Personalized Minimal Effective Concentration Therapy.

It has been recognized for literally centuries that patients should be given only the amount of medication necessary to treat disease(s) or relieve symptoms. It is also well known that this amount can vary greatly between patients or even over time in the same patient. The ability to identify this amount, that is, to "personalize" dosing, requires a reliable measure of a patient's response to treatment. The development of analytical methods for the accurate measurement of pharmacologically meaningful drug concentrations in physiologic fluids, combined with mathematical methods for reliable prediction of how dosing changes affect these concentrations, has led to the development of therapeutic drug management (TDM) for more effective individualization of dosing. Using TDM, clinicians modify dosing to achieve concentrations or exposures (ie, AUC) found to be effective in patients with similar clinical attributes and conditions. These concentrations, called therapeutic (or target) concentrations or exposure ranges (TRs), are specific to both disease/condition and patient population. TDM is routinely used by many clinicians to adjust dosing of a wide range of medications for maximal efficacy and limited toxicity, thereby improving clinical outcomes. Failure to properly perform TDM or to appreciate the limitations of TDM have, however, contributed to the delayed acceptance of TDM by clinicians. This Commentary briefly discusses the limitations and the benefits of TR-guided TDM, and then discusses immunosuppressant drugs and anticancer medications as examples of drugs that require clinicians to change their prescribing practices from giving all patients the same or maximal tolerated doses, to instead adjusting individual doses to achieve minimal effective concentrations identified using circulating tumor- or graft-derived DNA or copy number instability rather than published TRs.

Donor-derived cell-free DNA as a diagnostic tool in transplantation.

There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. Biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and prevent immune activation is also important. There is robust clinical evidence from a large number of published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. Dd-cfDNA indicates graft cell death without being rejection specific. It can be determined in plasma through droplet digital PCR using preselected SNPs or next generation sequencing. Changes in recipient cfDNA (e.g., by infection) can affect the results of dd-cfDNA fractional determination. This limitation can be overcome using absolute dd-cfDNA quantification. The combination of fractional and absolute determination including total cfDNA is recommended for meaningful interpretation of the results. The value proposition for the patient includes earlier transplant injury detection and intervention, less full blown rejection risk, an alternative to invasive biopsies, and personalized immunosuppression with potential for improved long-term outcome. Transplant physicians benefit from better immunosuppressive guidance and having an alternative when biopsies are refused or contraindicated. Further advantages are improved biopsy interpretation, less trial and error changes in immunosuppression, and less time dealing with complications. The laboratory medicine specialist can provide more effective services. Hospital management and insurance companies could benefit from more cost-effective surveillance of transplant recipients. Potential cost savings would result from fewer biopsies as a result of the tests' high negative predictive value, fewer re-transplantations, and less organ failure with return to dialysis. A pathway to implementation and metrics is suggested to measure the effectiveness of dd-cfDNA testing.

Liquid biopsies: donor-derived cell-free DNA for the detection of kidney allograft injury.

In kidney transplantation, the use of minimally invasive damage biomarkers that are more sensitive and specific than plasma creatinine will be crucial to enable early, actionable detection or exclusion of structural kidney damage due to acute or chronic rejection. Donor-derived cell-free DNA (dd-cfDNA), which can be quantified, for example, through next-generation sequencing, droplet digital PCR and quantitative PCR, is a candidate biomarker with great potential for enabling comprehensive monitoring of allograft injury. dd-cfDNA has a favourable overall diagnostic performance for the detection of rejection and its high negative predictive value might be especially useful for avoiding unnecessary biopsies. Elevated dd-cfDNA levels have been shown to be detectable before graft injury can be clinically identified using current diagnostic methods. Moreover, dd-cfDNA falls rapidly to baseline levels after successful treatment for rejection owing to its short half-life. dd-cfDNA can detect graft injury caused by immune activation owing to insufficient immunosuppression and might therefore also help guide immunosuppression dosing. The fractional abundance of dd-cfDNA can be affected by changes in the recipient cfDNA (for example, due to infection or physical exercise) but the use of absolute quantification of dd-cfDNA overcomes this limitation. Serial dd-cfDNA determinations might therefore facilitate cost-effective personalized clinical management of kidney transplant recipients to reduce premature graft loss.

Donor-Derived Cell-Free DNA Testing in Solid Organ Transplantation: A Value Proposition.

BACKGROUND: There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. More efficient biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and to prevent immune activation is also important. Donor-derived cell-free DNA (dd-cfDNA) has become available for comprehensive monitoring of allograft integrity. A value proposition concept was applied to assess the potential benefits of dd-cfDNA to stakeholders (patient, transplant physician, laboratory medicine specialist, hospital management, insurance companies) involved in solid organ transplantation care. CONTENT: There is robust clinical evidence from more than 48 published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. The value proposition framework was used to evaluate published key evidence regarding clinical validity, economic implications, and limitations of this approach. It has been shown that dd-cfDNA testing is essential for guiding earlier transplant injury intervention with potential for improved long-term outcome. SUMMARY: Monitoring dd-cfDNA offers a rapid and reproducible method to detect graft injuries at an early actionable stage without protocol biopsies and allows for more effective personalized immunosuppression. The appropriate use of dd-cfDNA testing can provide both clinical and economic benefits to all transplantation stakeholders.

Plasma EGFR mutation testing in non-small cell lung cancer: A value proposition.

Genomics-driven precision medicine using targeted therapies requires advanced molecular diagnostic tests. Decisions about the use and reimbursement for such tests are increasingly being made on the basis of more outcome-based and value-based approaches. The value proposition concept is a tool to assess the benefits of laboratory testing to each stakeholder of the care pathway with respect to outcomes. This concept was applied to the use of noninvasive plasma epidermal growth factor receptor (EGFR) mutation testing in patients with advanced or metastatic non-small cell lung cancer (NSCLC) to guide treatment with EGFR tyrosine kinase inhibitors (TKIs). Using the value proposition framework, we evaluated published key evidence regarding clinical validity, economic implications, and limitations of this approach. It has been shown that plasma EGFR mutation testing is essential for guiding clinical decisions regarding prediction of eligibility of individual patients for TKI treatment, real-time monitoring, or adjustment of treatment regimens and tracking resistance. The appropriate use of plasma EGFR mutation testing has been shown to deliver both clinical and economic benefits to stakeholders across the entire care pathway; especially in clinical situations where biopsy material is inadequate or unavailable and where it leads to fewer tissue biopsies.

Absolute quantification of donor-derived cell-free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study.

Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.3-fold and median dd-cfDNA(%) 2.0-fold higher (82 cp/mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy-proven rejection (BPR). dd-cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd-cfDNA(cp/mL) (AUC = 0.83) compared to dd-cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd-cfDNA(cp/mL), and 6.02 for dd-cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd-cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd-cfDNA(cp/mL) with lower tacrolimus levels (<8 µg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd-cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd-cfDNA(cp/mL) allowed for better discrimination than dd-cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.

Circulating Cell-Free DNA-Diagnostic and Prognostic Applications in Personalized Cancer Therapy.

Genomic analyses in oncologic care allow for the development of more precise clinical laboratory tests that will be critical for personalized pharmacotherapy. Traditional biopsy-based approaches are limited by the availability of sequential tissue specimens to detect resistance. Blood-based genomic profiling ("liquid biopsy") is useful for longitudinal monitoring of tumor genomes and can complement biopsies. Tumor-associated mutations can be identified in cell-free tumor DNA (ctDNA) from patient blood samples and used for monitoring disease activity. The US Food and Drug Administration approved a liquid biopsy test for EGFR-activating mutations in patients with non-small-cell lung cancer as a companion diagnostic for therapy selection. ctDNA also allows for the identification of mutations selected by treatment such as EGFR T790M in non-small-cell lung cancer. ctDNA can also detect mutations such as KRAS G12V in colorectal cancer and BRAF V600E/V600K in melanoma. Chromosomal aberration pattern analysis by low-coverage whole genome sequencing is a new, broader approach. Genomic imbalances detected in cell-free DNA (cfDNA) can be used to compute a copy number instability (CNI) score. In clinical studies, it was demonstrated that the change in CNI score can serve as an early predictor of therapeutic response to chemotherapy/immunotherapy of many cancer types. In multivariable models, it could be shown that the CNI score was superior to clinical parameters for prediction of overall survival in patients with head and neck cancer. There is emerging evidence for the clinical validity of ctDNA testing regarding identification of candidates for targeted therapies, prediction of therapeutic response, early detection of recurrence, resistance mutation detection, measuring genetic heterogeneity, tumor burden monitoring, and risk stratification. Improvement of sensitivity to detect tumors at very early stages is difficult due to insufficient mutant DNA fraction of ≤0.01%. Further developments will include validation in prospective multicenter interventional outcome studies and the development of digital platforms to integrate diagnostic data.

Cell-Free Plasma DNA for Disease Stratification and Prognosis in Head and Neck Cancer.

BACKGROUND: Clinicians face many challenges in disease stratification and outcome prediction in head and neck squamous cancer cell (HNSCC) patients. Given the limitations of currently used clinical scoring, repetitive biopsies, and imaging techniques, liquid biopsy approaches may provide valuable additional diagnostic and prognostic information. METHODS: A noninterventional, single-center observational study was performed with clinical data and plasma samples from HNSCC patients. Cell-free tumor DNA-derived copy number aberrations (CNAs) were determined in 116 patients by low-coverage next-generation sequencing (NGS). Significant CNAs were combined in a genome-wide copy number instability score (CNI), which was evaluated with respect to conventional clinical staging and patient outcome. RESULTS: Receiver-operating characteristic (ROC) curve analysis comparing the presurgery CNI in patients (n = 103) with that in tumor-free controls (n = 142) yielded an area under the ROC curve of 87.2% (95% CI, 79.4%-93.3%). At a specificity of 95%, the sensitivity to detect tumors varied between 46% (pT1) and 94% (pT4). A CNI above the median (i.e., >72) had a positive predictive value of 90% (95% CI, 79%-96%) for lymph node involvement (LNI), while the negative predictive value was 57% (95% CI, 43%-70%). For a CNI >72, overall survival (OS) was worse (hazard ratio, 4.89; 95% CI, 1.39-17.17; P = 0.01) with 62% and 90% survivors 3 years after surgery for a CNI >72 and ≤72, respectively. In multivariable models, the CNI was a superior predictor of OS compared to established disease features, including LNI. CONCLUSIONS: The CNI may assist in predicting LNI and prognosis in HNSCC with direct therapeutic implications concerning the need for neck dissection or more aggressive treatment.

Diagnostic value of alpha-1-fetoprotein (AFP) as a biomarker for hepatocellular carcinoma recurrence after liver transplantation.

BACKGROUND: Alpha-1-fetoprotein (AFP) is used to monitor progression, evaluate response to therapy and predict recurrence of hepatocellular carcinoma (HCC) in liver transplantation (LTx) patients. To date, the diagnostic value of serum AFP determinations for detecting tumor recurrence in HCC patients after LTx is unclear. OBJECTIVE: A retrospective, single-center, cross-sectional, non-interventional study was performed with the objective of determining post-transplant cut-off AFP values for detecting HCC recurrence post LTx. METHODS: Using receiver operating characteristic (ROC) analyses, post-transplant serum AFP values were evaluated against HCC recurrences in 63 HCC patients who had LTx between November 1995 and December 2011 at the University Medical Center Göttingen (UMG). Optimal and application-independent cut points for predicting tumor recurrence at 1, 3, and 5years after LTx were determined. RESULTS: Post-LTx serum AFP was found to represent an independent risk factor (predictor) for HCC relapse. Post-operative AFP cut-off values of 7µg/l, 6µg/l, and 6µg/l, respectively, were determined to be optimal at 1, 3, and 5years after LTx respectively for predicting a HCC relapse. Using these cut-off values, patients were correctly classified as relapse-positive with a diagnostic sensitivity of 79%, 81%, and 77%, and as relapse-free with a specificity of 82%, 79%, and 69%. The diagnostic accuracy measured by area under the curve (AUC) values ranged from 0.813 to 0.886. However, a limitation is that at a clinically relevant specificity of ≥95%, the analyses showed sensitivity values of only 50%, 52%, and 50%, respectively. CONCLUSION: Post-transplant serum AFP may have diagnostic value to detect HCC recurrence after LTx.

Using circulating cell-free DNA to monitor personalized cancer therapy.

High-quality genomic analysis is critical for personalized pharmacotherapy in patients with cancer. Tumor-specific genomic alterations can be identified in cell-free DNA (cfDNA) from patient blood samples and can complement biopsies for real-time molecular monitoring of treatment, detection of recurrence, and tracking resistance. cfDNA can be especially useful when tumor tissue is unavailable or insufficient for testing. For blood-based genomic profiling, next-generation sequencing (NGS) and droplet digital PCR (ddPCR) have been successfully applied. The US Food and Drug Administration (FDA) recently approved the first such "liquid biopsy" test for EGFR mutations in patients with non-small cell lung cancer (NSCLC). Such non-invasive methods allow for the identification of specific resistance mutations selected by treatment, such as EGFR T790M, in patients with NSCLC treated with gefitinib. Chromosomal aberration pattern analysis by low coverage whole genome sequencing is a more universal approach based on genomic instability. Gains and losses of chromosomal regions have been detected in plasma tumor-specific cfDNA as copy number aberrations and can be used to compute a genomic copy number instability (CNI) score of cfDNA. A specific CNI index obtained by massive parallel sequencing discriminated those patients with prostate cancer from both healthy controls and men with benign prostatic disease. Furthermore, androgen receptor gene aberrations in cfDNA were associated with therapeutic resistance in metastatic castration resistant prostate cancer. Change in CNI score has been shown to serve as an early predictor of response to standard chemotherapy for various other cancer types (e.g. NSCLC, colorectal cancer, pancreatic ductal adenocarcinomas). CNI scores have also been shown to predict therapeutic responses to immunotherapy. Serial genomic profiling can detect resistance mutations up to 16 weeks before radiographic progression. There is a potential for cost savings when ineffective use of expensive new anticancer drugs is avoided or halted. Challenges for routine implementation of liquid biopsy tests include the necessity of specialized personnel, instrumentation, and software, as well as further development of quality management (e.g. external quality control). Validation of blood-based tumor genomic profiling in additional multicenter outcome studies is necessary; however, cfDNA monitoring can provide clinically important actionable information for precision oncology approaches.

Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: A prospective, observational, multicenter cohort study.

BACKGROUND: Graft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection. METHODS AND FINDINGS: Traditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy-proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%-41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%-3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%-10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)-positive, rejection-free patients. LFTs had low overall correlations (r = 0.28-0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%-98.0%) and 92.9% (95% CI 89.3%-95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%-100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits. CONCLUSIONS: In this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further research, which should ideally include protocol biopsies, will be needed to establish the practical value of GcfDNA measurements in the management of LTx patients.

Do the European Medicines Agency Decisions Hurt Pediatric Melanoma Patients?

PURPOSE: US pediatric legislation was introduced in 1997 and was followed by European Union pediatric legislation that, since 2007, requires a European Medicines Agency (EMA)-approved pediatric investigation plan (PIP) for registration of new medicines unless they are PIP exempted. In 2008, the EMA decided that enough adolescent patients with melanoma existed and removed melanoma from the list of PIP-exempted diseases (class waiver list). We examined the logic and the results of this decision. METHODS: We analyzed the EMA class waiver decision, the melanoma PIP decisions, the wording of the European Union pediatric legislation, and melanoma trials listed in www.clinicaltrials.gov and www.clinicaltrialsregister.eu that recruit adults and minors or only minors. FINDINGS: There are 12 melanoma PIP decisions. Two apparently PIP-triggered melanoma trials were terminated in 2016 because of slow recruitment, and 4 are ongoing. Numerous non-PIP-driven trials are recruiting both adults and minors with melanoma worldwide, thus competing with PIP-triggered melanoma trials. IMPLICATIONS: Revoking the melanoma class waiver was not based on science but on flawed logic. It resulted in PIP-demanded pediatric trials that do not make medical sense, fail to recruit adequately, and prevent participants from more promising off-label treatment or treatment in clinically, scientifically, and ethically superior non-PIP-triggered studies. Institutional review boards and ethics committees should consult both www.clinicaltrials.gov and www.clinicaltrialsregister.eu for competing trials in the same population and reject or withdraw approval for questionable trials. A major revision or replacement of the European Union pediatric legislation is needed to prevent children from being enrolled in unnecessary, unfeasible, or scientifically invalid trials.

Graft-Derived Cell-Free DNA as a Marker of Transplant Graft Injury.

Although short-term success after solid organ transplantation is good, long-term graft and recipient survival are both not satisfactory. Despite therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs), both excessive and insufficient immunosuppression still do occur. There is a need for new biomarkers that, when combined with TDM, can be used to provide more effective and less toxic, personalized immunosuppression to improve long-term survival. Currently used methods are insufficient to rapidly, cost-effectively, and directly interrogate graft integrity after solid organ transplantation. However, because organ transplants are also genome transplants, measurement of graft-derived circulating cell-free DNA (GcfDNA) has shown promise as a way to improve both graft and recipient outcomes after solid organ transplantation through the early detection of severe graft injury, enabling an early intervention. A newly developed droplet digital polymerase chain reaction (ddPCR) method has advantages over expensive high-throughput sequencing methods to rapidly quantify GcfDNA percentages and absolute amounts. This procedure does not require donor DNA and therefore can be applied to any organ donor/recipient pair. The droplet digital polymerase chain reaction method allows for the early, sensitive, specific, and cost-effective direct assessment of graft integrity and can be used to define individual responses to ISDs including the minimal ISD exposures necessary to prevent rejection. This is especially important in patients undergoing ISD switches due to ISD toxicity, infections, or malignancies. Although prospective, multicenter clinical trials in liver, heart, and kidney transplantation have not been completed, early results suggest that GcfDNA can be combined with TDM to guide changes in immunosuppression to provide more effective, and less toxic treatment. Personalized immunosuppression will shift emphasis in transplantation from reaction to prevention and could improve outcome at lower health care costs.

The contributions of the European Medicines Agency and its pediatric committee to the fight against childhood leukemia.

BACKGROUND: Although the diagnosis of childhood leukemia is no longer a death sentence, too many patients still die, more with acute myeloid leukemia than with acute lymphoblastic leukemia. The European Union pediatric legislation was introduced to improve pharmaceutical treatment of children, but some question whether the European Medicines Agency (EMA) approach is helping children with leukemia. Some have even suggested that the decisions of EMA pediatric committee (PDCO) are counterproductive. This study was designed to investigate the impact of PDCO-issued pediatric investigation plans (PIPs) for leukemia drugs. METHODS: All PIPs listed under "oncology" were downloaded from the EMA website. Non-leukemia decisions including misclassifications, waivers (no PIP), and solid tumors were discarded. The leukemia decisions were analyzed, compared to pediatric leukemia trials in the database http://www.clinicaltrials.gov, and discussed in the light of current literature. RESULTS: The PDCO leukemia decisions demand clinical trials in pediatric leukemia for all new adult drugs without prioritization. However, because leukemia in children is different and much rarer than in adults, these decisions have resulted in proposed studies that are scientifically and ethically questionable. They are also unnecessary, since once promising new compounds are approved for adults, more appropriate, prioritized pediatric leukemia trials are initiated worldwide without PDCO involvement. CONCLUSION: EMA/PDCO leukemia PIPs do little to advance the treatment of childhood leukemia. The unintended negative effects of the flawed EMA/PDCO's standardized requesting of non-prioritized testing of every new adult leukemia drug in children with relapsed or refractory disease expose these children to questionable trials, and could undermine public trust in pediatric clinical research. Institutions, investigators, and ethics committees/institutional review boards need to be skeptical of trials triggered by PDCO. New, better ways to facilitate drug development for pediatric leukemia are needed.

Lack of correlation between Treg quantification assays in inflammatory bowel disease patients.

AIM: To compare the number of regulatory T-cells (Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR (qPCR) method in patients suffering from inflammatory bowel disease (IBD). METHODS: Tregs percentages obtained by both flow cytometry and qPCR methods in 35 adult IBD patients, 18 out of them with Crohn´s disease (CD) and 17 with ulcerative colitis (UC) were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index (HBI) for CD patients and the Simple Colitis Clinical Activity Index (SCCAI) for UC patients. The Treg percentages by flow cytometry were defined as CD4(+)CD25(high)CD127(low)FOXP3(+) cells in peripheral blood mononuclear cells, whereas the Treg percentages by qPCR method were determined as FOXP3 promoter demethylation in genomic DNA. RESULTS: We found an average of 1.56% ± 0.78% Tregs by using flow cytometry, compared to 1.07% ± 0.53% Tregs by using qPCR in adult IBD patients. There were no significant correlations between either the percentages of Tregs measured by flow cytometry or qPCR and the HBI or SCCAI questionnaire scores in CD or UC patients, respectively. In addition, there was no correlation between Treg percentages measured by qPCR and those measured by flow cytometry (r = -0.06, P = 0.73; Spearman Rho). These data suggest that, either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity. Until the cause(s) for these differences are more clearly defined, the results suggest caution in interpreting studies of Tregs in various inflammatory disorders. CONCLUSION: The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.

Use of graft-derived cell-free DNA as an organ integrity biomarker to reexamine effective tacrolimus trough concentrations after liver transplantation.

BACKGROUND: Immunosuppressant therapeutic ranges for transplant patients have traditionally been established by indirect clinical means. However, "liquid biopsy" methods measuring graft-derived cell-free DNA (GcfDNA) in blood directly interrogate donor organ integrity. This study was performed to determine whether GcfDNA quantification could be used to reexamine minimally effective trough tacrolimus (Tacro) concentrations in liver transplantation (LTx) patients. METHODS: As part of a large prospective study to demonstrate the ability of GcfDNA to identify early graft rejection, 10 adult white LTx patients [8 men, 2 women, 3 hepatitis C virus (HCV) positive; mean ± SD age (years) = 56 ± 9.4] had simultaneous GcfDNA and whole-blood trough Tacro concentrations measured between days 5 and 30 after LTx. Samples were analyzed using droplet digital polymerase chain reaction for GcfDNA and liquid chromatography tandem mass spectrometry for Tacro. GcfDNA and trough Tacro concentrations were then compared to identify Tacro concentrations associated with intact graft integrity. RESULTS: Although there were large individual differences, there was a highly significant (Fisher P = 0.00002) segregation between whole-blood Tacro concentrations of ≥8 µg/L and normal (≤10%) GcfDNA percentages. The best discrimination in this population between effective and ineffective trough Tacro concentrations was estimated to be at 6.8 µg/L (P < 10(-7)). Compared with HCV- patients (n = 7), the 3 HCV+ patients had more variable associations between GcfDNA percentages and Tacro concentrations. CONCLUSIONS: Direct measurement of graft integrity using GcfDNA was useful to confirm the lower limit of the therapeutic ranges for trough Tacro concentrations after LTx. It would probably be useful to do so also for other immunosuppressant drugs and after other solid organ transplants. The method might be especially useful to detect graft injury during immunosuppressant dose minimization strategies.

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue.

OBJECTIVES: FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC). DESIGN AND METHODS: Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n=6 ICC induced by thioacetamide and n=4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay. RESULTS: A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P<0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n=11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n=6) and in tumor tissue (4.14%, n=6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P<0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n=6) in comparison to controls (2.14%, n=4). CONCLUSION: FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.

Pharmacokinetics of meropenem in critically ill patients with severe infections.

BACKGROUND: Meropenem is an effective ß-lactam antibiotic that is frequently used to treat serious infections in both intensive care unit (ICU) and febrile neutropenic hematology/oncology (Hem/Onc) patients. Studies suggest that to be effective, meropenem concentrations must be maintained above the inhibitory concentrations for the majority of a dosing interval. However, the pharmacokinetics (PK) of meropenem seem to differ in critically ill patients compared with healthy or less ill subjects used to select labeled dosing regimens. OBJECTIVES: This study was designed to investigate meropenem PK in critically ill patients and to see how often standard dosing regimens produced adequate plasma concentrations. A secondary objective was to investigate how achieved concentrations were related to outcomes (morbidity and mortality) in these patients. METHODS: Meropenem plasma concentrations over time were measured using a high pressure liquid chromatography assay in febrile Hem/Onc and ICU patients who were treated with standard meropenem dosing schedules. Outcomes such as fever control and survival were assessed in these patients and compared with individual meropenem PK data and with recommended target concentrations. RESULTS: A total of 25 subjects including 10 febrile Hem/Onc and 15 ICU patients with a variety of serious illnesses and baseline renal function were studied. Mean peak concentrations were less variable than were pre-dose concentrations. Post peak and trough concentrations were often below recommended minimum inhibitory concentrations. Both clearance and volumes of distribution were greater than reported in less ill subjects, only in part explained by increased renal clearance. Therefore, serum concentrations often did not exceed recommended concentration targets even for moderately sensitive organisms. Inadequate concentrations were especially common in the mostly ill, febrile neutropenic Hem/Onc subjects and seemed to explain at least some therapeutic failures. Conversely, drug accumulation occurred in ICU subjects with decreased renal function. CONCLUSIONS: Standard meropenem dosing regimens were inadequate in many critically ill febrile, neutropenic Hem/Onc, and septic ICU patients. These data suggest a role for meropenem concentration monitoring in such patients.