Julia Beck

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.

Profile of the Circulating DNA in Apparently Healthy Individuals

BACKGROUND Circulating nucleic acids (CNAs) have been shown to have diagnostic utility in human diseases. The aim of this study was to sequence and organize CNAs to document typical profiles of circulating DNA in apparently healthy individuals. METHODS Serum DNA from 51 apparently healthy humans was extracted, amplified, sequenced via pyrosequencing (454 Life Sciences/Roche Diagnostics), and categorized by (a) origin (human vs xenogeneic), (b) functionality (repeats, genes, coding or noncoding), and (c) chromosomal localization. CNA results were compared with genomic DNA controls (n = 4) that were subjected to the identical procedure. RESULTS We obtained 4.5 x 10(5) sequences (7.5 x 10(7) nucleotides), of which 87% were attributable to known database sequences. Of these sequences, 97% were genomic, and 3% were xenogeneic. CNAs and genomic DNA did not differ with respect to sequences attributable to repeats, genes, RNA, and protein-coding DNA sequences. CNA tended to have a higher proportion of short interspersed nuclear element sequences (P = 0.1), of which Alu sequences were significant (P < 0.01). CNAs had a significantly lower proportion of L1 and L2 long interspersed nuclear element sequences (P < 0.01). In addition, hepatitis B virus (HBV) genotype F sequences were found in an individual accidentally evaluated as a healthy control. CONCLUSIONS Comparison of CNAs with genomic DNA suggests that nonspecific DNA release is not the sole origin for CNAs. The CNA profiling of healthy individuals we have described, together with the detailed biometric analysis, provides the basis for future studies of patients with specific diseases. Furthermore, the detection of previously unknown HBV infection suggests the capability of this method to uncover occult infections.

Next Generation Sequencing of Serum Circulating Nucleic Acids from Patients with Invasive Ductal Breast Cancer Reveals Differences to Healthy and Nonmalignant Controls

Circulating nucleic acids (CNA) isolated from serum or plasma are increasingly recognized as biomarkers for cancers. Recently developed next generation sequencing provides high numbers of DNA sequences to detect the trace amounts of unique serum biomarkers associated with breast carcinoma. Serum CNA of 38 women with ductal carcinoma was extracted and sequenced on a 454/Roche high-throughput GS-FLX platform and compared with healthy controls and patients with other medical conditions. Repetitive elements present in CNA were detected and classified, and each repetitive element was normalized based on total sequence count or repeat count. Multivariate regression models were calculated using an information-theoretical approach and multimodel inference. A total of 423,150 and 953,545 sequences for the cancer patients and controls, respectively, were obtained. Data from 26 patients with stages II to IV tumors and from 67 apparently healthy female controls were used as the training data set. Using a bootstrap method to avoid sampling bias, a five-parameter model was developed. When this model was applied to a validation data set consisting of patients with tumor stage I (n = 10) compared with healthy and nonmalignant disease controls (n = 87; 1,261,561 sequences) a sensitivity of 70% at a specificity of 100% was obtained. At a diagnostic specificity level of 95%, a sensitivity of 90% was calculated. Identification of specific breast cancer–related CNA sequences provides the basis for the development of a serum-based routine laboratory test for breast cancer screening and monitoring. Mol Cancer Res; 8(3); 335–42

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.

Prion interaction with the 37-kDa/67-kDa laminin receptor on enterocytes as a cellular model for intestinal uptake of prions.

Enterocytes, a major cell population of the intestinal epithelium, represent one possible barrier to the entry of prions after oral exposure. We established a cell culture system employing enterocytes from different species to study alimentary prion interaction with the 37-kDa/67-kDa laminin receptor LRP/LR. Human, bovine, porcine, ovine, and cervid enterocytes were cocultured with brain homogenates from cervid, sheep, and cattle suffering from chronic wasting disease (CWD), scrapie, and bovine spongiform encephalopathy (BSE), respectively. PrP(CWD), ovine PrP(Sc), and PrP(BSE) all colocalized with LRP/LR on human enterocytes. PrP(CWD) failed to colocalize with LRP/LR on bovine, porcine, and ovine enterocytes. Ovine PrP(Sc) colocalized with the receptor on bovine enterocytes, but failed to colocalize with LRP/LR on cervid and porcine enterocytes. PrP(BSE) failed to colocalize with the receptor on cervid and ovine enterocytes. These data suggest possible oral transmissibility of CWD and sheep scrapie to humans and may confirm the oral transmissibility of BSE to humans, resulting in zoonotic variant Creutzfeldt-Jakob disease. CWD might not be transmissible to cattle, pigs, and sheep. Sheep scrapie might have caused BSE, but may not cause transmissible spongiform encephalopathy in cervids and pigs. BSE may not be transmissible to cervids. Our data recommend the enterocyte model system for further investigations of the intestinal pathophysiology of alimentary prion infections.

FLT3-ITD and TLR9 use Bruton tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. Although many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton tyrosine kinase (BTK) in AML. BTK was frequently expressed, and its inhibition strongly impaired the proliferation and survival of AML cells also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics, and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context dependent. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive AML, BTK mediates FLT3-ITD-dependent Myc and STAT5 activation, and combined targeting of FLT3-ITD and BTK showed additive effects. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to nuclear factor κΒ and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and, for the first time, point to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA.

Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS) was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR). Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20). Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA) was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants.

Molecular genetics of coat colour variations in White Galloway and White Park cattle.

White Galloway cattle exhibit three different white coat colour phenotypes, that is, well marked, strongly marked and mismarked. However, mating of individuals with the preferred well or strongly marked phenotype also results in offspring with the undesired mismarked and/or even fully black coat colour. To elucidate the genetic background of the coat colour variations in White Galloway cattle, we analysed four coat colour relevant genes: mast/stem cell growth factor receptor (KIT), KIT ligand (KITLG), melanocortin 1 receptor (MC1R) and tyrosinase (TYR). Here, we show that the coat colour variations in White Galloway cattle and White Park cattle are caused by a KIT gene (chromosome 6) duplication and aberrant insertion on chromosome 29 (Cs29 ) as recently described for colour-sided Belgian Blue. Homozygous (Cs29 /Cs29 ) White Galloway cattle and White Park cattle exhibit the mismarked phenotype, whereas heterozygous (Cs29 /wt29 ) individuals are either well or strongly marked. In contrast, fully black individuals are characterised by the wild-type chromosome 29. As known for other cattle breeds, mutations in the MC1R gene determine the red colouring. Our data suggest that the white coat colour variations in White Galloway cattle and White Park cattle are caused by a dose-dependent effect based on the ploidy of aberrant insertions and inheritance of the KIT gene on chromosome 29.

Chromosomal Instability in Cell-Free DNA Is a Serum Biomarker for Prostate Cancer

BACKGROUND Genomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate. METHODS DNA was extracted from serum of 204 patients with prostate cancer (Gleason score 2-10), 207 male controls, and patients with benign hyperplasia (n = 10) and prostatitis (n = 10). DNA was amplified by use of random primers, tagged with molecular identifiers, sequenced on a SOLID system, and aligned to the human genome. We evaluated the number of sequence reads of cfDNA in sliding 100-kbp intervals for variation from controls. We used chromosomal regions with significant variations in alignment hits for their ability to segregate patients and matched controls. RESULTS Using ROC curves to assess diagnostic performance, we evaluated the number of regions in a first subset (n = 177), with variations in alignment hits alone, provided an area under the curve (AUC) of 0.81 (95% CI 0.7-0.9, P < 0.001). Using 5 rounds of 10-fold cross-validation with the full data set, we established a final model that discriminated prostate cancer from controls with an AUC of 0.92 (0.87-0.95), reaching a diagnostic accuracy of 83%. Both benign prostatic hypertrophy and prostatitis could be distinguished from prostate cancer by use of cfDNA, with an accuracy of 90%. CONCLUSIONS Assessment of a limited number of chromosomal structural instabilities by use of massive parallel sequencing of cfDNA was sufficient to distinguish between prostate cancer and controls. This large cohort demonstrates the utility of cfDNA in prostate cancer recently established in other malignant neoplasms.

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.