ALBERT

Description: Background Follicular lymphoma (FL) is a B-cell lymphoma whose cytogenetic hallmark is the translocation t(14;18)(q32;q21) which juxtaposes the BCL2 oncogene to the immunoglobulin heavy chain locus (IGHV). FLs maintain key features of normal germinal center reactions, such as ongoing somatic hypermutation (SHM) of IGHV genes and selection for a functional B-cell receptor. SHM is mediated by activation-induced cytidine deaminase (AID) leading to single nucleotide exchange in IGHV genes and to a much lesser extent in non-IG genes. It was our objective to investigate the clonal evolution of t(14,18) FL from primary to relapse tumors simultaneously on several genetic and epigenetic levels. Methods We studied paired primary and relapsed tumors from 33 patients with t(14;18)-positive FL (76 samples: 25 pairs; 6 trios and 2 quadruples). In a core set of 19 patients we performed Sanger and next generation sequencing of the clonal VHDHJH rearrangements of the IGHV locus. We performed deep sequencing of 9 genes (BCL2, BCL6, MYC, RHOH, PAX5, IRF4, C2TA, REL and PIM1) targeted by aberrant SHM (aSHM) in lymphoma for 69 FL samples including the complete core set. We furthermore analyzed mutations in the coding regions of 10 candidate driver genes of lymphomagenesis (BCL2, MLL2, CREBBP, TNFRSF14, EZH2, EP300, MEF2B, BCL6, MYC, TP53) by deep sequencing. In addition to genetic analyses, evolutionary patterns of DNA-methylation were addressed by Illumina 27k arrays. Results We found strong evidence for ongoing selection against replacement mutations in the IGHV genes both in complementarity determining regions and framework regions, consistent with ongoing dependence of FL on a functional B-cell receptor and stimulation by antigens. Using mean normalized Hamming distance as a quantitative measure for the evolutionary divergence of paired samples (IGHV-divergence) and analyzing phylogenetic trees we classified the patterns of evolution into 3 categories: “No Evolution” (shared IGHV sequences in primary and relapse tumors), “Sequential Evolution” (relapse sequences emerge out of primary ones), “Divergent Evolution” (sequences of primary and relapse sample appear disjoint). We observed a mutation frequency of 62.0 per 100 kb in aSHM targets. These mutations were strongly enriched at the WRCY/RGYW target motifs characteristic for the SHM/AID machinery (OR=3.4; p

Description: Minimal residual disease (MRD) assessment via next generation sequencing (NGS) of immunoglobulin (Ig) and T-cell receptor (TR) gene rearrangements for lymphoid malignancies is currently under extensive development. NGS MRD has a potential to overcome the limitations of current techniques; laboriousness and difficult interpretation of qPCR for Ig/TR and low sensitivity of flow cytometry. However, amplicon-based NGS MRD has potential pitfalls that have to be addressed before it can be safely introduced for clinical decision making. Multi-center concordance in the experimental setting, quality control and interpretation of the results need to be achieved in order to surpass the advantages of qPCR, which is currently rigorously standardized within the EuroMRD consortium. Our aim was to test the stability and reproducibility of an optimized Ig heavy chain (IGH) based NGS approach for MRD assessment in a multi-center setting within the EuroClonality NGS Consortium on two different sequencing platforms. A one-step PCR library preparation approach was tested in seven institutions (Kiel, Salamanca, Milano, Bristol, London, Prague, Torino). Serial dilutions (10-1 to 10-5) of diagnostic DNA into polyclonal DNA as well as follow-up samples of 30 B-cell precursor ALLs with known complete IGH rearrangements were sequenced on the MiSeq. Serial dilutions of five different diagnostic ALL samples and libraries from polyclonal control were sequenced in parallel on both the MiSeq and Ion Torrent platforms. All samples were spiked with pre-defined copy numbers of five reference IGH sequences as a calibrator. FR2 primers, harboring platform-specific sequencing adapters, were used during the one-step PCR with 500ng of DNA per sample (75,000 copies). Negative and positive controls (27 pooled B-cell lines) were used for testing assay stability and reproducibility among the labs. Purpose-built bioinformatics methods were applied to analyze data. MRD results were compared to results of EuroMRD-based qPCR results. A total of 333 libraries were sequenced in 29 deep sequencing runs producing 194 million reads. The IGH gene rearrangements of all 27 pooled positive B-cell line controls were identified in all centers. NGS MRD analysis in 116 ALL follow-up samples revealed MRD positivity in 69/116 samples vs. 66/116 samples in qPCR, with discrepancies concerning samples with low MRD (R2=0.81). The dilution experiments gave similar results for both platforms, with a minimum sensitivity of 10-4 (as currently required by most treatment protocols using qPCR) for all tested assays. The correlation between MRD levels obtained by the two NGS platforms was good (R2=0.84). Ratios of reads containing reference IGH sequences were highly consistent in intra- and inter-laboratory analyses, independent of the total number of reads in the sample. When comparing platforms, in 10-1 dilution samples sequenced on MiSeq the ratio of reads harboring reference sequences was 2.1 to 2.7 times lower than in remaining dilutions, while on the Ion Torrent it was only 0.9 to 1.3 times, reflecting the competition with the leukemic clone. The correlation of the amounts of spiked-in sequences with the representation of reads harboring these sequences was slightly better for the Ion Torrent (R2=0.88) than for the MiSeq (R2=0.79). Amplification efficiency of each primer was checked by analyzing libraries from healthy polyclonal control. All primer sequences were present in all samples on both platforms, however, the differences between four libraries prepared from the same sample sequenced on the MiSeq were 2.6 times higher than in one library from this sample sequenced in five replicates on the Ion Torrent. The newly developed IGH assay shows robust intra and inter-laboratory reproducibility, which is the first step towards the safe use of this new MRD technique in a multi-center setting. The distribution of reference sequences and sequences of primers confirmed that the main source of differences between platform strategies is the library preparation and not the platform itself. Using the same amount of DNA, the sensitivity of the method is similar to qPCR. The performance and costs of the assay are similar for both the MiSeq and Ion Torrent. MRD analysis via NGS has therefore a great potential to replace qPCR as the gold standard for MRD-guided therapy in ALL, provided that thorough standardization can be achieved. Support: NV15-30626A, GBP302/12/G101. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics..

Description: INTRODUCTION: T-prolymphocytic leukemia (T-PLL) is a rare T cell malignancy with an aggressive clinical course. Preliminary clinical data suggest that allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in a proportion of patients. However, direct evidence that graft-versus-leukemia (GVL) activity is indeed effective in T-PLL is lacking. We sought to investigate GVL in T-PLL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions (immunosuppression tapering, donor lymphocyte infusions (DLI), chronic graft-versus-host disease (cGvHD)), and T cell receptor (TCR) repertoire diversity alterations after alloSCT. METHODS: The study sample consisted of 10 consecutive patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma (TRG) gene rearrangements. Data interpretation followed EuroMRD guidelines. In selected patients, TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS). TRBV-TRBD-TRBJ gene rearrangements were amplified according to BIOMED2 protocol on genomic DNA. PCR products were sequenced on Illumina's MiSeq platform. NGS data were analyzed through a purpose-built bioinformatics immunoprofiler (EuroClonality-NGS consortium). Rearrangements with very similar junctional amino acid sequences and identical TRBV and TRBJ gene usage were defined as clonotypes. RESULTS: Patients underwent alloSCT in remission after first-line (8) or salvage (2) alemtuzumab-based therapy. 5 patients were allografted with an unrelated donor, 4 with a related donor, and one received haploidentical alloSCT. Conditioning was fludarabine with cyclophosphamide and/or total body irradiation-based. All patients had a cytological complete response (CR) after alloSCT. 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 81+ and 12+ months post transplant), whilst MRD re-increased in 3 patients after 5-28 months despite ongoing cGvHD in one of them. NGS of the TCR repertoire was performed longitudinally in 3 patients with the longest follow-up. A total of 104 samples (blood (n=91) and BM (n=10) plus 3 donor blood samples) were sequenced. The sample at diagnosis showed one or two major clonotypes in all 3 sequenced T-PLLs, predominantly reflecting a mono- or biallelic leukemic TRB gene rearrangement. Kinetics of this leukemic clonotype followed kinetics of RQ-PCR MRD measurement, demonstrating that NGS can be used to quantify MRD in T cell lymphoma. Immediately after transplantation, the TRB repertoire was heavily skewed in all 3 patients, but recovered over time. Also in all 3 patients, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. This corresponded to the donor clonotype repertoire and disappeared with increasing MRD levels. In each of the samples after alloSCT, several expanded non-leukemic clonotypes were observed at a maximum frequency from 3% to 54% of total TRB sequences. However, during MRD response, novel dominant clonotypes that could explain a clonal GVL effect did not emerge. Figure 1 shows an example of a patient repeatedly showing MRD response to immune interventions. Currently, 5 patients are alive and in cytological CR (4-81 months after alloSCT), translating into a median relapse-free survival of 40 months. CONCLUSIONS: The MRD responses to immune interventions observed here provide the first direct evidence for the efficacy of GVL in T-PLL. However, the GVL effect in T-PLL appears to be often only limited or transient. It also does not seem to be caused by the emergence of novel dominant T cell clones but is rather relying on a poly-/oligoclonal T cell response. Figure 1. Example for MRD kinetics in relation to immune interventions. Figure 1. Example for MRD kinetics in relation to immune interventions. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The current gold standard molecular method for minimal residual disease (MRD) assessment is RQ-PCR for detection of leukemia-associated immunoglobulin (IG) and T cell receptor (TR) rearrangements, which must be identified beforehand in the diagnostic material. Conventional screening of IG/TR rearrangements in the diagnostic sample consists of several multiplex PCRs followed by Sanger sequencing. Recently, the EuroClonality-NGS Consortium (www.euroclonalityngs.org, coordinated by AW Langerak) developed a set of assays for next-generation sequencing-based marker identification in lymphoid malignancies (Brüggemann, Haematologica, 2017). We have been using this approach in routine diagnostics since 2016 and herein we provide an overview of markers detected in acute lymphoblastic leukemia (ALL) patients. Material & Methods Between 02/2017 and 06/2018, 471 ALL samples were screened employing the EuroClonality-NGS assay. The assay consists of 5-8 multiplex PCRs, depending on the type of malignancy [IGH-VJ, IGH-DJ, TRB-VJ, TRB-DJ, TRG, TRD, and IGK-VJ-Kde (and intron-Kde) tubes] followed by a 2nd-step PCR in which barcodes and sequencing adaptors are introduced. The resulting libraries were sequenced on the Illumina MiSeq, producing 2×250bp reads, aiming to reach at least 3000 reads per sequencing library. Rearrangements with an abundance ≥ 5% were considered to be leukemia-associated. Out of the 471 investigated samples, 453 were diagnostic (dx) and 18 were relapse (rel) samples. If available, bone marrow samples (BM) were investigated (n=370), otherwise peripheral blood (PB) samples were used (n=101). For B-ALL patients (n=344) all PCRs were performed, whereas for T-ALL (n=127) the IGH-VJ and both IGK tubes were omitted. Results At least one leukemia-associated marker was detected in 244/253 (96%) B-ALL dx BM samples (Fig. 1A), 237 (94%) of patients carried two or more rearrangements. On average we detected 5.1 markers per patient (range 0-10). The majority of patients carried at least one complete IGH (168; 66%), TRG (155; 61%), TRD (154; 61%), or IGK (142; 56%) rearrangement (Fig. 1B). In dx BM T-ALL samples at least one leukemic marker was detected in 88/100 (88%) cases, and in 80 cases (80%) two or more were detected (Fig. 1C). On average 3.6 markers per patient were detected (range 0-10). The majority of patients carried TRG (67; 67%), TRD (65; 65%), or complete TRB (52; 52%) rearrangements (Fig. 1D). In dx PB samples at least 1 marker was detected in 74/75 (98%) B-ALL (on average 5.2 markers per patient), and 23/25 (92%) T-ALL samples (on average 3.7 markers per patient). Out of 18 rel samples (17 BM, 1 PB; 16 B-ALL, 2 T-ALL), 14 (78%) carried at least 1 rearrangement and 13 (72%) at least two rearrangements. On average 3.7 markers per patient were detected. Conclusions The EuroClonality-NGS assays detected at least 1 leukemic marker in 443/471 (94%) patients in our cohort. The number of patients with no marker was higher in the T-ALL cohort (15/127; 12%) compared to the B-ALL cohort (13/344; 4%). Only 3/100 (3%) dx PB samples had no marker detected, which is surprising considering the usually lower leukemic infiltration of PB in B-ALL, but can be attributed to the higher sensitivity of NGS. Besides higher sensitivity, NGS-based marker identification brings along other benefits: the turn-around time is shorter and it is possible to detect multiple markers per tube without laborious cloning, or splitting the multiplex PCRs into singleplex. Overall, EuroClonality-NGS assays have been shown to be robust, sensitive, and routinely applicable in a clinical diagnostic setting. Disclosures Goekbuget: Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:PRMA: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Regeneron: Research Funding; Affimed: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy.