Immunoglobulin Gene Rearrangements Research Articles

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).

Cutaneous lymphoproliferative disorders: Back to the future.

In the 1980s, immunohistochemistry and clonality analyses became instrumental in the recognition and definition of new types of cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL) and the development of new classifications. By accepting loss of pan-T-cell antigens and clonal T-cell receptor gene rearrangements as important criteria to differentiate between benign and malignant T-cell proliferations, and monotypic immunoglobulin light-chain expression and clonal immunoglobulin gene rearrangements as crucial criteria to distinguish between benign and malignant B-cell proliferations, many cases, until then diagnosed as cutaneous lymphoid hyperplasia or pseudolymphoma, were reclassified as primary cutaneous CD4+ small/medium T-cell lymphoma (PCSM-TCL) or primary cutaneous marginal zone lymphoma (PCMZL), respectively. However, in recent years there is growing awareness that neither these immunohistochemical criteria nor demonstration of T-cell or B-cell clonality is specific for malignant lymphomas. In addition, many studies have reported that these low-grade malignant CTCL and CBCL have an indolent clinical behavior and an excellent prognosis with disease-specific survival rates of or close to 100%. As a result, recent classifications have downgraded several low-grade malignant cutaneous lymphomas to lymphoproliferative disorder (LPD). Both the 5th edition of the WHO classification (2022) and the 2022 International Consensus Classification (ICC) of mature lymphoid neoplasms reclassified PCSM-TCL as primary cutaneous CD4+ small/medium T-cell LPD and primary cutaneous acral CD8+ T-cell lymphoma as primary cutaneous acral CD8+ T cell LPD. While the 2022 ICC introduced the term "primary cutaneous marginal zone LPD," in the 5th edition of the WHO classification PCMZL is maintained. In this review we describe the background and rationale of the continually changing terminology of these conditions and discuss the clinical consequences of downgrading malignant lymphomas to LPDs.

Assessment of Bone Marrow Involvement in B-Cell non-Hodgkin Lymphoma Using Immunoglobulin Gene Rearrangement Analysis with Next-Generation Sequencing.

Assessment of bone marrow involvement (BMI) in non-Hodgkin lymphoma (NHL) is crucial for determining patient prognosis and treatment strategy. We assessed the prognostic value of next-generation sequencing (NGS)-based immunoglobulin (Ig) gene clonality analysis as an ancillary test for BMI evaluation in NHL. A retrospective cohort of 124 patients newly diagnosed with B-cell NHL between 2019 and 2022 was included. NGS-based Ig clonality analysis was conducted using LymphoTrak IGH FR1 Assay and IGK Assay (Invivoscribe Technologies, San Diego, CA, USA) on BM aspirate samples, and the results were compared with those of histopathological BMI (hBMI). Among the 124 patients, hBMI was detected in 16.9% (n = 21). The overall agreement of BMI between Ig clonality analyses and histopathological analysis for IGH, IGK, and either IGH or IGK was 86.3%, 92.7%, and 90.3%. The highest positive percent agreement was observed with clonal rearrangements of either IGH or IGK gene (90.5%), while the highest negative percent agreement was observed with clonal rearrangement of IGK gene (96.1%). For the prediction of hBMI, positive prediction value ranged between 59.1% and 80.0% and the negative prediction value ranged between 91.3% and 97.9%. NGS-based clonality analysis is an analytic platform with a substantial overall agreement with histopathological analysis. Assessment of both IGH and IGK genes for the clonal rearrangement analysis could be considered for the optimal diagnostic performance of BMI detection in B-cell NHL.

Clinical significance of bone marrow involvement by immunoglobulin gene rearrangement in de novo diffuse large B-cell lymphoma: a multicenter retrospective study.

Bone marrow (BM) involvement is an indicator of a poor prognosis in diffuse large B-cell lymphoma (DLBCL); however, few studies have evaluated the role of immunoglobulin gene rearrangement (IgR) in detecting BM involvement. We evaluated the clinical characteristics and treatment outcomes of patients with DLBCL based on histological BM involvement or positive BM IgR using polymerase chain reaction or next-generation sequencing. We also investigated the role of consolidative upfront autologous hematopoietic stem cell transplantation (ASCT) in patients with DLBCL and BM involvement. Among 624 patients, 123 (19.7%) with histological BM involvement and 88 (17.5%) with positive IgR in histologically negative BM had more advanced disease characteristics. Overall (OS) and progression-free (PFS) survival was better for patients with negative BM histology and negative IgR than that in patients with histological BM involvement (P = 0.050 and P < 0.001, respectively) and positive IgR with negative BM histology (P = 0.001 and P = 0.005, respectively). Survival rates did not differ among 82 (13.1%) patients who were treated with upfront ASCT and had histological BM involvement or positive IgR with negative BM histology. The survival outcomes were worse for patients who were not treated with upfront ASCT and for those with histological BM involvement or positive IgR, than for those with negative BM histology and negative IgR. Patients diagnosed with DLBCL and BM involvement based on histology or IgR had aggressive clinical features and poor survival. Upfront ASCT mitigated poor prognosis due to BM involvement.

Comparison of Measurable Residual Disease in Pediatric B-Lymphoblastic Leukemia Using Multiparametric Flow Cytometry and Next-Generation Sequencing.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Molecular B-cell clonality assay in minor salivary glands as a useful tool for the lymphoma risk assessment in Sjögren's syndrome

ObjectivesNon-Hodgkin's lymphoma (NHL) risk assessment is crucial in Sjögren's syndrome (SS). We studied the prevalence of clonal immunoglobulin gene rearrangements in minor salivary glands (MSG) and their correlations with lymphoma occurrence and with previously established NHL predictors. MethodsMolecular B-cell expansion was studied in fresh-frozen MSG of 207 patients with either suspected SS or with suspected lymphoma during SS, using a standardised multiplex PCR assay combined with heteroduplex analysis by microcapillary electrophoresis. The assignation of clonal cases was based on EuroClonality consortium guidelines. ResultsAmong 207 studied patients, 31 (15%) had MSG monoclonal B-cell infiltration. Monoclonality was significantly more frequent in patients with SS (28/123, 22.8%) compared with patients without SS (3/84, 3.6%, P<0.001). Monoclonal B-cell infiltration in MSG of SS patients correlated significantly with ongoing salivary gland NHL, salivary gland swelling, CD4+ T-cell lymphopenia, rheumatoid factor (RF) activity, low complement levels and type 2 mixed cryoglobulinemia. The accumulation of biological risk factors was associated with a higher rate of MSG B-cell monoclonality given that patients with only positive RF had no probability of MSG B-cell monoclonality, RF-positive patients with 1 or 2 other risk factors had a 25.0% and 85.7% probability of MSG B-cell monoclonality, respectively. ConclusionThe detection of MSG monoclonal B-cell expansion by this easy-to-perform molecular assay is useful, both at the time of diagnosis and during the course of SS. Monoclonal B-cell expansion is associated with a subset of SS patients presenting either ongoing lymphoma or other established lymphoma predictive factors.

Transbronchial cryobiopsy using an ultrathin cryoprobe with a guide sheath for the diagnosis of pulmonary mucosa-associated lymphoid tissue lymphoma.

Pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma is difficult to diagnose and relatively rare. Tissue sampling through transbronchial biopsy is often inadequate, necessitating surgical lung biopsy. However, a recently developed technique, transbronchial lung cryobiopsy (TBLC), has shown promise for obtaining larger specimens. A 1.1 mm cryoprobe has recently become available, and its usefulness has been increasingly reported. Use of a conventional cryoprobe for TBLC in diagnosing pulmonary MALT lymphoma has been previously reported; however, there are no reports on the use of a 1.1 mm ultrathin cryoprobe and guide sheath (GS). We aimed to assess the effectiveness and safety of using a 1.1 mm ultrathin cryoprobe in combination with a GS for diagnosing pulmonary MALT lymphoma using a simpler and safer method. We retrospectively analyzed the findings for four patients showing characteristic computed tomography (CT) findings of MALT lymphoma, including peripheral pulmonary lesions, air bronchogram nodules, and bronchiectasis, at our hospital. Each patient underwent endobronchial ultrasound (EBUS) with a GS, followed by TBLC using a 1.1 mm cryoprobe. Morphological diagnosis, immunohistochemical examination, and molecular testing were performed on the biopsy specimens to establish the diagnosis. Complications during the procedure were also monitored. We obtained 8-16 biopsy specimens in all four cases using a cryoprobe. Histopathological analysis of two cases revealed the infiltration of small lymphocytes with numerous lymphoepithelial lesions, confirming MALT lymphoma. Immunohistochemical examination further demonstrated B-cell lymphocyte proliferation and light-chain restriction, confirming monoclonality and providing a definitive diagnosis. In the remaining two cases, histopathological evidence of pulmonary MALT lymphoma was lacking. However, molecular testing using polymerase chain reaction to analyze immunoglobulin gene rearrangements revealed B-cell clonality, which supported the diagnosis. Molecular testing proved particularly useful when histopathological diagnosis alone was inconclusive. No complications such as pneumothorax or hemorrhage occurred during the procedure. The combination of a GS and EBUS facilitated specimen collection at the same location as EBUS, with the GS providing compression hemostasis and eliminating the need for an additional hemostatic device. Therefore, TBLC with a GS is a useful and safe method for diagnosing pulmonary MALT lymphomas and reproducibly yielded sufficient quantities of good-quality biopsy specimens.

Diagnostic and Prognostic Value of Circulating Cell-Free DNA in Cerebrospinal Fluid and Plasma in Newly Diagnosed Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous non-Hodgkin lymphoma. The clinical risk assessment for central nervous system (CNS) recurrence is still challenging in DLBCL due to the limited conventional biomarkers and techniques available. Circulating cell-free DNA (cfDNA), which reflects comprehensive tumor genetic profiles, has emerged as a potential novel strategy in the era of precision medicine. However, the clinical value of cfDNA detected in different samples in newly diagnosed DLBCL in China remains elusive. This study aims to explore the diagnostic and prognostic value of cfDNA detection in cerebrospinal fluid (CSF) and plasma in DLBCL. Methods:Next-generation sequencing (NGS) was performed on cfDNA derived from CSF, plasma, encompassing single nucleotide variants (SNV), insertion/deletions, copy number variants (CNV), IG related fusions, and clonotypic immunoglobulin gene rearrangements，alongside systemic tumor tissues in newly diagnosed DLBCL patients. Results:We recruited 30 patients, among whom CSF cfDNA was detected in 19 patients, with 6 (31.6%) being positive for SNV only. The positive rate of CSF cfDNA increased to 36.8% when considering both SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement. Notably, cfDNA was detected in 100% of CSF samples from 3 patients with known CNS involvement, and in 4 patients without known CNS involvement based on absent evidence of abnormal CSF cytology, flow cytometry, or radiography. The positive rates of SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement in CSF were 80%, 40%, and 40%, respectively, relatively confirmed by available paired tumor tissues or bone marrow samples. CfDNA was detected in 100% of plasma samples. When accounting for both SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement, the positive rate increased to 76.7% from 73.3%, which was much higher than paired peripheral blood genomic DNA (20%). The cfDNA detected in plasma exhibited high concordance rates with matched systemic tumor tissues or bone marrow samples, with concordance rates of 100%, 76.9%, and 76.5% for SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement, respectively. Interestingly, cfDNA was detected in plasma samples of 2 patients while tumor tissues' genomic DNA was negative. In total, 24 gene alterations were identified in CSF, 172 in plasma, and 160 in tumor tissue or bone marrow. Commonly mutated genes in plasma and CSF cfDNA included PIM1 (36.7% vs. 14.2%), MYD88 (33.3% vs. 28.6%), TET2 (30% vs. 28.6%), and CD79B (26.7% vs. 28.6%). The most common types of immunoglobulin gene rearrangement were IGK in different samples, and the common IG related fusions were IGH:: BCL6, IGH:: MYC, and IGH:: BCL2. Conclusion:Our study suggests that plasma cfDNA exhibits a high concordance rate with tumor tissues and can aid in the precise diagnosis and measurable residual disease monitoring. CSF cfDNA is more sensitive than conventional methods for diagnosing CNS invasion and could serve as a more accurate biomarker for predicting the risk of CNS recurrence among patients with newly diagnosed DLBCL, helping to select patients who may require enhanced CNS prophylaxis. Moreover, synchronous detection of SNV, CNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement could be helpful in improving the positive rate of cfDNA.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones &amp;gt;5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

Minimal residual disease detection by next-generation sequencing of different immunoglobulin gene rearrangements in pediatric B-ALL

While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. This study is to evaluate the prognosis of IGH and IGK/IGL rearrangement-based MRD detected by next-generation sequencing in B-ALL at the end of induction (EOI) and end of consolidation (EOC). IGK/IGL rearrangements identify 5.5% of patients without trackable IGH clones. Concordance rates for IGH and IGK/IGL are 79.9% (cutoff 0.01%) at EOI and 81.0% (cutoff 0.0001%) at EOC, respectively. Patients with NGS-MRD < 0.01% at EOI or <0.0001% at EOC present excellent outcome, with 3-year event-free survival rates higher than 95%. IGH-MRD is prognostic at EOI/EOC, while IGK-MRD at EOI/EOC and IGL-MRD at EOI are not. At EOI, NGS identifies 26.2% of higher risk patients whose MRD < 0.01% by flow cytometry. However, analyzing IGK/IGL along with IGH fails to identify additional higher risk patients both at EOI and at EOC. In conclusion, IGH is crucial for MRD monitoring while IGK and IGL have relatively limited value.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88 L265P (&gt;95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of &gt;1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88 L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88 WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study

AbstractKMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

Usefulness and Limitations of Current Diagnostic Strategies for Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma: Lessons Learned From a Large Cohort.

The pathologic diagnosis of pulmonary extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is challenging. To evaluate the diagnostic usefulness and limitations of current diagnostic strategies for pulmonary MALT lymphoma. A retrospective review of 120 cases of pulmonary MALT lymphoma from 2014 through 2021 was performed. Clinicoradiologic presentations overlapped with previous observations in patients with MALT lymphoma, such as a wide age range, female predominance, frequent association with autoimmune disease or immunodeficiency, and broad imaging findings. The histopathologic diagnosis was based on a combination of morphology, immunohistochemistry, and demonstration of B-cell lineage clonality. Two-thirds (76 of 113) of MALT lymphomas had lymphoplasmacytoid cytomorphology. Occasionally, MALT lymphomas were associated with granulomas/giant cells (29%, 35 of 120) or immunoglobulin deposition disease (21%, 25 of 120), including light chain/heavy chain deposition disease, amyloidosis, and/or crystal storing histiocytosis. While CD5, CD10, Bcl-2, and Bcl-6 rarely revealed aberrancies, aberrant CD43 expression either on B-cells or on plasma cells was detected in 42% (27 of 64) of cases, including cases for which proof of clonality could not be obtained. κ/λ in situ hybridization was particularly useful for tumors with lymphoplasmacytoid morphology but performed poorly in lymphomas having no plasmacytic differentiation. κ/λ immunohistochemistry showed no additional usefulness when applied together with κ/λ in situ hybridization. Immunoglobulin gene rearrangement studies by polymerase chain reaction achieved high detection rates of clonality in all cytomorphologic subgroups. Our study offers a practical evaluation of common diagnostic tests in pulmonary MALT lymphoma. We offer recommendations for a diagnostic workup that takes into consideration the usefulness and the specific limitations of the various diagnostic strategies.

Gastrointestinal ALK-positive anaplastic large cell lymphoma: a clinicopathological analysis of five cases

Objective: To investigate the clinicpathological characteristics of ALK-positive anaplastic large cell lymphoma (ALCL) of the gastrointestinal tract, and to discuss its diagnosis and differential diagnosis. Methods: Five cases of gastrointestinal ALK-positive ALCL diagnosed and treated in Xijing Hospital of the Fourth Military Medical University, between 2011 and 2019 were collected. There were three male and two female patients, aged 5-42 years (mean 25 years). These patients clinically presented with fever and night sweats, weight loss, abdominal pain, abdominal mass, ulcers, bleeding, or intestinal obstruction, and underwent surgical resection of the tumors or endoscopic biopsy. The clinical manifestations, auxiliary examinations, histopathological characteristics, immunophenotypes and genetic alterations were analyzed. Results: In this cohort, one case was common type, two cases were monomorphic variant of common type, and two cases were small cell variant. The tumor cells in all cases expressed ALK, CD30, and one or more T lymphocyte markers, while all the markers of B lymphocyte and plasmacyte were negative. Clonality analysis showed that two cases had clonal T cell receptor (TCR) and immunoglobulin (Ig) gene rearrangement, one case had no clonal TCR but Ig gene rearrangement, and one case had no clonal TCR and Ig gene rearrangements. During the 4 to 67 months' follow-up, two patients died of the disease, two were alive with free of disease and one had a relapse. Conclusions: ALK-positive ALCL of the gastrointestinal tract is extremely rare, and has poor prognosis. Lymphoma originating from this site with CD30 and ALK-positive phenotypes may be considered to be ALCL; however differentiation from other tumors that had anaplastic features, expressed CD30 and or ALK, in particular, ALK positive large B-cell lymphoma is necessary.

Molecular pathology assists the diagnosis of lymphoepithelial sialadenitis, Sjögren's syndrome and extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue

Background/purposeLymphoepithelial sialadenitis (LESA), Sjögren's syndrome (SS), and salivary MALT lymphoma are diseases characterized by lymphoepithelial lesions, and the differential diagnosis between them in the salivary glands is challenging. This study aimed to explore clinicopathological and genetic characteristics of the three diseases. Materials and methodsWe retrospectively analyzed the clinical features, the histomorphology, immunohistochemistry, and genetic profiling by polymerase chain reaction (PCR) and next-generation sequencing (NGS). ResultsThere included 68 LESAs, 25 SSs, and 62 MALT lymphomas. Ten cases relapsed in total, and 3 of MALT lymphomas died due to high-level transformation. Immunohistochemical double staining showed FCRL4 cells co-expressed Pax-5 and Ki-67, suggesting FCRL4 cells were proliferative B-cells. The expression level of the FCRL4 was significantly higher in MALT lymphoma than LESA and SS. The detection rates of clonal IGH, IGK, and IGL gene rearrangements in MALT lymphoma with a sensitivity of 83.33%. Monoclonal immunoglobulin gene rearrangements were confirmed in five suspected patients by NGS (100%). ConclusionFCRL4 B cells might play an important role in the formation of lymphoepithelial lesions and might be as a diagnostic positive marker of salivary MALT lymphoma. The application of multiple detection methods could significantly improve the diagnostic accuracy for MALT lymphomas from LESA and SS.

Identification of Immunoglobulin Gene Rearrangement Biomarkers in Multiple Myeloma through cfDNA-Based Liquid Biopsy Using tchDNA-Seq.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of pathogenic CD138+ plasma cells (PPCs) in bone marrow (BM). Recent years have seen a significant increase in the treatment options for MM; however, most patients who achieve complete the response ultimately relapse. The earlier detection of tumor-related clonal DNA would thus be very beneficial for patients with MM and would enable timely therapeutic interventions to improve outcomes. Liquid biopsy of "cell-free DNA" (cfDNA) as a minimally invasive approach might be more effective than BM aspiration not only for the diagnosis but also for the detection of early recurrence. Most studies thus far have addressed the comparative quantification of patient-specific biomarkers in cfDNA with PPCs and BM samples, which have shown good correlations. However, there are limitations to this approach, such as the difficulty in obtaining enough circulating free tumor DNA to achieve sufficient sensitivity for the assessment of minimal residual disease. Herein, we summarize current data on methodologies to characterize MM, and we present evidence that targeted capture hybridization DNA sequencing (tchDNA-Seq) can provide robust biomarkers in cfDNA, including immunoglobulin (IG) rearrangements. We also show that detection can be improved by prior purification of the cfDNA. Overall, liquid biopsies of cfDNA to monitor IG rearrangements have the potential to provide important diagnostic, prognostic, and predictive information in patients with MM.

Impact of T-cell Receptor Status on Mutational Landscape and Outcome in T-ALL.

Impact of T-cell Receptor Status on Mutational Landscape and Outcome in T-ALL.

Research Topic: Measurable Residual Disease in Hematologic Malignancies. Can digital droplet PCR improve measurable residual disease monitoring in chronic lymphoid malignancies?

Minimal/measurable residual disease (MRD) monitoring is progressively changing the management of hematologic malignancies. The possibility of detecting the persistence/reappearance of disease in patients in apparent clinical remission offers a refined risk stratification and a treatment decision making tool. Several molecular techniques are employed to monitor MRD, from conventional real-time quantitative polymerase chain reaction (RQ-PCR) to next generation sequencing and digital droplet PCR (ddPCR), in different tissues or compartments through the detection of fusion genes, immunoglobulin and T-cell receptor gene rearrangements or disease-specific mutations. RQ-PCR is still the gold standard for MRD analysis despite some limitations. ddPCR, considered the third-generation PCR, yields a direct, absolute, and accurate detection and quantification of low-abundance nucleic acids. In the setting of MRD monitoring it carries the major advantage of not requiring a reference standard curve built with the diagnostic sample dilution and of allowing to reduce the number of samples below the quantitative range. At present, the broad use of ddPCR to monitor MRD in the clinical practice is limited by the lack of international guidelines. Its application within clinical trials is nonetheless progressively growing both in acute lymphoblastic leukemia as well as in chronic lymphocytic leukemia and non-Hodgkin lymphomas. The aim of this review is to summarize the accumulating data on the use of ddPCR for MRD monitoring in chronic lymphoid malignancies and to highlight how this new technique is likely to enter into the clinical practice.

Angioimmunoblastic T-Cell Lymphoma Diagnosed From Serous Effusion by Integration of Cytologic Features and Ancillary Studies.

To explore the approach to the diagnosis of malignant serous effusion (SE) caused by angioimmunoblastic T-cell lymphoma (AITL). The clinical, cytomorphologic, immunophenotypic, and molecular features of 6 patients were summarized. Clinically, SE caused by AITL was predominant in middle-aged and older male patients with multiple SEs and lymphadenopathy. Cytomorphology showed small to medium-sized, irregular lymphocytes with clear cytoplasm and mixed with various inflammatory cells and apoptosis. Hodgkin/Reed-Sternberg-like cells were detected in 2 of 6 cases. Furthermore, 2 patterns of cytomorphology were described for the first time. Flow cytometry revealed abnormal T-cell populations with loss of surface CD3 (3/4 cases) and CD7 (3/4 cases). In addition, B-cell populations lacking surface immunoglobulin (Ig) were identified in 2 of 4 cases. Immunocytochemical staining revealed expression of at least 2 T follicular helper markers. Epstein-Barr virus-encoded RNA (EBER)-positive cells were demonstrated in 4 of 5 cases. Clonal T-cell receptor γ chain rearrangement was detected in 6 cases, and 3 of them had concomitant clonal immunoglobulin gene rearrangement. Moreover, 2 cases revealed discrepant findings regarding IgH/Igκ rearrangements in cytohistologic correlation. This study broadens the morphologic spectrum of malignant SE caused by AITL and provides diagnostic criteria in routine practice.