. In contrast to enalapril, the mean AUC0–24h and Cmax of enalaprilat in *15 noncarriers was significantly higher than those in *15 carriers (P = 0.040 and P = 0.027, respectively) in the single-dose phase. There were no significant differences in enalaprilat's AUCss or Cmaxss between the 2 groups in the multiple-dose phase. For the 3 groups classified according to the effect of A388G variant in all subjects homozygous for 521T (TT), *1a/*1a, *1a/*1b, and *1b/*1b, no significant difference was found in AUC0–24h, Cmax, and Tmax of enalapril and enalaprilat. ConclusionsIn this small population of healthy Chinese men, the OATP1B1*15 allele and T521C variant appeared to be an important determinant of the pharmacokinetics of enalapril. There were significant differences between the *15 carriers and noncarriers in enalapril's AUCss and enalaprilat's AUC0–24h, Cmax, and Rac. However, there were no significant differences in enalapril's AUC0–24, Cmax, or enalaprilat's AUCss, Cmaxss between the *15 carriers and noncarriers.

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7-10 days.

Detection of HIV drug resistance mutations in pregnant women receiving single dose Nevirapine in south India

Single dose of Nevirapine to prevent mother to child transmission of HIV is the commonest preventive regimen in resource-limited countries. The objective of this study was to detect drug-resistant virus after single dose of Nevirapine (sdNVP) provided to delivering HIV seropositive (HIV+ve) women and to evaluate the time taken for its decay. Of the 36 consenting HIV+ve pregnant women enrolled into the study, the mean hemoglobin and total lymphocyte counts were 10.8 g/dl and 1843 cells/mm 3 , respectively. Mean CD4 counts in 64% of women was 363 cells/mm 3 and mean viral load for 16/36 women was 28,143 copies/ml of plasma. Nevirapine-resistance mutations were detected in 28% of women at delivery; using OLA (Oligonucleotide Ligation Assay). K103N mutations were seen in 19.4% of women while the Y181C mutation was seen in 5%. Both the mutations were detected in 2.7% of women. Sequential blood samples collected at delivery, 7-10 days, 6 weeks, 4 months, 6 months and one year postpartum showed that 81% of K103N mutations and 66.7% of Y181C mutations were detected at 6 weeks postpartum . Wild-type virus had replaced the mutants by one year postpartum in all women except one. These observations are relevant for future treatment with antiretroviral therapy in these women for their HIV disease.

High and Increasing Oxa-51 DNA Load Predict Mortality in Acinetobacter baumannii Bacteremia: Implication for Pathogenesis and Evaluation of Therapy

BackgroundWhile quantification of viral loads has been successfully employed in clinical medicine and has provided valuable insights and useful markers for several viral diseases, the potential of measuring bacterial DNA load to predict outcome or monitor therapeutic responses remains largely unexplored. We tested this possibility by investigating bacterial loads in Acinetobacter baumannii bacteremia, a rapidly increasing nosocomial infection characterized by high mortality, drug resistance, multiple and complicated risk factors, all of which urged the need of good markers to evaluate therapeutics.Methods and FindingsWe established a quantitative real-time PCR assay based on an A. baumannii-specific gene, Oxa-51, and conducted a prospective study to examine A. baumannii loads in 318 sequential blood samples from 51 adults patients (17 survivors, 34 nonsurvivors) with culture-proven A. baumannii bacteremia in the intensive care units. Oxa-51 DNA loads were significantly higher in the nonsurvivors than survivors on day 1, 2 and 3 (P = 0.03, 0.001 and 0.006, respectively). Compared with survivors, nonsurvivors had higher maximum Oxa-51 DNA load and a trend of increase from day 0 to day 3 (P<0.001), which together with Pitt bacteremia score were independent predictors for mortality by multivariate analysis (P = 0.014 and 0.016, for maximum Oxa-51 DNA and change of Oxa-51 DNA, respectively). Kaplan-Meier analysis revealed significantly different survival curves in patients with different maximum Oxa-51 DNA and change of Oxa-51 DNA from day 0 to day 3.ConclusionsHigh Oxa-51 DNA load and its initial increase could predict mortality. Moreover, monitoring Oxa-51 DNA load in blood may provide direct parameters for evaluating new regimens against A. baumannii in future clinical studies.

Response to Dasatinib In Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Correlates with p-Lyn and p-Syk

AbstractAbstract 2457 Background:Preclinical studies have shown that CLL cells overexpress Lyn kinase, and in vitro inhibition of Lyn kinase leads to apoptosis of the CLL cells (Contri, J Clin Invest 2005). In 2007, based on this evidence, we initiated a phase II study in patients with previously treated CLL/SLL using dasatinib as a single agent. Preliminary clinical results of this study were reported in 2008. Methods:Patients were required to be over 18 years of age, have a pathologically confirmed diagnosis of CLL/SLL by flow cytometry or immunohistochemistry, and have failed either 1 course of treatment with a fludarabine-containing regimen or 2 non-fludarabine containing regimens. The starting dose of dasatinib was 140 mg daily by mouth. In the first week of therapy sequential blood samples were tested for Lyn kinase protein, p-Lyn, Syk protein, p-Syk, indicators of apoptosis (caspase 3 and Tunel assay), and gene expression profiling among the 3 patients that agreed to participate in this aspect of the study. The final clinical results, with over 1 year follow-up of all patients, showed that among the 15 patients enrolled the major toxicity encountered was myelosuppression: grade 3 + 4 neutropenia in 10 subjects and grade 3 + 4 thrombocytopenia in 6 subjects. Other toxicities included one subject with grade 3 diarrhea, 2 patients with grade 2 pleural effusions, and 1 patient had a transiently prolonged QTc of 516 ms. There were no fatal events, and all toxicities were reversible. In the final analysis partial responses (PR) by NCI-WG criteria were achieved in 3 of the 15 patients (20% with 90% CI 6%-44%). An additional 1 patient would have qualified for an NCI-WG PR except for myelosuppression. Among the 15 patients, 9 had nodal responses (2 CR and 7 PR) by physical exam (PE), 5 without a 50% reduction in lymphocytosis. CT scans confirmed nodal responses in 4 of the 9 patients. Among the 5 patients with del (11q), all 5 had nodal responses by PE. The median time to treatment failure, time to progression, and overall survival were 6.7 months, 7.5 months, and 27 months, respectively. The relationships between clinical response, Lyn kinase, Syk kinase, and apoptosis were analyzed using mononuclear cells from 3 patients at baseline, and at 3, 6, and 24 hours after dosing with dasatinib. For the one of these 3 patients that responded to dasatinib, the high baseline level of p-Lyn was dramatically suppressed at 6 hours correlating with suppression of p-Syk. For the other 2 patients, who did not have a clinical response, there was minimal suppression of p-Lyn and no suppression of p-Syk at 6 hours. Microarray analysis of mRNA from these samples showed that within 6 hours there was significant (p<.05) down-regulation of genes for spleen tyrosine kinase, FK506 binding protein, early growth response 1, NLR family CARD 3, and NOTCH, while genes for GNAS and NFKB2 were up-regulated. Conclusions:While our in vitro data is very preliminary, it suggests that the activity of dasatinib in CLL may result from the suppression of p-Lyn and inhibition of p-Syk. Disclosures:Amrein: Bristol-Myers Squibb: Honoraria. Off Label Use: Dasatinib used in CLL is off-label use. We show that some patients have a clinical response to the drug in this setting.

Molecular mechanisms of metastasis in breast cancer—clinical applications

The metastatic cascade is a series of biological processes that enable the movement of tumor cells from the primary site to a distant location and the establishment of a new cancer growth. Circulating tumor cells (CTCs) have a crucial role in tumor dissemination. The role of CTCs in treatment failure and disease progression can be explained by their relation to biological processes, including the epithelial-to-mesenchymal transition and 'self seeding', defined as reinfiltration of the primary tumor or established metastasis by more aggressive CTCs. CTCs are a unique and heterogeneous cell population with established prognostic and predictive value in certain clinical situations. The possibility of collecting sequential blood samples for real-time monitoring of systemic-therapy efficacy presents new possibilities to evaluate targeted therapies based on the genomic profiling of CTCs and to improve the clinical management of patients by personalized therapy. Interruption of the metastatic cascade via the targeting of CTCs might be a promising therapeutic strategy.

Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients

BackgroundAn unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic.MethodologyThe role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs.Principal Findings13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters.ConclusionsDisease severity was associated with pronounced impairment of host immune response.

N-carbamylglutamate Augments Ureagenesis and Reduces Ammonia and Glutamine in Propionic Acidemia

The objective of this study was to determine whether N-carbamylglutamate (NCG) reduces plasma levels of ammonia and glutamine and increases the rate of ureagenesis in patients with propionic acidemia (PA). Identical 4-hour studies were performed before and immediately after a 3-day trial of oral NCG in 7 patients with PA. An oral bolus of [(13)C]sodium acetate was administered at the start of each study, and sequential blood samples were obtained to measure [(13)C]urea, ammonia, urea, and amino acids. With longitudinal mixed-effects linear regression, peak [(13)C]urea increased after treatment with NCG (from 2.2 to 3.8 microM; P < .0005). There were concomitant decreases in mean plasma ammonia (59-43 microM; P < .018) and glutamine (552-331 microM; P < .0005). NCG augments ureagenesis and decreases plasma ammonia and glutamine in patients with PA. The drug may serve as an important therapeutic adjunct in the treatment of acute hyperammonemia in this disorder.

Phase II study of irinotecan plus panitumumab as second-line therapy for patients with advanced esophageal adenocarcinoma (EAC).

TPS206 Background: Esophageal carcinoma is highly lethal and the incidence is increasing, especially the subtype of EAC. Our group demonstrated that overexpression of the epidermal growth factor receptor (EGFR) correlates with lower survival in EAC. Panitumumab (P) is a high affinity fully human IgG2 monoclonal antibody directed against human EGFR. Cetuximab combined with irinotecan (I) is effective for second-line treatment of advanced colorectal cancer (CRC). P alone is active in EGFR expressing CRC. Our group has empiric evidence for the activity of cetuximab + I. As such, we composed this phase II study of P + I as second-line therapy for advanced EAC. Methods: The primary endpoint is overall response (OR) as measured by RECIST (imaging every 2 cycles). Secondary endpoints include overall survival, safety/toxicity and correlative studies. Patients with a history of one prior treatment will be treated with P 9 mg/m2 on day 1 and I 125 mg/m2 on days 1 and 8 of each 21 day cycle to a maximum of 6 cycles. Inclusion criteria include confirmed EAC, measurable disease, no prior I or P, PS <2 and normal organ function. Exclusion criteria include use of CYP 3A4 inhibitors, prior anti-EGFR therapy, uncontrolled intercurrent illness. A Simon two-stage design uses power of 80% and a type I error of 0.05. 18 patients will be tested in the first stage, and the trial will be terminated if 2 or fewer respond. In the second stage, a total of 43 patients will be studied. Correlative Studies: Research blood and tissue will be obtained pre-treatment and during the third week of cycle 2. Blood will be studied for the role of FcgR receptors on the effector cell surface leading to antibody-directed cellular cytotoxicity. Tumor tissues will be analyzed for pharmacodynamic (PD) and pharmacogenomic (PG) effects by measurement of EGFR pathway components using FISH and IHC and by kras mutation analysis. Enrollment: The study recently opened at UPMC, and two patients are enrolled. Significance: Along with the primary endpoint of OR, the goal of this trial is to utilize sequential tumor and blood sampling to study PD and PG correlates. The goal is to improve patient selection for targeted therapies in future trials. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen

Phase I/II trial of association of sorafenib in combination with temozolomide in patients with metastatic melanoma: Looking for predictive markers of efficacy.

8552 Background: The poor activity of treatments (tt) against metastatic melanoma (MM) mainly relies in the absence of predictive markers of efficacy that could allow selection of responsive pts. This phase I/II clinical trial of sorafenib (S) + temozolomide (T) was done with an intense translational research program in order to evaluate both safety and efficacy and to identify early markers of treatment benefit. Methods: 63 pts with MM were included in the dose-escalating phase I (n = 15) and the phase II (n = 48). Sequential blood and tumor samples were harvested to study baseline parameters and intra-individual variations of biomarkers during the treatment. Tumor sequencing of 44 exons in 12 genes were done in 37 pts. CGH arrays, RNA expression profiling, immunostaining (P-ERK, P-MEK and Ki67) were performed at d0 and 21. Functional imaging using dynamic contrast enhanced ultrasonography (DCE-US) was performed at days 0, 7, 15, and every month. Results: 54 pts weretreated at the MTD: 400 mg of S bid and 150 mg/m2 of T every other week. Half of them had received ≥ 2 lines of prior chemotherapies and 37% had high LDH. Median number of cycles was 4 (1-22). Toxicity was manageable and required tt interruption for only 3 pts. On 49 evaluable pts, 4 had PR (8%), 19 SD (39%) and 26 PD (53%) for a treatment benefit in 47%. Six months PFS and overall survival rates were respectively 15.2% and 60.6%. DCE-US functional imaging parameters before treatment, at day 7 or day 15 were significantly associated with tt efficacy (OR or SD vs. PD) at 2, 4 or 6 months (p < 0.05). NKG2D ligand, soluble MICA, was found in 80% of pts with PR or SD vs. 3% of PD pts and was also significantly associated with clinical benefit (p < 0.05). On 37 tumors tested, mutations were found in BRAF (62%), STK11 (25%), NRAS (8%), TP53 (6%) CDKN2A (3%), CTNNB1 (3%) without correlation to clinical response. CGH array and RNA expression profiling results are pending. Conclusions: This study confirms the safety and efficacy of the association of S and T in pts with MM. Translational research identified early markers associated with treatment benefit that could allow selection of patients for more personalized and efficient treatment. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bayer

Correlation of Parasite Load Determined by Quantitative PCR to Clinical Outcome in a Heart Transplant Patient with Disseminated Toxoplasmosis

Disseminated toxoplasmosis is a life-threatening infection in transplant recipients, which results either from reactivation of latent infection or from organ-transmitted primary infection. Preventive measures and diagnostic screening methods differ between countries and are related to the seroprevalence of Toxoplasma spp. in the general population. Here we report a case of disseminated toxoplasmosis in a heart transplant recipient with previous immunity that occurred after cotrimoxazole prophylaxis for the prevention of Pneumocystis jirovecii pneumonia was stopped. Quantitative PCR proved useful for the diagnosis and monitoring of Toxoplasma infection. Decreasing parasitic burdens in sequential samples of cerebrospinal fluid, blood, and bronchoalveolar lavage fluid correlated with a favorable outcome and allowed modulation of the immunosuppressive drug regimen. The duration of anti-Toxoplasma treatment and the need for maintenance prophylaxis are discussed, as well as prophylaxis for solid-organ transplant recipients. Although a rare event in heart transplant recipients, Toxoplasma reactivation must be investigated promptly, since early treatment improves the prognosis.

Detection of Aspergillus species in bone marrow transplant patients

Invasive aspergillosis is a severe complication of cytotoxic chemotherapies and bone marrow transplantation (BMT). The aim of this study was to assess the utility of a real-time PCR assay for the early diagnosis of Aspergillus species in blood samples from BMT patients. Blood specimens (n = 993) from patients (n = 82) scheduled for BMT were collected prior to transplant and for 100 days post transplantation. The specimens were later tested using an Aspergillus-specific real-time PCR assay. Cultures of clinical samples, along with sonography and computerized tomographic scans, were performed as standard of care. Aspergillus DNA was positive in 94 sequential blood samples from 13 patients with clinical and radiological signs of infection. Samples from three of these patients were PCR-positive for Aspergillus in the first week of admission, prior to transplantation. Four patients with aspergillosis were cured with antifungal agents and nine died. An additional 12 patients without clinical signs of infection were PCR-positive on one occasion each, while two patients with clinical signs of infection were PCR-negative. Compared to routine methods of aspergillosis diagnosis, the respective sensitivity, specificity, negative, and positive predictive values of the PCR method by patient were 86.6%, 82%, 96.5% and 52%. The results show that Aspergillus infections in the blood of bone marrow transplant patients can be dectected by PCR methods. Early detection of Aspergillus infections by PCR has the potential to positively impact patient mortality rate and provide cost savings to hospitals.