OBJECTIVE:Traditional rejection surveillance after heart transplantation (HTx) is based on endomyocardial biopsies (EMBs), which are invasive, expensive, and associated with complications. Monitoring using cell-free DNA (cfDNA) is promising, but most studies report only on the donor fraction (DF) as the percentage of donor-derived cfDNA (dd-cfDNA) relative to total cfDNA. We evaluated the performance of dd-cfDNA to detect rejection. METHODS:HTx patients were prospectively enrolled in a multicenter study, and blood samples were collected concurrently with EMB. Dd-cfDNA was quantified using droplet digital PCR (ddPCR). Rejection was defined by EMB results and compared to nonrejection EMB. Patients with symptomatic rejection were studied as a subgroup, and test performance was determined using receiver operation characteristic analysis. RESULTS:We included 94 patients (70 adults and 24 children), which resulted in 1007 EMB and blood samples. In 19 patients, there were 32 rejection episodes >14 days past HTx, with 15 of them being symptomatic. In receiver operation characteristic analysis, dd-cfDNA and DF could discriminate quiescence from rejection with an area under the curve (AUC) of 0.68 and 0.65, respectively. Dd-cDNA at a threshold of 25 copies/ml showed an AUC of 0.87 to detect symptomatic rejection, significantly better than DF (AUC of 0.75). CONCLUSIONS:dd-cfDNA found good discrimination between cardiac recipients with and without rejection. Absolute quantification of dd-cfDNA with ddPCR is a fast and effective method to monitor graft health. Analyzing absolute dd-cfDNA levels helps identify other factors, besides rejection, that may influence cfDNA levels, potentially reducing the need for EMB.

In this prospective study we investigated a cohort after heart transplantation with a novel PCR-based approach with focus on treated rejection. Blood samples were collected coincidentally to biopsies, and both absolute levels of dd-cfDNA and donor fraction were reported using digital PCR. 52 patients (11 children and 41 adults) were enrolled (NCT03477383, clinicaltrials.gov), and 557 plasma samples were analyzed. 13 treated rejection episodes >14 days after transplantation were observed in 7 patients. Donor fraction showed a median of 0.08% in the cohort and was significantly elevated during rejection (median 0.19%, p < 0.0001), using a cut-off of 0.1%, the sensitivity/specificity were 92%/56% (AUC ROC-curve: 0.78). Absolute levels of dd-cfDNA showed a median of 8.8 copies/mL and were significantly elevated during rejection (median 23, p = 0.0001). Using a cut-off of 7.5 copies/mL, the sensitivity/specificity were 92%/43% for donor fraction (AUC ROC-curve: 0.75). The results support the feasibility of this approach in analyzing dd-cfDNA after heart transplantation. The obtained values are well aligned with results from other trials. The possibility to quantify absolute levels adds important value to the differentiation between ongoing graft damage and quiescent situations.

Background: Heart transplantation (HTx) relies on an endomyocardial biopsy for rejection surveillance. Cell-free DNA (cfDNA) is released into the circulatory system from the cells of the recipient (recipient-derived rd-cfDNA) as well as from the graft (donor-derived dd-cfDNA) and is determined by the analysis of a blood test. There is growing evidence that the donor fraction (DF, the ratio of dd-cfDNA to all cfDNA) corresponds to the integrity of the graft. However, it is important that rd-cfDNA levels vary greatly in different, sometimes clinically silent circumstances, which in turn makes DF the sole measure of graft status vulnerable to bias. Methods: Using a digital PCR-based method that allowed us to quantify separate values of rd-cfDNA, dd-cfDNA, and DF, we investigated a prospective cohort of 52 patients during their first year after HTx. Results: We found median DF levels of approximately 0.1%, which is in accordance with the literature. We present patients with various clinical scenarios after HTx, including an uncomplicated clinical course, several infections (viral and bacterial), acute and chronic rejection, and false-negative and false-positive rejections. Conclusions: Quantification of circulating cfDNA reflects a live-view of cell turnaround in the recipient and graft. Measuring the DF alone may not be sufficient to fully understand the complex biology of cfDNA after HTx.

OBJECTIVE:Cell-free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR-based approach allowing us to separately quantify absolute values of dd-cfDNA and recipient-derived cfDNA (rd-cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. METHODS:We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false-negative and false-positive rejections in six patients (five adults, one child). RESULTS:There was a substantial discrepancy between relative (DF) and absolute cfDNA-levels in several clinical situations. Rd- and dd-cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. CONCLUSIONS:Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection.

Introduction:Myeloproliferative neoplasm (MPN) is a heterogenous group of hematological malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F is the most frequent driver mutation in all three entities, but in PMF and ET mutations in CALR and MPL are also frequent. Mutations seen in additional genes are also often the same regardless of subtype of MPN. The aim of this study was to analyze a population based MPN cohort for genetic variants with prognostic value that can guide clinical decisions.Methods:MPN patients from Western Sweden diagnosed between 2008-2013 (n=248) were screened for mutations in 54 genes associated with myeloid malignancy.Results:Mutations in the genes SRSF2 and U2AF1 correlated significantly with impaired overall survival but did not correlate to increased risk for vascular events, neither before nor after diagnosis. Rather, mutations in these genes showed an association with disease transformation. Several recurrent gene variants with allele frequency close to 50% were confirmed to be germline. However, none of these variants was found to have an earlier onset of MPN.Discussion:In conclusion, we identified gene mutations to be independent markers of impaired survival in MPN. This indicates the need for more individualized assessment and treatment of MPN patients and a wider gene mutation screening already at diagnosis. This could ensure the identification of patients with high-risk mutations early on. In addition, several genetic variants were also identified as germline in this study but gave no obvious clinical relevance. To avoid conclusions from non-informative genetic variants, a simultaneous analysis of normal cell DNA from patients at diagnosis should be considered.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Allomap test was defined as ≥32 in patients ≤6 months and ≥34 in patients > 6 months post-HT.Positive Allosure test was defined as ≥0.12%.Positive biopsy included ISHLT Grades ≥1B.Results: 320 heart transplant recipients enrolled to this study.Mean age was 57.5 §14.0, 231 were males (72.0%).There was a total of 523 Heart-Care blood tests drawn and 148 biopsies were performed (Figure).Of 82 patients with elevated Allomap and Allosure, only 7 (8.5%) of them had a confirmed rejection episode.Among 71 patients with elevated Allosure with normal Allomap, only 2 (2.8%) patients had confirmed rejection.2 patients (4.3%) with negative Allomap and Allosure had rejection.During the study period 55 patients (17.2%) were admitted to the hospital for a total of 67 admissions.17 patients were admitted for rejection (5.3%).None of the patients died during the study period.Conclusion: Transitioning from invasive endomyocardial biopsy monitoring to a non-invasive HeartCare protocol in HT recipients seems feasible based on the high negative value of the combined Allomap and Allosure tests.However, further study is needed to follow the long-term outcomes and presence of rejection in patients post-transplant with this protocol.

INTRODUCTION:Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR.METHODS:Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs.RESULTS:The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA.CONCLUSION:DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.

normothermic temperatures, however, may aggravate graft injury, especially in endothelial cells.We hypothesized that replacing CSS with hypothermic, oxygenated perfusion (HOPE) provides cardioprotection by preserving the vasculature through the production of nitric oxide.Methods: Following anaesthesia, diaphragm transection and circulatory arrest in male Wistar rats to simulate DCD conditions, hearts underwent 21 min of warm, in-situ ischemia.Hearts were then subjected to either 30 min of CSS, HOPE, or HOPE with the presence of L-NAME (nitric oxide synthase inhibitor).Afterwards, hearts were reperfused ex-situ for 60 min under oxygenated, normothermic conditions.Results: Compared to CSS, HOPE hearts demonstrated higher cardiac function (determined by cardiac output, left ventricular work, as well as contraction and relaxation rates) after 60 min of reperfusion.Furthermore, preliminary results indicate a higher coronary vascular resistance at the end of the hypothermic perfusion period in hearts with L-NAME compared to HOPE alone.Early reperfusion coronary flow, an indicator of vascular function, tended to be higher in hearts subjected to HOPE compared to hearts treated with L-NAME or to the current clinical scenario (CSS).Conclusion: Preservation of vascular and contractile function with HOPE appears superior to the current clinical protocol (CSS).The increase in coronary flow during early reperfusion in HOPE hearts was abolished with the addition of L-NAME, indicating that the beneficial vascular effects of HOPE could be mediated by the production of nitric oxide.Consequently, we believe that HOPE holds great potential for preservation of cardiac grafts obtained with DCD.

Polycythaemia vera (PV) patients have an overall comparatively favourable prognosis, but disease progression is very heterogeneous and life-threatening thrombosis and bleedings are frequent complications in untreated disease. Moreover, transformation to more severe secondary myelofibrosis and acute myeloid leukaemia can occur. The aim of this study was to identify gene mutations that could be used together with clinical data as prognostic markers to guide treatment decisions in PV patients. A well-characterized WHO-defined cohort of PV patients was used. Clinical data and blood values were evaluated and a myeloid sequencing panel was used to screen for additional mutations other than the diagnostic JAK2 V617F and JAK2 exon 12 mutations. In 78% of the PV patients, at least one mutation additional to JAK2 V617F was detected. Additional mutations in genes coding for epigenetic modifiers, like TET2, DNMT3A and ASXL1, were most frequent. When correlated to overall survival, mutations in ASXL1 were significantly associated with inferior survival. In an attempt to obtain prognostic guidance in a larger number of patients, the presence of ASXL1 mutations was combined with age and vascular complications prior to diagnosis. Based on these data we were able to define three risk groups that predicted survival.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

Transcriptional studies of the human heart provide insight into physiological and pathophysiological mechanisms, essential for understanding the fundamental mechanisms of normal cardiac function and how they are altered by disease. To improve the understanding of why men and women may respond differently to the same therapeutic treatment it is crucial to learn more about sex-specific transcriptional differences. In this study the transcriptome of right atrium and left ventricle was compared across sex and regional location. Paired biopsies from five male and five female patients undergoing aortic valve replacement or coronary artery bypass grafting were included. Gene expression analysis identified 620 differentially expressed transcripts in atrial and ventricular tissue in men and 471 differentially expressed transcripts in women. In total 339 of these transcripts overlapped across sex but notably, 281 were unique in the male tissue and 162 in the female tissue, displaying marked sex differences in the transcriptional machinery. The transcriptional activity was significantly higher in atrias than in ventricles as 70% of the differentially expressed genes were upregulated in the atrial tissue. Furthermore, pathway- and functional annotation analyses performed on the differentially expressed genes showed enrichment for a more heterogeneous composition of biological processes in atrial compared with the ventricular tissue, and a dominance of differentially expressed genes associated with infection disease was observed. The results reported here provide increased insights about transcriptional differences between the cardiac atrium and ventricle but also reveal transcriptional differences in the human heart that can be attributed to sex.

Purpose Donor-derived cell-free DNA (dd-cfDNA) as a highly sensitive marker of rejection after heart transplantation (HTx) has gained emerging interest. Recent studies are based on sequencing techniques and use fractional abundance (dd-cfDNA as a fraction of total cfDNA) as their outcome. Here we present patient examples of the BIODRAFT-study (NCT03477383) based on PCR-techniques. Methods Blood samples are taken prospectively in parallel with endomyocardial biopsies (EMB) during the first year after HTx. dd-cfDNA is analyzed using targeted preamplification of 35 single nucleotide polymorphisms followed by digital droplet-PCR. Outcome is fractional abundance as well as total number of DNA copies, both from the donor and the recipient. Results 71 patients (57 adults, 14 children) are so far included and more than 500 blood samples analyzed. In otherwise stable patients, both fractional abundance and total DNA copies seem to follow biopsy patterns with respect to rejection. However, we could also identify different scenarios in which the distribution of cfDNA seems more complicated than hitherto described: Patients with primary graft failure, clinically silent CMV-infection or mild right heart failure show elevated levels of dd-cfDNA, thus complicating the interpretation of results. Some patients present with markedly elevated levels of their own cfDNA without obvious clinical reasons. Conclusion Besides the well-known concept of fractional abundance, our approach gives results that allow following absolute DNA copy numbers of both donor and recipient. This widens the horizon of cfDNA as a marker of graft injury after HTx, and gives an outlook to clinical scenarios with possible false positive (mimicking rejection) and, even worse, false negative results: high levels of recipient-cfDNA could mask ongoing rejection if only fractional abundance can be reported. The biology of cell-free DNA is complex and seems not yet fully understood. Donor-derived cell-free DNA (dd-cfDNA) as a highly sensitive marker of rejection after heart transplantation (HTx) has gained emerging interest. Recent studies are based on sequencing techniques and use fractional abundance (dd-cfDNA as a fraction of total cfDNA) as their outcome. Here we present patient examples of the BIODRAFT-study (NCT03477383) based on PCR-techniques. Blood samples are taken prospectively in parallel with endomyocardial biopsies (EMB) during the first year after HTx. dd-cfDNA is analyzed using targeted preamplification of 35 single nucleotide polymorphisms followed by digital droplet-PCR. Outcome is fractional abundance as well as total number of DNA copies, both from the donor and the recipient. 71 patients (57 adults, 14 children) are so far included and more than 500 blood samples analyzed. In otherwise stable patients, both fractional abundance and total DNA copies seem to follow biopsy patterns with respect to rejection. However, we could also identify different scenarios in which the distribution of cfDNA seems more complicated than hitherto described: Patients with primary graft failure, clinically silent CMV-infection or mild right heart failure show elevated levels of dd-cfDNA, thus complicating the interpretation of results. Some patients present with markedly elevated levels of their own cfDNA without obvious clinical reasons. Besides the well-known concept of fractional abundance, our approach gives results that allow following absolute DNA copy numbers of both donor and recipient. This widens the horizon of cfDNA as a marker of graft injury after HTx, and gives an outlook to clinical scenarios with possible false positive (mimicking rejection) and, even worse, false negative results: high levels of recipient-cfDNA could mask ongoing rejection if only fractional abundance can be reported. The biology of cell-free DNA is complex and seems not yet fully understood.

Successful hematopoietic stem and progenitor cell (HSPC) transplantation rests upon reliable methods for their enumeration in sources such as cord blood (CB). Methods used today are costly, time consuming and exhaust the limited number of cells needed for transplantation. The aim of this study was to analyze if surplus plasma from CB contains biomarkers that can predict HSPC content in CB. Frozen, surplus plasma from 95 CB units was divided into two groups based on CD34+ cell concentration. Birth weight, gestation age, gender, mode of delivery, collection volume, nucleated cell count and colony forming unit assay results were available. Samples were analyzed with a proximity ligation assay covering 92 different proteins. Two-group t-test with p-values adjusted for false discovery rate (FDR) identified 5 proteins that significantly differed between the two groups. CDCP1 was the most significant (FDR adjusted p-value 0.006). Correlation with CDCP1 concentration was most significant for CD34+ concentration and nucleated cell count. Multivariate analysis showed that CD34 and gender seemed to influence the level of CDCP1. In conclusion, CDCP1 was identified as a potential biomarker of HSPC content in CB. The finding also warrants further investigation for a possible role of CDCP1 in regulating HSPC presence in CB.

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0.02% was achieved. The assay was linear in MRD range (0.03% to 1%) with good precision [CV, 4.1% (2.2% to 5.7%) at VAF 1% and 13.3% (8.8% to 19.4%) at VAF 0.1%], and Low relative bias [7.9% (2.5% to 15.3%) at VAF 1%]. When applied to six childhood AML cases and compared with multiparameter flow cytometry for MRD analysis, deep sequencing showed concordance and superior sensitivity. Further high concordance was found with expression of fusion transcripts RUNX1-RUNX1T1 and KMT2A-MLLT10. The deep sequencing assay also detected mutations in blood when VAF in bone marrow exceeded 0.1% (n = 19). In conclusion, deep sequencing enables reliable detection of Low levels of residual leukemic cells. Introduction of this method in patient care will allow for highly sensitive MRD surveillance in virtually every patient with AML.

Mutations in NPM1 can be used for minimal residual disease (MRD) analysis in acute myeloid leukemia (AML). We here applied a newly introduced method, deep sequencing, allowing for simultaneous analysis of all recurrent NPM1 insertions and thus constituting an attractive alternative to multiple PCRs for the clinical laboratory. We retrospectively used deep sequencing for measurement of MRD pre- and post-allogeneic hematopoietic stem cell transplantation (alloHCT). For 29 patients in morphological remission at the time of alloHCT, the effect of deep sequencing MRD on outcome was assessed. MRD positivity was defined as variant allele frequency ≥0.02%. Post-transplant MRD status was significantly and independently associated with clinical outcome; 3-year relapse-free survival 20% vs 85% (p < .001), HR 45 (95% CI 2-1260), and overall survival 20% vs 89% (p < .001), HR 49 (95% CI 2-1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML.

Donor-derived cell-free DNA (dd-cfDNA) has gained emerging interest as a highly specific marker of rejection after heart transplantation (HTx). However, the method is not established, in part due to technical issues: Sequencing techniques come with high cost and time delay, while PCR-based methods struggle with a low yield of cfDNA-harvest. Hitherto there has been a focus on either relative (dd-cfDNA versus all cfDNA, including recipient-derived cfDNA [rd-cfDNA] + dd-cfDNA) or absolute measurements of dd-cfDNA alone. We propose a novel approach involving a targeted pre-amplification step before digital droplet PCR using probe-based SNP-assays is performed, allowing for the measurement of both relative and absolute values of dd-cfDNA at the same time. This approach is currently being evaluated in an ongoing, prospective single-center study of HTx recipients.