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Clonal hematopoiesis (CH) denotes somatic mutations in genes related to myeloid neoplasms present at any variant allele frequency (VAF). Clonal hematopoiesis is associated with increasing age and with a factor 6 increase in the risk of developing therapy-related myeloid neoplasms (tMNs) following autologous stem cell transplantation (ASCT). However, the impact of specific mutations on progression from CH to tMN has yet to be unraveled, and it remains unclear whether mutations directly impact or even drive the development of tMN. We performed deep sequencing in longitudinal samples from a cohort of 12 patients with either multiple myeloma or lymphoma who developed tMN following ASCT. Nine patients had one or more mutations that could be tracked longitudinally. Seven patients had clonal expansion from time of ASCT to diagnosis of tMN. Of these, six patients had CH at VAF

This work was partially supported by intramural funds of Univerity of Pisa and DKFZ; by Fondo de Investigaciones Sanitarias (Madrid, Spain) [PI12/02688 to J. S., PI17/02276 to J.S.]; by Instituto de Salud Carlos III, co-funded by FEDER funds —a way to build Europe—[PI14-00613 to V.M.] and by Agency for Management of University and Research Grants (AGAUR) of the Catalan Government (Barcelona, Spain) [2017SGR723 to V.M.]. Open Access funding enabled and organized by Projekt DEAL., Telomeres are involved in processes like cellular growth, chromosomal stability, and proper segregation to daughter cells. Telomere length measured in leukocytes (LTL) has been investigated in different cancer types, including multiple myeloma (MM). However, LTL measurement is prone to heterogeneity due to sample handling and study design (retrospective vs. prospective). LTL is genetically determined; genome-wide association studies identified 11 SNPs that, combined in a score, can be used as a genetic instrument to measure LTL and evaluate its association with MM risk. This approach has been already successfully attempted in various cancer types but never in MM. We tested the "teloscore" in 2407 MM patients and 1741 controls from the International Multiple Myeloma rESEarch (IMMeNSE) consortium. We observed an increased risk for longer genetically determined telomere length (gdTL) (OR = 1.69; 95% CI 1.36-2.11; P = 2.97 x 10(-6) for highest vs. lowest quintile of the score). Furthermore, in a subset of 1376 MM patients we tested the relationship between the teloscore and MM patients survival, observing a better prognosis for longer gdTL compared with shorter gdTL (HR = 0.93; 95% CI 0.86-0.99; P = 0.049). In conclusion, we report convincing evidence that longer gdTL is a risk marker for MM risk, and that it is potentially involved in increasing MM survival., Univerity of Pisa, Helmholtz Association, Instituto de Salud Carlos III PI12/02688 PI17/02276, Instituto de Salud Carlos III, European Commission, FEDER funds-a way to build Europe PI14-00613, Agency for Management of University and Research Grants (AGAUR) of the Catalan Government (Barcelona, Spain) 2017SGR723, Projekt DEAL

Clonal hematopoiesis of indeterminate potential (CHIP) is suspected of being a risk factor for patients with cancer. This study aimed to assess the clinical consequences of CHIP in patients with lymphoma intended for high-dose chemotherapy and autologous stem-cell transplantation (ASCT) in a population-based setting. We identified 892 lymphoma patients who had undergone stem cell harvest at all transplant centers in Denmark. A total of 565 patients had an available harvest sample, which was analysed for CHIP by next-generation sequencing, and the median follow-up was 9.1 years. Of the patients who were intended for immediate ASCT, 25.5% (112/440) carried at least one CHIP mutation. In contrast to previous single-center studies CHIP was not associated with inferior overall survival (OS) in multivariate analyses. However, patients with mutations in genes of the DNA repair pathway (PPM1D, TP53, RAD21, BRCC3) had a significant inferior OS (HR after 1 year of follow-up 2.79, 95% confidence interval 1.71–4.56; p < 0.0001), which also was evident in multivariate analysis (p = 0.00067). These patients had also increased rates of therapy-related leukemia and admission to intensive care. Furthermore, in patients who did not undergo immediate ASCT, a significant inferior OS of individuals with DNA repair mutations was also identified (p = 0.003).

Therapy-related myeloid neoplasms (tMN) develop after exposure to cytotoxic and radiation therapy, and due to their adverse prognosis, it is of paramount interest to identify patients at high risk. The presence of clonal hematopoiesis has been shown to increase the risk of developing tMN. The value of analyzing hematopoietic stem cells harvested at leukapheresis before autologous stem cell transplantation (ASCT) with next-generation sequencing and immunophenotyping represents potentially informative parameters that have yet to be discovered. We performed a nested case-control study to elucidate the association between clonal hematopoiesis, mobilization potential, and aberrant immunophenotype in leukapheresis products with the development of tMN after ASCT. A total of 36 patients with nonmyeloid disease who were diagnosed with tMN after treatment with ASCT were included as case subjects. Case subjects were identified from a cohort of 1130 patients treated with ASCT and matched with 36 control subjects who did not develop tMN after ASCT. Case subjects were significantly poorer mobilizers of CD34+ cells at leukapheresis (P = .016), indicating that these patients possess inferior bone marrow function. Both clonal hematopoiesis (odds ratio, 5.9; 95% confidence interval, 1.8-19.1; P = .003) and aberrant expression of CD7 (odds ratio, 6.6; 95% confidence interval, 1.6-26.2; P = .004) at the time of ASCT were associated with an increased risk of developing tMN after ASCT. In conclusion, clonal hematopoiesis, present at low variant allele frequencies, and aberrant CD7 expression on stem cells in leukapheresis products from patients with nonmyeloid hematologic cancer hold potential for the early identification of patients at high risk of developing tMN after ASCT.

Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL), are generally considered mutually exclusive, but several reports have suggested that they coexist in a small subgroup of patients. In this study, we retrospectively searched for CALR mutations in 136 suspected MPN patients with low allelic burden (≤5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutations were identified, of whom 10 were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR-mutated MPN cases. Type 1 is generally considered the most common mutation, but, interestingly, our double-mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of coexisting JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F.

The prognosis of children with acute myeloid leukemia (AML) is improving but still unsatisfactory. We and others have shown that measurable residual disease (MRD) as measured with multiparameter flow cytometry (MFC) is a very strong prognostic factor, however this technique cannot reliably identify low-risk cases. An alternative approach for MRD analysis is quantification of leukemia-specific transcripts using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). This approach has shown strong prognostic value in adult AML but its significance in childhood AML is less studied. In this retrospective study, we evaluated early treatment response with RT-qPCR and MFC in parallel to determine treatment kinetics and the relationship between results of the two methods. The study included 15 children (7 females, 8 males, median age 6 years (range 1-16)), diagnosed 2004-2011 with de novo AML with a quantifiable fusion transcript (8 with RUNX1-RUNX1T1, one with CBFB-MYH11 and 6 with KMT2A-MLLT3), all treated in the NOPHO AML-2004 trial. MRD analyses were performed on samples from day 15 from start of the first induction course and just before the first consolidation course. MRD analysis with MFC was performed in bone marrow samples by identifying leukemia-associated immunophenotypes (LAIP), and reported as percent of viable cells. Fusion transcripts were analyzed with RT-qPCR in bone marrow and blood according to the Europe against cancer program, and reported as percentage of diagnostic level in bone marrow. Results of all MRD analyses were reported to clinicians but not used for treatment decisions. All patients achieved complete remission. 9 patients relapsed (median time from diagnosis 12 months, range 8-16); 5 with RUNX1-RUNX1T1 and 4 with KMT2A-MLLT3. When we compared results of RT-qPCR in blood and bone marrow day 15 and before consolidation, there was a high correlation; 0.93, p0.1%. These analyses confirmed the absence of immature cells with LAIP and detected either more mature myeloid cells or immature cells without LAIP. We hypothesize that fusion transcripts can be present in mature cells not detected as MRD using MFC. This was supported by analyzing sorted immature and mature cells from bone marrow samples at diagnosis of AML (n=4, adults). In CD34-CD117+ cells, leukemic transcripts (RUNX1-RUNX1T1/CBFB-MYH11/mutated NPM1) were 67±10% (mean±SEM) of the level in the most immature cells, CD34+CD117+, from the same patients and in mature granulocytes (CD34-CD117-, high SSC) 88±9%. In conclusion, RT-qPCR and MFC provide different information during induction treatment. RT-qPCR has a stronger ability to detect remaining leukemic burden, but whether this burden is clinically relevant remains to be shown. The impact of fusion transcript levels on relapse risk is currently investigated in the NOPHO-DBH AML-2012 trial in which MFC is used for risk stratification. Disclosures No relevant conflicts of interest to declare.

Over the past four decades, remarkable progress has been made in the treatment and prognosis of multiple myeloma (MM), although it remains an incurable disease. Chemotherapy resistance is a major hurdle for treatment efficacy. Drug resistance can be innate and so driven by genes involved in the drug metabolism pathways. We performed an association study of 71 germline variants within the major genes in those pathways (ABCB1, ABCC2, ABCG2, and their regulators NR1I2/PXR and NR1I3/CAR) in the International Multiple Myeloma rESEarch (IMMEnSE) consortium, consisting of 1365 MM cases with survival information recruited in 5 European countries. Two of the SNPs showed a significant association with the survival of MM patients, namely rs2235013, located in ABCB1 [Hazard ratio (HR) = 1·52, 95% confidence interval (CI) = 1·18-1·95, P = 0·00087], and rs4148388, located in ABCC2 (HR = 2·15, 95% CI = 1·44-3·22, P = 0·0001). ABCC2 plays an essential role in transporting various anticancer drugs, including several used against MM, out of the cell. In silico analyses predict that the variant alleles of four SNPs in linkage disequilibrium with ABCC2-rs4148388 are associated with increased gene expression. Overexpression of ABCC2 increases drug clearance and therefore may induce drug resistance mechanisms. In conclusion, we found a promising association between ABCC2-rs4148388 and MM outcome that is supported by a plausible biological explanation., This work was partially supported by intramural funds of DKFZ and University of Pisa. This work was also partially supported by grant from FIBAO foundation and from the Instituto de Salud Carlos III (Madrid, Spain; PI17/02276)., info:eu-repo/semantics/publishedVersion

Background: Somatic driver mutations in hematopoietic cells may lead to clonal hematopoiesis of indeterminate potential (CHIP). In patients with lymphoma CHIP has been associated with increased risk of therapy-related myeloid neoplasms (tMN) and inferior survival after autologous stem cell transplantation as demonstrated in a large single center study and in a case-control study (Gibson CJ et al., JCO 2017 and Berger G et al., Blood 2018). Here, we investigated the clinical impact of clonal hematopoiesis in a nation-wide population-based cohort of Danish lymphoma patients undergoing autologous transplant with prospective data from four national patient registries. Methods: Patients with lymphoma who had undergone leukapheresis at all danish transplant centers from 2000 to 2012 were identified. DNA and RNA was extracted from mobilized peripheral blood products. Targeted sequencing of all samples was performed using an Illumina TruSeq Custom Amplicon panel (Illumina, San Diego, CA, USA) designed to cover >95% of mutations associated with CHIP (ASXL1, ASXL2, BCOR, BRCC3, CBL, CREBBP, DNMT3A, ETV6, GNB1, IDH1, IDH2, JAK2, KRAS, NRAS, PPM1D, RAD21, SF3B1, SRSF2, TET2, TP53). To allow detection of low-level mutations and secure variant calling, unique molecular identifiers (UMI's) were used. Filtering of variants was done by stringent criteria consistent with earlier studies. Assessment of mutations was performed blinded to the patients' clinical data. Prospective clinical patient data was obtained for all patients from four national registries, including the Danish Lymphoma Registry (diagnosis, involvement, lymphoma treatment, relapse and death), the Danish National Patient Registry (hospital admission diagnoses and treatments), the Danish Cancer Registry (primary and secondary cancer diagnoses) and the Danish Pathology Database (histopathological examinations and diagnoses), respectively. Results: Samples from 574 patients were included. The median age was 55.5 years (IQR: 45.3 - 62.2) and the median follow-up time for survivors was 9.2 years (IQR: 7.1 - 11.2). The lymphoma subtypes were typical of patients selected for autologous transplantation; diffuse large B-cell lymphoma (191 pts), follicular lymphoma (102 pts), mantle cell lymphoma (88 pts), Hodgkin's lymphoma (80 pts), peripheral T-cell lymphoma (77 pts) and other histologies (36 pts). Of the 574 patients analyzed, 191 (33.3%) of the patients had somatic mutations meeting CHIP criteria (total mutations called=210). The most commonly mutated genes were DNMT3A (n=59, 28%), TET2 (n=48, 23%), PPM1D (n=34, 16%), ASXL1 (n=21, 10%) and TP53 (n=18, 8%). As expected CHIP mutations were more frequent in patients above 60 years (p=0.002). Prevalence of CHIP was associated with an inferior overall survival (p=0.004) and event-free survival (p=0.03). It was also associated with increased risk of biopsy-confirmed tMN (p=0.03) and higher probability of receiving blood transfusions after autologous transplant (p=0.027). Especially patients with mutations in DNA damage response genes PPM1D and TP53 (found in 48 pts, 8.3%) had a significantly increased risk of adverse outcomes. Both overall survival and event-free survival were significantly poorer with the presence of DNA damage pathway mutations (p The impact of low-level mutations and statistical modelling of interactions between parallel outcomes will be presented at the meeting. Conclusion: To our knowledge this is the first population-based study of clonal hematopoiesis in patients with lymphoma. We find that CHIP and particularly mutations in DNA damage response genes (PPM1D/TP53) are associated with increased mortality, which confirms findings from single center studies. These data support the evaluation of CHIP for risk assessment in lymphoma patients before high-dose chemotherapy. Our study also identifies increased rates of several clinically relevant adverse outcomes (severe infections, blood transfusions and secondary cancers) in lymphoma patients with clonal hematopoiesis. Figure 1. Figure 1. Disclosures Grønbæk: Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

Summary Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of

Recent studies have identified SOX11 as a novel diagnostic marker for mantle cell lymphoma (MCL). We quantified SOX11 by a truly mRNA specific qPCR assay in longitudinal peripheral blood samples from 20 patients and evidenced a close relationship of SOX11 expression and clinical status of the patients. In eight patient courses we validated the expression of SOX11 using t(11;14) and demonstrated positive correlation of SOX11 and t(11;14) levels. To the best of our knowledge this is the first report stating that quantification of SOX11 can be used as an minimal residual disease marker equal to the key translocation t(11;14) in MCL.