Philipp Kanzow

Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury

BACKGROUND Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR). METHODS Plasma samples were collected from stable recipients after liver (LTx, n = 10), kidney (KTx, n = 9), and heart (HTx, n = 8) transplantation as well as from 7 additional patients directly after LTx. Known single-nucleotide polymorphisms were selected for high minor allelic frequencies, of which 41 hydrolysis probe assays were established. Plasma cfDNA was preamplified, followed by conventional real-time PCR to define informative (heterologous) SNPs, which were then used for quantification (percentage) of graft-derived cfDNA (GcfDNA) using ddPCR. RESULTS Mean recovery was 94% (SD, 13%) with an imprecision of 4%-14% with the use of controls with 2% minor allele. GcfDNA in stable patients was <6.8% (LTx), <2.5% (KTx), and <3.4% (HTx). On the day of LTx, GcfDNA was approximately 90% and by day 10 it was <15% in complication-free LTx recipients. In 2 patients with biopsy-proven rejection, GcfDNA increased to >60%, whereas in 1 patient with cholestasis no increase was found. CONCLUSIONS A novel, cost-effective, rapid technique was developed to quantify GcfDNA in transplant recipients. This technique embodies a promising, potentially universal biomarker for early detection of rejection, which could enable more effective therapeutic interventions.

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 &mgr;g/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 &mgr;g/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.

Using circulating cell-free DNA to monitor personalized cancer therapy

Abstract 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.

Etiology and pathogenesis of dental erosion

The condition of dental erosion is defined as acid-related loss of tooth structure which does not involve microorganisms. Depending on the origin of the acid, extrinsic (usually caused by acids in food) and intrinsic (caused by endogenous acid) erosion can be distinguished. The presence and severity of erosive defects depend on various parameters such as nutrition, saliva, general diseases, and mechanical stress by abrasion and attrition. As an example, dietary habits which involve frequent intake of acidic food and beverages, occupational acid exposure, as well as certain drugs or diseases that affect saliva flow rate are accompanied by an increased risk of erosive dental hard tissue defects. By a thorough clinical examination and an accurate anamnesis, various erosion-related risk factors can be identified and strategies to reduce or eliminate these factors be identified.

Understanding the management and teaching of dental restoration repair: Systematic review and meta-analysis of surveys

OBJECTIVES Repair instead of complete replacement is recommended to manage partially defective restorations. It is unclear if and why such treatment is taught at dental schools or practiced by dentists. We aimed to identify barriers and facilitators for repairs using a systematic review and meta- and qualitative analysis. SOURCES Electronic databases (PubMed, CENTRAL, Embase, PsycINFO) were searched. STUDY SELECTION Quantitative studies reporting on the proportion of (1) dentists stating to perform repairs, (2) dental schools teaching repairs, (3) failed restorations having been repaired were included. We also included qualitative studies on barriers/facilitators for repairs. Random-effects meta-analyses, meta-regression and a thematic analysis using the theoretical domains framework were conducted. DATA 401 articles were assessed and 29, mainly quantitative, studies included. 7228 dentists and 276 dental schools had been surveyed, and treatment data of 30,172 restorations evaluated. The mean (95% CI) proportion of dentists stating to perform repairs was 71.5% (49.7-86.4%). 83.3% (73.6-90.0%) of dental schools taught repairs. 31.3% (26.3-36.7%) of failed restorations had been repaired. More recent studies reported significantly more dentists to repair restorations (p=0.004). Employment in public health practices and being the dentist who placed the original restoration were facilitators for repairs. Amalgam restorations were repaired less often, and financial aspects and regulations came as barriers. CONCLUSIONS While most dentists state to perform repairs and the vast majority of dental schools teach repairs, the proportion of truly repaired restorations was low. A number of interventions to implement repair in dental practice can be deduced from our findings. CLINICAL SIGNIFICANCE Partially defective restorations are common in dental practice. While repairs are taught and dentists are aware of the recommendation towards repairs, the actually performed proportion of repairs seems low.

Chapter 7 – Graft-derived cell-free DNA as a marker of graft integrity after transplantation

Genome transplant dynamics is a particularly promising new approach for the detection of graft injury based on the determination of graft-derived circulating cell-free DNA (cfDNA) in the blood of the recipient. An increase of donor DNA is an early indication of organ damage. A novel potential routine assay for graft-derived circulating cfDNA quantification has been developed using droplet digital polymerase chain reaction for the determination of the donor/recipient circulating cfDNA ratio. This method is very cost-effective and provides results on the same day. Monitoring graft-derived cfDNA has the advantage that it directly interrogates the health of the donor organ, and it allows early detection of transplant injury (“liquid biopsy”). The detection of subclinical rejection would be desirable to allow early intervention. Undiagnosed chronic damage can result in chronic rejection. The determination of graft-derived circulating cfDNA may complement or possibly replace other approaches for post-transplant monitoring, and it may improve the chances of long-term graft survival. This method will be helpful to individualize immunosuppressive regimens. Personalized immunosuppression will in the future shift emphasis from reaction to prevention, which could make immunosuppressive drugs safer and more effective and also reduce the cost of health care.

Contemporary teaching of restoration repair at dental schools in Germany – Close to universality and consistency

OBJECTIVES To identify potential changes in various aspects of teaching and to ascertain whether previously found inconsistencies in the teaching of criteria, indications and operative techniques for the repair of defective composite restorations at German dental schools have been resolved. METHODS A validated questionnaire was used to gain the information sought. It was sent to all dental schools in Germany (n = 30). Whenever possible, data were compared to previous studies conducted in 2000 and 2009. Statistical analysis was performed using Fishers exact tests (p < 0.05). RESULTS Twenty-nine schools responded to the survey - a response rate of 97%. All respondents indicated positive experiences with the repair of restorations. The teaching of repairs in 2018 (90%) was found to be comparable to the findings from the 2009 survey (88%, p = 1.000), but significantly increased since the 2000 survey (50%, p = 0.006). Main reasons reported for teaching repairs are tooth substance preservation (97%) and reduction of pulpal damage (79%). Main clinical indications are marginal defects and secondary caries. When performing repairs, almost all dental schools were found to teach both mechanical and adhesive substrate surface conditioning. Marked variation was observed in the method of mechanical surface treatment, with air abrasion having gained widespread popularity. The average expected longevity of repairs was 7.4 ± 3.0 years. CONCLUSIONS The teaching of the repair of resin composite restorations is widespread in dental schools in Germany. Aspects of this teaching were found to be more consistent between dental schools than in previous surveys, albeit variation in operative techniques still exists. CLINICAL SIGNIFICANCE Graduates from dental schools in Germany may be found to be well equipped with the knowledge and skills to perform repairs of defective resin based composite restorations in clinical practice.

Graft-Derived Cell-Free DNA (GcfDNA) as a Sensitive Measure of Individual Graft Integrity After Liver Transplantation.: Abstract# A7

A8 Exosomes in Human Bile for the Assessment of Transplanted Liver in the Early Postoperative Period. D. Yoshii,1 M. Mitsuhashi,2 T. Murokawa,1 K. Asonuma,1 Y. Inomata.1 1Transplantation and Pediatric Surgery, Kumamoto University Hospital, Kumamoto, Japan; 2Hitachi Chemical Research Center, Irvine. Background: After liver transplantation, transplanted liver undergoes various clinical problems. It is not always easy to make an accurate diagnosis to develop an appropriate therapeutic plan. Liver biopsy is the last resort for diagnosis, but it is invasive, and often it is not conclusive. Thus, alternative noninvasive methods are highly anticipated. Exosomes are membranous nanovesicles of 40-100nm in size, and are secreted by a variety of cell types into blood, urine, ascites, etc. More interestingly, because exosomes are known to contain proteins, mRNAs, and microRNAs, exosomes have become an interesting biomarker in many research fi elds. The aim of this study is to explore the values of yet-to-be characterized exosomes in the bile in patients with liver transplantation. Methods: From November 2012 to June 2013, we recruited 16 cases of liver transplant recipients after institutional review board approval. Using the external drainage stent tube, which is routinely placed in every case in our facility, bile was collected at POD1-7, 14, 21, 28, or everyday in some cases. Exosomes were isolated by the fi lter device, and poly(A)+ mRNAs were purifi ed by oligo(dT)-immobilized microplate and cCNA was synthesized on the same plate. The cDNAs were used for real time polymerase chain reaction (PCR) to quantify mRNAs, such as housekeeping gene (ACTB), albumin(ALB), interleukin (IL) (1β, 2, 6, 8, 10), TNFα, FasL, IFNγ, HGF, VEGF, CD16, PRG2 and DEFA3. Results and discussion: ACTB mRNA was successfully detected from the majority of bile samples, suggesting that the bile contained exosomes, and mRNAs were securely present inside exosome. Similarly, liver-specifi c ALB mRNA was also detected in the bile exosomes, suggesting that the bile contained liver-derived exosomes. Three patients diagnosed as acute rejection expressed various mRNAs, and especially the levels of IL-8 mRNA were very prominent. Moreover, eosinophil-derived PRG2 was increased at the time of rejection, which corresponded to the eosinophil-infi ltration, a typical pathological landmark of acute rejection. Conclusions: This study suggests that biliary exosomal mRNAs are promising biomarkers for the assessment of pathological conditions of transplanted livers. DISCLOSURES: Mitsuhashi, M.: Other, Hitachi Chemical Research Center, employee of R&D company developing of diagnostics. Abstract# A9 Monocyte Presentation of Donor Antigen Predicts Acute Cellular Rejection (ACR) After Liver or Intestine Transplantation (ITx, LTx). C. Ashokkumar, M. Ningappa, B. Higgs, Q. Sun, R. Sindhi. Surgery, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA. Background: Enhanced donor alloantigen presentation by CD14+monocytes predicts or associates with ACR in small cohorts of pediatric LTx and ITx. Purpose: To evaluate the performance of monocyte alloantigen presentation for predicting biopsy-proven ACR in training-set-validation-set testing of 67 pediatric LTx and ITx. Methods: The frequency of recipient CD14+monocytes which presented donor and HLA-non-identical third-party alloantigen was measured with fl ow cytometry and expressed as a ratio termed the monocyte antigen presenting index (mAPI). An mAPI >1 implied increased donor antigen presentation relative to presentation of third-party alloantigen. A threshold mAPI associated with ACR was developed in 35 cross-sectional training set samples from children with ITx (n=11) and LTx (n=24) using logistic regression and applied to 32 samples from 26 independent LTx and 6 independent ITx recipients. Test results were also correlated with allospecifi c CD154+T-cytotoxic memory cells (CD154+TcM), which predict ACR with high sensitivity and specifi city. Results: Mean age was 5.9 years (range 0.6 to 21, n=67). Distribution of male: female gender was 38: 29, caucasian: other race was 58: 9, LTx: ITx was 50: 17, and rejector: non-rejector status was 31: 19 for LTx and 9: 8 for ITx recipients. Rejectors were sampled at a mean interval of 5.4 days (range 0 to 38) before biopsy-proven ACR. A signifi cantly greater mAPI was seen among rejectors compared with non-rejectors 0.6±0.7 vs 4.4±6.2, p=0.003. For predicting ACR, the performance of an mAPI ≥ 1.2, developed in 35 training set samples was measured as sensitivity, specifi city, positive predictive value and negative predictive value. Test performance in the training set was 75%, 95%, 92% and 82%, respectively. Performance in 32 validation set samples was 73%, 81%, 67% and 85%, respectively. Monocyte antigen presentation correlated signifi cantly (Spearman r=0.423, p=0.022) with allospecifi c CD154+TcM in 29 of 32 validation samples. Conclusion: Enhanced donor alloantigen presentation by recipient monocytes predicts or associates with ACR with clinically relevant performance in training-set-validation-set testing of pediatric liver or intestine transplant recipients. This test system may have clinical utility in recipients treated with Tand B-cell ablative immunosuppressive regimens. DISCLOSURES: Ashokkumar, C.: Other, Plexision, Consultant. Sindhi, R.: Other, Plexision, Unpaid Consultlant. A9 Monocyte Presentation of Donor Antigen Predicts Acute Cellular Rejection (ACR) After Liver or Intestine Transplantation (ITx, LTx). C. Ashokkumar, M. Ningappa, B. Higgs, Q. Sun, R. Sindhi. Surgery, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA. Background: Enhanced donor alloantigen presentation by CD14+monocytes predicts or associates with ACR in small cohorts of pediatric LTx and ITx. Purpose: To evaluate the performance of monocyte alloantigen presentation for predicting biopsy-proven ACR in training-set-validation-set testing of 67 pediatric LTx and ITx. Methods: The frequency of recipient CD14+monocytes which presented donor and HLA-non-identical third-party alloantigen was measured with fl ow cytometry and expressed as a ratio termed the monocyte antigen presenting index (mAPI). An mAPI >1 implied increased donor antigen presentation relative to presentation of third-party alloantigen. A threshold mAPI associated with ACR was developed in 35 cross-sectional training set samples from children with ITx (n=11) and LTx (n=24) using logistic regression and applied to 32 samples from 26 independent LTx and 6 independent ITx recipients. Test results were also correlated with allospecifi c CD154+T-cytotoxic memory cells (CD154+TcM), which predict ACR with high sensitivity and specifi city. Results: Mean age was 5.9 years (range 0.6 to 21, n=67). Distribution of male: female gender was 38: 29, caucasian: other race was 58: 9, LTx: ITx was 50: 17, and rejector: non-rejector status was 31: 19 for LTx and 9: 8 for ITx recipients. Rejectors were sampled at a mean interval of 5.4 days (range 0 to 38) before biopsy-proven ACR. A signifi cantly greater mAPI was seen among rejectors compared with non-rejectors 0.6±0.7 vs 4.4±6.2, p=0.003. For predicting ACR, the performance of an mAPI ≥ 1.2, developed in 35 training set samples was measured as sensitivity, specifi city, positive predictive value and negative predictive value. Test performance in the training set was 75%, 95%, 92% and 82%, respectively. Performance in 32 validation set samples was 73%, 81%, 67% and 85%, respectively. Monocyte antigen presentation correlated signifi cantly (Spearman r=0.423, p=0.022) with allospecifi c CD154+TcM in 29 of 32 validation samples. Conclusion: Enhanced donor alloantigen presentation by recipient monocytes predicts or associates with ACR with clinically relevant performance in training-set-validation-set testing of pediatric liver or intestine transplant recipients. This test system may have clinical utility in recipients treated with Tand B-cell ablative immunosuppressive regimens. DISCLOSURES: Ashokkumar, C.: Other, Plexision, Consultant. Sindhi, R.: Other, Plexision, Unpaid Consultlant. Abstract# A10 Lymphocytes Can Play The Trojan-Horse Role in Polyoma BKV Infection. D. Morsy, L. Tibbles. Internal Medicine, University of Calgary, Calgary, AB, Canada. A10 Lymphocytes Can Play The Trojan-Horse Role in Polyoma BKV Infection. D. Morsy, L. Tibbles. Internal Medicine, University of Calgary, Calgary, AB, Canada. BKV nephropathy (BKN) is currently the leading cause of early renal allograft loss. The process starts after immune suppression following renal transplantation, which causes BK virus (BKV) reactivation, resulting in lytic injury of renal tubular epithelial cells followed by tubular infl ammation, and allograft destruction. Although the role played by lymphocytes in BKN pathogenesis remains largely uncharacterized, both cellular and humoral immunity were demonstrated to be involved in BKV early elimination. Our laboratory is interested in characterizing the role played by lymphocytes in BKV pathogenesis. Using real-time polymerase chain reaction (rtPCR) and immunoblotting, we demonstrated that BKV can infect human B and T cell lines. BKV was also shown to infect and replicate in primary lymphocytes from peripheral blood at higher effi ciency than cell lines. Viral replication was associated with remarkable changes in cell shape and behaviour, with cells acquiring a refractile, fusiform appearance and becoming more adhesive. B lymphocytes infected with BKV differentiated into both memory and plasma cells, but a signifi cant proportion of these differentiated cells were not specifi c for BKV proteins. These observations may provide a clue into the accumulation of plasma cells in renal transplants affected by BK nephropathy. The potential transmission of BKV virions between immune lymphocytes was confi rmed by rt-PCR of CFSE-labelled BKV infected and non-infected purifi ed lymphocytes that were p