Analysis of measurable residual disease by IG/TR gene rearrangements: quality assurance and updated EuroMRD guidelines.

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

Introduction: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Methods: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. Results: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Discussion: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

NGS better discriminates true MRD positivity for the risk stratification of childhood ALL treated on an MRD-based protocol.

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

cfDNA-Based NGS IG Analysis in Lymphoma.

Liquid biopsy is a novel diagnostic approach at first developed to characterize the molecular profile of solid tumors by analyzing body fluids. For cancer patients, it represents a noninvasive way to monitor the status of the solid tumor with respect to representative biomarkers. There is growing interest in the utilization of circulating tumor DNA (ctDNA) analysis also in the diagnostic and prognostic fields of lymphomas. Clonal immunoglobulin (IG) gene rearrangements are fingerprints of the respective lymphoid malignancy and thus are highly suited as specific molecular targets for minimal residual disease (MRD) detection. Tracing of the clonal IG rearrangement patterns in ctDNA pool during treatment can be used for MRD assessment in B-cell lymphomas. Here, we describe a reproducible next-generation sequencing assay to identify and characterize clonal IG gene rearrangements for MRD detection in cell-free DNA.

Prognostic value of low-level MRD in adult acute lymphoblastic leukemia detected by low- and high-throughput methods.

Persistence of minimal residual disease (MRD) after induction/consolidation therapy in acute lymphoblastic leukemia is the leading cause of relapse. The GMALL 07/2003 study used MRD detection by real-time quantitative polymerase chain reaction of clonal immune gene rearrangements with 1 × 10-4 as discriminating cutoff: levels ≥1 × 10-4 define molecular failure and MRD-negativity with an assay sensitivity of at least 1 × 10-4 defining complete molecular response. The clinical relevance of MRD results not fitting into these categories is unclear and termed "molecular not evaluable" (MolNE) toward MRD-based treatment decisions. Within the GMALL 07/03 study, 1019 consecutive bone marrow samples after first consolidation were evaluated for MRD. Patients with complete molecular response had significantly better outcome (5-year overall survival [OS] = 85% ± 2%, n = 603; 5-year disease-free survival [DFS] = 73% ± 2%, n = 599) compared with patients with molecular failure (5-year OS = 40% ± 3%, n = 238; 5-year DFS = 29% ± 3%, n = 208), with patients with MolNE in between (5-year OS = 66% ± 4%; 5-year DFS = 52% ± 4%, n = 178). Of MolNE samples reanalyzed using next-generation sequencing (NGS), patients with undetectable NGS-MRD (n = 44; 5-year OS = 88% ± 5%, 5-year DFS = 70% ± 7%) had significantly better outcome than those with positive NGS-MRD (n = 42; 5-year OS = 37% ± 8%; 5-year DFS = 33% ± 8%). MolNE MRD results not just are borderline values with questionable relevance but also form an intermediate-risk group, assignment of which can be further improved by NGS.

Immune responses against shared antigens are common in esophago-gastric cancer and can be enhanced using CD40-activated B cells.

BACKGROUND: Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA). METHODS: RNA expression was assessed by NanoString in tumor samples of 41 treatment-naïve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing, in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion, clonality and cytotoxic activity of expanded T cells. RESULTS: 68.3% of patients expressed ≥5 TAAs simultaneously with coregulated clusters, which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ≥1 TAA were detectable in 75.0% and 53.7% of patients, respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal, could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare. CONCLUSIONS: We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA.

Immune Gene Rearrangements: Unique Signatures for Tracing Physiological Lymphocytes and Leukemic Cells.

The tremendous diversity of the human immune repertoire, fundamental for the defense against highly heterogeneous pathogens, is based on the ingenious mechanism of immune gene rearrangements. Rearranged immune genes encoding the immunoglobulins and T-cell receptors and thus determining each lymphocyte's antigen specificity are very valuable molecular markers for tracing malignant or physiological lymphocytes. One of their most significant applications is tracking residual leukemic cells in patients with lymphoid malignancies. This so called 'minimal residual disease' (MRD) has been shown to be the most important prognostic factor across various leukemia subtypes and has therefore been given enormous attention. Despite the current rapid development of the molecular methods, the classical real-time PCR based approach is still being regarded as the standard method for molecular MRD detection due to the cumbersome standardization of the novel approaches currently in progress within the EuroMRD and EuroClonality NGS Consortia. Each of the molecular methods, however, poses certain benefits and it is therefore expectable that none of the methods for MRD detection will clearly prevail over the others in the near future.

UMIc: A Preprocessing Method for UMI Deduplication and Reads Correction.

A recent refinement in high-throughput sequencing involves the incorporation of unique molecular identifiers (UMIs), which are random oligonucleotide barcodes, on the library preparation steps. A UMI adds a unique identity to different DNA/RNA input molecules through polymerase chain reaction (PCR) amplification, thus reducing bias of this step. Here, we propose an alignment free framework serving as a preprocessing step of fastq files, called UMIc, for deduplication and correction of reads building consensus sequences from each UMI. Our approach takes into account the frequency and the Phred quality of nucleotides and the distances between the UMIs and the actual sequences. We have tested the tool using different scenarios of UMI-tagged library data, having in mind the aspect of a wide application. UMIc is an open-source tool implemented in R and is freely available from https://github.com/BiodataAnalysisGroup/UMIc.

Thymic Hyperplasia with Lymphoepithelial Sialadenitis (LESA)-Like Features: Strong Association with Lymphomas and Non-Myasthenic Autoimmune Diseases.

Thymic hyperplasia (TH) with lymphoepithelial sialadenitis (LESA)-like features (LESA-like TH) has been described as a tumor-like, benign proliferation of thymic epithelial cells and lymphoid follicles. We aimed to determine the frequency of lymphoma and autoimmunity in LESA-like TH and performed retrospective analysis of cases with LESA-like TH and/or thymic MALT-lymphoma. Among 36 patients (21 males) with LESA-like TH (age 52 years, 32-80; lesion diameter 7.0 cm, 1-14.5; median, range), five (14%) showed associated lymphomas, including four (11%) thymic MALT lymphomas and one (3%) diffuse large B-cell lymphoma. One additional case showed a clonal B-cell-receptor rearrangement without evidence of lymphoma. Twelve (33%) patients (7 women) suffered from partially overlapping autoimmune diseases: systemic lupus erythematosus (n = 4, 11%), rheumatoid arthritis (n = 3, 8%), myasthenia gravis (n = 2, 6%), asthma (n = 2, 6%), scleroderma, Sjögren syndrome, pure red cell aplasia, Grave's disease and anti-IgLON5 syndrome (each n = 1, 3%). Among 11 primary thymic MALT lymphomas, remnants of LESA-like TH were found in two cases (18%). In summary, LESA-like TH shows a striking association with autoimmunity and predisposes to lymphomas. Thus, a hematologic and rheumatologic workup should become standard in patients diagnosed with LESA-like TH. Radiologists and clinicians should be aware of LESA-like TH as a differential diagnosis for mediastinal mass lesions in patients with autoimmune diseases.

Next-Generation Sequencing Technology to Identify Minimal Residual Disease in Lymphoid Malignancies.

Next-generation sequencing (NGS) of immunoglobulin (IG) and T cell receptor (TR) rearrangements represents a modern alternative to classical RQ-PCR-based minimal residual disease (MRD) detection. The same primer sets and conditions can be used for all patients, which is undoubtedly one of the most important benefits of NGS, not only reducing the labor required to perform the analysis but also enabling the assay to comply with the upcoming EU IVD regulation. So far, only one standardized academic protocol for this task has been published, developed, and validated within the EuroClonality-NGS working group. In this chapter we describe the materials and methods for amplicon library preparation for sequencing on Illumina MiSeq, and the bioinformatic pipeline for this protocol.

Tumor and microenvironment response but no cytotoxic T-cell activation in classic Hodgkin lymphoma treated with anti-PD1.

Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics.

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.

Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study.

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.