Immunoglobulin Heavy Chain Gene Research Articles

880: PULMONARY AMYLOIDOSIS ASSOCIATED WITH B CELL LYMPHOPROLIFERATIVE DISORDER: A CASE REPORT

Introduction: Amyloidosis is caused by the deposition of misfolded autologous proteins in various organs and can be systemic or localized. AL amyloidosis is often associated with plasma cell disorder but here we report a rare case of Isolated Nodular Pulmonary Amyloidosis associated with a B cell Lymphoproliferative Disorder. Description: An 89 year old female with a past medical history of pulmonary arterial hypertension presented to the clinic with chronic stable exertional dyspnea and no associated symptoms. Four years prior, a computed tomography (CT) of the chest showed multiple bilateral calcified and noncalcified lung nodules with no further work-up. Chest X-ray performed at current presentation revealed extensive pulmonary reticulonodular shadowing at the peripheries with extensive fibrotic changes and scattered nodules. Subsequent CT chest showed interval increase of several bilateral nodules. A PET-CT showed multiple mildly hypermetabolic nodules. A CT guided needle biopsy of an FDG avid right upper lobe nodule measuring 1.5 x 1.4 cm was performed. Pathology was significant for homogenous acellular amorphous material with an entrapped plasma cell infiltrate. Congo red stain confirmed the presence of apple green birefringence diagnostic of amyloidosis. Immunohistochemistry showed a mixture of kappa-positive and lambda-positive cells, with kappa-positive cell predominance suggestive of an atypical plasma cell population. Clonal gene rearrangements were detected in both the immunoglobulin heavy chain and light chain gene which highly suggested the presence of a B-cell Lymphoproliferative Disorder. The patient was subsequently referred to oncology for further management. Discussion: Pulmonary Amyloidosis is an incidental radiological finding with patients being asymptomatic. CT chest may show lung nodules with/without calcifications. Biopsy is required for diagnosis with radiology guided fine needle aspiration. Isolated pulmonary amyloidosis has a good prognosis but should trigger evaluation for autoimmune disorders and B cell lymphoproliferative disorders as in this case with early referral and treatment of the underlying disorder.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Comparing Data Generated Using Multiparametric Flow Cytometry, Capillary Electrophoresis, and Next-Generation Sequencing in a Cohort of B-Cell Lymphoproliferative Disorder Samples

Background: Determining whether suspected lymphoid proliferation disorder (LPD) samples are benign or malignant remains a significant challenge for the hematopathologist. For the vast majority of LPDs, the current gold standard includes assessment of clinical characteristics, cytomorphology, and immunophenotyping by flow cytometry; however, 10-15% of LPDs require further resolution by molecular methods, such as PCR-based capillary electrophoresis or next-generation sequencing, to rule out clonality based on V-D-J rearrangements in the immunoglobulin heavy-chain (IGH) gene. Invivoscribe (San Diego, CA) has developed multiple complementary methodologies to evaluate LPDs; including multiparametric flow cytometry (MFC), capillary electrophoresis (CE), and next-generation sequencing (NGS). To compare performance between these three methods, a cohort of 21 clinical samples were tested in a pilot study. Method: The CAP/CLIA Hematolymphoid Screening assay, a 10-color flow panel, was used to identify all major types of hematopoietic neoplasia according to the 2006 Bethesda Consensus and 2017 WHO Classification. For molecular testing, DNA was extracted from peripheral blood (PB), then PCR-based assays were used to determine presence or absence of clonality. The Research Use Only (RUO) molecular assays were designed for CE (ABI 3500xL) and NGS (MiSeqTM) detection, utilizing framework multiplex PCR master mixes targeting the conserved framework regions FR1, FR2, or FR3 within the VH and the JH regions of the IGH gene. Twenty-one (21) de-identified PB samples were collected from informed consensus donors (LabPMM, San Diego, CA) or from residual samples (Lahey Hospital, Burlington, MA) and tested under an IRB approved protocol. An aliquot of each PB sample was used for MFC evaluation, then the remaining DNA was extracted and tested using the CE and NGS methods. Test results were reported as "present abnormal" or "normal" for MFC and "clonal" or "non-clonal" for molecular assays based on pre-defined criteria. Results: The MFC assay identified 12 out of 21 (57%) samples as "present abnormal" and 9 out of 21 (43%) samples as "normal." Both molecular assays (CE and NGS) reported 12 out of 21 (57%) samples as "clonal" and 9 out of 21 (43%) samples as "non-clonal." The results between CE and NGS assays were 100% in agreement for clonality detection. Comparisons between the CE and MFC assays again resulted in 100% agreement; all 12 "present abnormal" samples by MFC were reported as "clonal" by CE, while all 9 "normal" samples by MFC were "non-clonal" by CE. In addition to presence of clonality, the NGS method was able to detect the prevalence of a specific clonal V-D-J rearrangement sequence (expressed as % reads), which ranged from 10% to 99% in the 12 clonal samples. This pilot study demonstrated excellent concordance (21/21) between these 3 methods (MFC, CE and NGS). Accordingly, MFC, CE and NGS are all suitable methods for the evaluation and stratification of B-cell LPD samples. The combined use of all 3 methods provides a comprehensive assessment of clonality: including surface markers identified by MFC, DNA rearrangement lengths from CE, and the specific DNA rearrangement sequences from NGS. In addition, the NGS method allows for tracking of these clonal sequences in relation to disease status. Conclusion: This pilot study demonstrated 100% concordance between 3 different methods (MFC, CE, and NGS) when evaluating samples from LPD subjects. Depending on laboratory capabilities and the type of information desired, any one of these technologies can provide appropriate results when studying clonality. The alignment between MFC and NGS allows the combined use of both methods to provide rapid diagnosis via the MFC service and identification of specific biomarkers for subsequent tracking of these clonal populations in clinical studies.

T Cell Epitope Prediction within the Clonotypic Immunoglobulin Heavy and Light Chains in Patients with Multiple Myeloma

Despite remarkable therapeutic advances in recent years, multiple myeloma (MM) remains incurable, hence representing an unmet clinical need. Therapeutic approaches based on induced T cell anti-tumor immunity towards cancer eradication show promising results in many hematologic malignancies, including multiple myeloma (MM), highlighting the need for comprehensive understanding of the implicated mechanisms. Similar to other mature B cell malignancies, immunoglobulin (IG) gene rearrangements in MM lead to the expression of unique, novel peptide sequences that can be used as neoantigens, conceivably representing a potential attractive target for cancer-specific immune responses. Here we sought to explore this possibility by performing ad hoc prediction of putative T-cell class I/II neoepitopes contained within the MM clonotypic IG gene rearrangements. To that end, we investigated 16 newly diagnosed patients with MM from whom we isolated the peripheral blood and/or bone marrow mononuclear cell (PBMC/BMMC) fraction as well as CD138+ myeloma cells, CD4+ and CD8+ T cells. Clonotypic IG heavy and light chain gene rearrangements were RT-PCR amplified on RNA extracted from the PBMC/BMMC fraction using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. The corresponding sequences were determined by bidirectional Sanger sequencing. Moreover, T cell receptor beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4+ and CD8+ T cells and then subjected to paired-end next generation sequencing (NGS). The deduced amino acid sequences of the IG heavy and light chain variable/constant domains were subsequently parsed in peptides and subjected to bioinformatics analysis for the identification of putative T-cell class I/II neoepitopes using the NetMHCpan and NetMHIICpan softwares. The rank score was calculated, considering the 4-digit HLA-A, -B, -C and DRB1 typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Prediction of the binding specificity of T Cell Receptors (TR) to MHC-peptide complexes (pMHCs) was performed by the ERGO-II tool. The rank score was calculated, considering the peptides resulted by NetMHCpan/ NetMHIICpan, the HLA, and the clonotypes for each patient (AUC>0.8). Exact matches to germline and/or proteome databases were excluded. Overall, 757,263 TRΒ distinct clonotypes were assessed (median= 18,244 /sample). All patients displayed oligoclonal T cell expansions in both CD4+ and CD8+ T cells, albeit these were significantly (p<0.001) more pronounced in the latter. Both T cell subpopulations displayed skewed TRBV and TRBJ gene repertoires (7 TRBV genes and 3 TRBJ genes accounting for about 40% and 59% of the repertoire, respectively). Overall, 1,219 predicted neoepitopes were identified. All patients had predicted CD4+ and CD8 + T-cell epitopes within the MM clonotypic IG, either in the heavy chain (16/16 pts, n=459 epitopes) or the light chain (16/16 pts, n=760 epitopes). There was no statistically significant difference in the rank score of peptides involving the complementarity determining region 3 (CDR3) vs. all other IG regions. Interestingly, 998/1,219 (82%) peptides with strong binding affinity resulted from IG gene sequence motifs outside the CDR3. Most of these peptides (853/1,219, 75%) resulted from somatic hypermutations (SHM) across the IG variable domain. Overall, 51,574 clonotypes of CD4+ T cells and 35,751 clonotypes of CD8+ T cells were in silico predicted by ERGO to have strong binding affinity. Relevant to mention, 528 clonotypes with high binding score were found to be shared between CD4+ T cells of all patients and 209 clonotypes between CD8+ T cells of all patients and, thus, were deemed as public. In conclusion, in silico prediction identified a significant number of putative T-cell class I/II neoepitopes contained within the clonotypic IG of MM patients. Many of the identified peptides derive from SHM, implying that the SHM which shapes the MM BcR IG repertoire may produce immunogenic CD4+/CD8+ T cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.

Measurable residual disease in hairy cell leukemia: Technical considerations and clinical significance.

Hairy cell leukemia (HCL) is a rare type of chronic lymphoid leukemia originating from a mature B lymphocyte. A diagnosis of HCL is based on cytology, confirmed by multiparametric flow cytometry (MFC) studies using anti-B-cell monoclonal antibodies, together with a panel of antibodies more specific to HCL, such as CD11c, CD25, CD103 and CD123. Recently, the BRAF V600E mutation has been described as a disease-defining genetic event. Measurable residual disease (MRD) is defined as the lowest level of HCL cells that can be detected accurately and reproducibly using validated methods; as MRD negativity is associated with high rates of durable complete response, by clearing MRD, the long-term outcome may be improved in patients with advanced HCL. MRD is typically detected using bone marrow, and in some cases, peripheral blood; however, in HCL, discrepancies frequently exist between MRD results obtained from blood, bone marrow aspirate and core biopsy. Among the methods used for MRD detection, MFC appears to be a more sensitive technique than immunohistochemistry. Molecular tests are also used, such as real-time quantitative PCR for unique immunoglobulin heavy chain (IgH) gene rearrangements and PCR techniques with clone specificity for BRAF V600E. Clone-specific PCR (spPCR) is able to detect one HCL cell in 106 normal cells, and is particularly suitable for patients found to be negative for MRD by MFC. Recently, the Hairy Cell Leukemia Consortium created a platform to work on a definition for MRD, and establish the optimal time point, tissue type and method for measuring MRD. This.

Human type I IFN deficiency does not impair B cell response to SARS-CoV-2 mRNA vaccination.

Inborn and acquired deficits of type I interferon (IFN) immunity predispose to life-threatening COVID-19 pneumonia. We longitudinally profiled the B cell response to mRNA vaccination in SARS-CoV-2 naive patients with inherited TLR7, IRF7, or IFNAR1 deficiency, as well as young patients with autoantibodies neutralizing type I IFNs due to autoimmune polyendocrine syndrome type-1 (APS-1) and older individuals with age-associated autoantibodies to type I IFNs. The receptor-binding domain spike protein (RBD)-specific memory B cell response in all patients was quantitatively and qualitatively similar to healthy donors. Sustained germinal center responses led to accumulation of somatic hypermutations in immunoglobulin heavy chain genes. The amplitude and duration of, and viral neutralization by, RBD-specific IgG serological response were also largely unaffected by TLR7, IRF7, or IFNAR1 deficiencies up to 7 mo after vaccination in all patients. These results suggest that induction of type I IFN is not required for efficient generation of a humoral response against SARS-CoV-2 by mRNA vaccines.

An improved, chromosome-level genome of the giant panda (Ailuropoda melanoleuca)

BackgroundThe iconic giant panda (Ailuropoda melanoleuca), as both a flagship and umbrella species endemic to China, is a world famous symbol for wildlife conservation. The giant panda has several specific biological traits and holds a relatively small place in evolution. A high-quality genome of the giant panda is key to understanding the biology of this vulnerable species. FindingsWe generated a 2.48-Gb chromosome-level genome (GPv1) of the giant panda named “Jing Jing” with a contig N50 of 28.56 Mb and scaffold N50 of 134.17 Mb, respectively. The total length of chromosomes (n = 21) was 2.39-Gb, accounting for 96.4% of the whole genome. Compared with the previously published four genomes of the giant panda, our genome is characterized by the highest completeness and the correct sequence orientation. A gap-free and 850 kb length of immunoglobulin heavy-chain gene cluster was manually annotated in close proximity to the telomere of chromosome 14. Additionally, we developed an algorithm to predict the centromere position of each chromosome. We also constructed a complete chromatin structure for “Jing Jing”, which includes inter-chromosome interaction pattern, A/B compartment, topologically associated domain (TAD), TAD-clique and promoter-enhancer interaction (PEI). ConclusionsWe presented an improved chromosome-level genome and complete chromatin structure for the giant panda. This is a valuable resource for the future genetic and genomic studies on giant panda.

Role of immunoglobulin heavy and light chain gene rearrangement analysis in differentiating between benign and malignant bone marrow B-cell lymphoid aggregates

Lymphoid aggregates are found in a minority of bone marrow biopsy and aspirate specimens, and when present, the distinction between benign and malignant aggregates can represent a diagnostic challenge. Morphologic and immunophenotypic evaluation of the aggregates can aid in that distinction but in a few cases, detection of immunoglobulin heavy chain (IGH) and kappa light chain (IGK) gene rearrangements may be needed to rule in or out a malignant disease process. We studied the role of testing for IGH/IGK rearrangements in the distinction between benign and malignant B cell-predominant lymphoid aggregates. Only a few studies have addressed this issue and most lacked an adequate number of cases for establishing statistical significance. Our study retrospectively evaluated 120 bone marrow aspirate and biopsy specimens, 79 cases originally diagnosed with benign lymphoid aggregates [4,5], and 41 demonstrating a B-cell lymphoma with malignant aggregates. Immunohistochemical stains were performed on all cases in our study and flow cytometry results were available in the vast majority of cases (98%). All patients included in our study but 9 had at least 2 years of clinical follow-up information. Of the malignant lymphoma cases, IGH/IGK rearrangements were demonstrated by polymerase chain reactionin 60% of the cases. Moreover, clonal rearrangements were identified in 15% of the cases with benign aggregates. After at least 2 years of follow-up, only one case with a positive clonality study occurring in the setting of morphologically benign-appearing bone marrow lymphoid aggregates experienced a relapse of non-Hodgkin lymphoma. Molecular analysis of the IGH and IGK genes may have utility in confirming the presence of malignancy in bone marrow aspirates and biopsy specimens. False-negative results, however, are possible due to testing limitations and sampling issues. Moreover, patients with conditions such as autoimmune disorders or infectious diseases are shown to also develop clonal B-cell lymphoid aggregates. As a result, we recommend a thorough morphological examination, informative immunohistochemical staining, accurate flow cytometric analysis, and current IGH/IGK rearrangement testing when evaluating bone marrow specimens containing B cell-predominant lymphoid aggregates, with the knowledge that molecular clonality results should be carefully interpreted in the context of morphological and immunophenotypic findings to prevent misdiagnosis.

Primary Vitreoretinal Lymphoma: Current Diagnostic Laboratory Tests and New Emerging Molecular Tools.

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations.

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.

Diffuse large b-cell lymphoma of the ocular adnexa with flow cytometry analysis and review of the literature.

The aim of this study was to evaluate the clinicopathological features and flow cytometry (FCM) of tumor tissues in ocular adnexal diffuse large B-cell lymphoma (DLBCL). This retrospective, multicenter case study was designed to evaluate the clinical and immunohistochemical features of tumors. DLBCL was diagnosed based on histopathology, immunoglobulin (Ig) heavy chain gene rearrangement, and FCM in all surgically removed periocular tumor tissues. This study involved assessing percentages (%) of B-cell/T-cell markers, a natural killer cell marker, and cell-surface Ig kappa/lambda (κ/λ) expression measured by FCM analysis in tumor tissues. Eleven DLBCL patients (4 men and 7 women) with 11 tumors were enrolled in this study. The median age at the time of initial presentation was 73 years. The tumor cells were immunohistochemically positive for cluster of differentiation (CD) 20, while CD5 was negative in all 8 cases tested. At the time of ophthalmic diagnosis, two cases already showed systemic dissemination of DLBCL throughout the body. FCM of tumor tissues detected a high percentage of B-cell markers including CD19 and CD20 in all 11 tumors. One case with high CD10 levels in FCM was histologic transformation from follicular lymphoma. One case with a relatively low CD20 population involved a history of systemic treatments including intravenous rituximab. Although caution should be exercised when interpreting the data, FCM is useful for not only supportive diagnosis complementary to immunohistochemistry, but also facilitates a better understanding of immunopathology including histologic transformation of follicular lymphoma to DLBCL in the ocular adnexa.

5′ Rapid amplification of cDNA ends (5′RACE): A simpler method to analyze immunoglobulin genes and discover the value of the light chain in chronic lymphocytic leukemia

The mutational status of the variable region of the immunoglobulin heavy chain gene (IGHV) is a very important biomarker for chronic lymphocytic leukemia (CLL) patients. However, the routine detection of IGHV mutational status is time-consuming and costly. Therefore, we performed 5′ Rapid amplification of cDNA ends (5′ RACE) in 81 CLL patients who previously underwent detection using Biomed-2. The agreement rate of these two methods was 93.8 %. Regarding the discordant cases, 5′ RACE was more sensitive to identify unproductive and multiple rearrangements. Furthermore, 5′ RACE can also be used to simultaneously sequence light chains. In most CLL cases, the mutational statuses of heavy and light chains are concordant, except in IGLV3–21. Most IGLV3–21 (24/25) rearrangement shared a similar LCDR3 (QVWDSSSDHPWV) and harbored a single point mutation, namely, IGLV3–21R110. Compared to mutated-CLL non IGLV3–2R110, IGLV3–21R110-CLL exhibited a shorter overall survival (OS) and time to first treatment (TTFT) (p = 0.05, p < 0.0001, respectively) even though 75 % (18/24) of these patients expressed mutated heavy chains. Altogether, IGLV3–21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and emphasizes the important value of the light chain. This study is the first to use 5′ RACE to detect the mutational status of IGH in CLL. Here, 5′ RACE was a reliable and effective method to test the mutational status of heavy and light chains. In addition, 5′ RACE can be combined with other assays in the NGS workflow to obtain more detailed insight into subclonal architecture and intraclonal diversity.

Intraepithelial cytotoxic lymphocytes are associated with a poor prognosis in feline intestinal T-cell lymphoma.

The expression of cytotoxic molecules in feline intestinal T-cell lymphoma cells was examined immunohistochemically using endoscopic samples of 50 cases. Cases included 14 large-cell lymphomas (LCLs) and 36 small-cell lymphomas (SCLs). Most LCL and some SCL exhibited marked erosion and villous atrophy. Clonal T-cell receptor (TCR) gene rearrangement was detected in 10/14 (71%) LCL cases and 33/36 (92%) SCL cases. No clonal immunoglobulin heavy chain (IgH) gene rearrangement was detected. Immunohistochemically, all cases were positive for CD3 and negative for CD79α, CD30, CD56, and Foxp3. LCLs were positive for CD8 in 13/14 cases (93%), T-cell intracellular antigen 1 (TIA1) in 14/14 cases (100%), and granzyme B in 6/14 cases (43%). SCLs were positive for CD8 in 28/36 cases (78%), TIA1 in 33/36 cases (92%), and granzyme B in 2/36 cases (6%). TIA1- and granzyme B-positive neoplastic lymphocytes were predominantly observed in the mucosal epithelium of 10/50 cases (20%) and 6/50 cases (12%), respectively. No significant differences in survival time were found based on cell size or epitheliotropism. However, cases with TIA1+ and/or granzyme B+ neoplastic lymphocytes predominantly in the mucosal epithelium had significantly shorter survival times (P < .05), suggesting that mucosal epithelium infiltration of neoplastic cells with a cytotoxic immunophenotype is a negative prognostic factor. Therefore, intraepithelial cytotoxic lymphocytes may be associated with mucosal injury and impaired intestinal function, leading to a poor prognosis in cats with intestinal T-cell lymphoma.

High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic B cell recirculation between ectopic lymphoid structures.

B cells play a central role in Sjögren's syndrome (SS) whereby autoreactive B-cells populate ectopic germinal centres (GC) in SS salivary glands (SG) and undergo somatic hypermutation (SHM) and class-switch recombination of the immunoglobulin genes. However, the capacity of specific B cell clones to seed ectopic GC in different SG and undergo clonal diversification is unclear. To unravel the dynamics of B cell recirculation among minor SG biopsies, we investigated the immunoglobulin heavy chain (IgH) gene usage and the pattern of SHM using a high-throughput sequencing approach. We generated ~166,000 reads longer than 350bp and detected 1631 clonotypes across eight samples from four different SS patients, all characterised by the presence of functional ectopic GC as demonstrated by the expression of activation-induced cytidine deaminase. A large number of shared clonotypes were observed among paired mSG biopsies from each patient but not across different patients. Lineage tree analysis revealed significant clonal expansion within the mSG with the identification of shared dominant B cell clones suggestive of extensive recirculation across different SG. Several shared clonotypes with high proliferating capacity displayed IgH-VH gene usage common in autoreactive B cells, including VH1-69, which is typical of rheumatoid factor+ B cells representing potential lymphoma precursors. The complex dynamic recirculation of B cells that we observed within ectopic GC responses linked with their ability to independently proliferate, undergo ongoing SHM and Ig class-switching within individual glands may explain the difficulty in achieving consistent eradication of ectopic GCs following B cell depleting agents reported in different studies.

Recurrent Novel P2RY8/IGH Translocations in B-Lymphoblastic Leukemia/Lymphoma.

Translocations involving the immunoglobulin heavy chain (IGH) locus are common abnormalities in B-lymphoblastic leukemia/lymphoma (B-ALL) and multiple myeloma. These rearrangements result in a juxtaposition of IGH enhancers to the vicinity of oncogenes, such as MYC and CRLF2, leading to the upregulation of oncogenes. Here, we identified recurrent novel P2RY8/IGH translocations in three B-ALL patients by transcriptome sequencing. Noncoding exon 1 of P2RY8 was translocated to different sites of the IGH gene, resulting in transcripts of P2RY8/IGHM, P2RY8/IGHV, and P2RY8/IGHD. However, a high expression level of truncated P2RY8 was observed in the patients compared with healthy donors, which might be related to the aggressive clinical course and inferior outcome. In summary, we described recurrent novel P2RY8/IGH translocations with high expression levels of P2RY8, which may contribute to the guidelines for clinical diagnosis and treatment.

P1245: CLINICAL IMPACT OF IMMUNOGLOBULIN HEAVY CHAIN REPERTOIRE IN MANTLE CELL LYMPHOMA: A STUDY FROM THE FONDAZIONE ITALIANA LINFOMI (FIL) PHASE III MCL0208 TRIAL.

Background: Mantle cell lymphoma (MCL) is characterized by a highly restricted immunoglobulin heavy chain (IGH) gene repertoire with stereotyped IGHV CDR3s, strongly implying a role for antigen-driven selection. Nonetheless, differently from chronic lymphocytic leukemia, where the prognostic role of IGH parameters is known since decades, published data are less consistent in MCL. Recently, LRPAP1 autoantibodies were detected in 13% of patients from the European MCL Network trials and were associated with superior outcome, while the autoreactivity of IGHV 4-34 antibodies is largely known. Aims: We aimed at characterizing the IGH VDJ repertoire and its putative clinical relevance in a large, prospective, trial of the Fondazione Italiana Linfomi (FIL) enrolling younger MCL patients. Methods: IGH clonal rearrangements detection was performed by Sanger sequencing on bone marrow or peripheral blood samples collected for minimal residual disease purposes in FIL MCL0208 trial (NCT02354313). This is a prospective, randomized phase III trial comparing lenalidomide maintenance vs observation after an intensive cytarabine containing chemo-immunotherapy followed by autologous transplantation in frontline <66 year-old MCL patients. IMGT/V-QUEST tool was used to annotate IGHV-D-J and mutational status (cut-off 98%) according to ERIC guidelines. Finally, these biological data were integrated with published clinical, pathological, and mutational data from the trial (current median follow-up 38 months). Results: A clonal IGH productive rearrangement was detected in 209/300 patients (70%), resulting in 159 (76%) unmutated (UM) and in 50 (24%) mutated (M) cases, as expected. Overall, VDJ usage included 29 IGHV families, with IGHV 3-21 (#45, 22%), 4-34 (#33, 16%), 3-7 (#15, 7%), 3-23 and 5-51 (#12 each, 6%), 4-39 and 4-59 (#10 each, 5%) as the most frequent groups, together representing the 66% of the analyzed series (Figure 1A). Interestingly, patients carrying IGHV 3-21 were often characterized by low Ki-67 index (Ki-67 <30%: 86% vs 64% of other cases, p<0.01) and low risk MIPI score (73% vs 43%, p=0.001), while no case of blastoid morphology was registered among the IGHV 4-34 patients (vs 89%, p<0.05). Moreover, by grouping together these two rearrangements, statistically significant associations were found with several biomarkers of favorable prognosis (low Ki-67, p<0.01; non-blastoid, p<0.05; absence of TP53 aberrations, p<0.05, or KMT2D mutations, p<0.05, and low-risk MIPI, p<0.0001). Accordingly, both IGHV 3-21 and 4-34 patients showed better long-term outcomes, with longer PFS and OS if compared with all the other cases, both singularly and when grouped together (3 years PFS: 75% vs 53%, p<0.05; 3 years OS: 91% vs 76%, p<0.01, Figure 1B, C). Finally, these results were independent from IGHV mutational status (p=0.09) and lenalidomide maintenance (p=0.46). Image:Summary/Conclusion: This is the first study assessing the clinical impact of VDJ IGH repertoire in a large prospective trial in MCL. As expected, 76% of patients resulted UM, with 3-21 and 4-34 representing more than one third of all the IGHV rearrangements. Notably, in our series, no significant correlation between mutational status and outcome was recorded. On the other hand, patients carrying either IGHV 3-21 or 4-34 rearrangements were characterized by favorable clinical and biological known prognosticators and were associated with both PFS and OS improvement, if compared to other patients. Deeper translational insights are needed to clarify the biological (autoreactive?) basis of this observation.

P1274: EARLY DETECTION OF NON-HODGKIN LYMPHOMA IN PREDISPOSED PATIENT GROUPS THROUGH IMMUNOGENETIC SEQUENCING

Background: Since chronic lymphocytic leukemia development can be detected up to 16 years prior to diagnosis through immunogenetic sequencing, we aim to evaluate whether early detection through immunogenetic sequencing translates to other types of non-Hodgkin lymphoma (NHL). A particularly interesting group with potential clinical utility consists of patients with underlying conditions that confer an increased risk of lymphoma and leukemia. Generally, this group is characterized by dysregulation of the immune system, in the form of autoimmune diseases, primary immunodeficiencies, immunosuppressive treatment and several infectious diseases. Aims: To evaluate the capacity of immunogenetic sequencing for early detection of NHL and leukemia in patient groups at increased risk. Methods: We selected two patient groups with an increased risk of NHL and leukemia: solid organ transplant recipients and patients with auto-immune disease. For each group, we selected patients who had developed NHL and controls with the same underlying condition matched on sex, age and sampling window. Currently, we have included 16 cardiac and 10 renal transplant recipients and 10 primary Sjögren’s syndrome (SjS) patients (in total 18 cases and 18 controls). A total of 114 longitudinal pre-diagnostic samples were included in the study, taken a median of 1.2 years before diagnosis (IQR 3.5 years). Genomic DNA was isolated from the PBMC samples of the participants and the immunoglobulin heavy chain (IGH) was sequenced on the Illumina Miseq platform. All PBMC samples were taken after SjS diagnosis or solid organ transplantation and prior to NHL diagnosis. Results: In 7 out of 18 NHL patients we identified clonotypes in prediagnostic PBMC samples that were present at an abnormal frequency compared to the healthy background clonotypes. Notably, distribution of the IGH gene repertoire in controls was normal. Prevalence of IGH gene repertoire skewing was particularly pronounced among SjS patients (3 out of 5 patients) and cardiac transplant recipients (4 out of 8 patients) compared to renal transplant recipients (1 out of 5 patients). IGH gene repertoire skewing was restricted to samples taken within 5 months to two years before diagnosis for the majority of the cases. In solid organ transplant recipients with an abnormal clonotype, the degree of skewing of the IGH gene repertoire increased over time towards the moment of NHL diagnosis or remained stable at elevated levels. For the SjS patients, two out of the three abnormal clonotypes utilized the IGHV1-69/IGHJ4 or IGHV4-59/IGHJ5 rearrangements stereotypic for rheumatoid factors, which have previously been associated with MALT lymphoma development in SjS. Unbiased marker identification has yielded putative prediagnostic lymphoma markers for 7 out of 18 patients. Image:Summary/Conclusion: We conducted a pilot study investigating a novel method of early detection of NHL development in 36 patients at increased risk of hematological malignancy. In total, we observed an abnormal distribution of the IGH repertoire between 5 months to 2 years prior to NHL diagnosis in 7 out of 18 patients, compared to 0 out of 18 of the matched controls. While our results suggest potential merit for early detection of NHL development through immunogenetic sequencing, the current cohort size does not yet permit drawing definitive conclusions. Therefore, inclusion of additional patients will be a priority during the continuation of the study.

Immunoglobulin heavy-chain loci in ancient allotetraploid goldfish

As an ancient allotetraploid species, goldfish (Carassius auratus) have two sets of subgenomes. In this study, immunoglobulin heavy-chain (IGH) genes were cloned from the red crucian carp (Carassius auratus red var.), and the corresponding loci were identified in the gynogenetic diploid red crucian carp (GRCC) genome as well as the genomes of three other goldfish strains (Wakin, G-12, and CaTCV-1). Examination showed that each goldfish strain possessed two sets of parallel IGH loci: a complete IGHA locus and a degenerated IGHB locus that was nearly 40 × smaller. In the IGHA locus, multiple τ chain loci were arranged in tandem between the μ&δ chain locus and the variable genes, but no τ-like genes were found in the IGHB locus.

Development of B-cell maturation antigen (BCMA)-specific CD8+ cytotoxic T lymphocytes using induced pluripotent stem cell technology for multiple myeloma.

2542 Background: A strategy for reversal of T cell exhaustion is reprograming of antigen-specific CTL to early lineage memory T cells with selective anti-tumor activities. To accomplish this goal, we epigenetically reprogrammed BCMA-specific CD8+ CTL to a pluripotent state through key defined transcription factors, established “induced Pluripotent Stem Cells (iPSC)” exhibiting transcriptional and epigenetic features, re-differentiated them back into antigen-specific CTL and evaluated their properties and functional activities against multiple myeloma (MM). Methods: Functionally activeIFN-g producing HLA-A2 heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ CTL were applied for iPSC via transduction of four reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC). Upon characterization of the BCMA-specific iPSC with high pluripotency potential, embryoid body was formed from the iPSC and further polarized into mesoderm layer development as evidenced by upregulation of transcriptional regulators (ABCA4, BMP10, CDH5, FOXF1, HAND1, PLVAP, SNAI2, TBX3). Next, BCMA-specific embryoid body-derived hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell differentiation in the presence of Fc-DLL4 signaling and rectonectin. Results: Our RNAseq analyses demonstrated unique transcriptional profiles of HPC from different iPSC clones committing to CD8+ T cells or other cell lineages (monocytes/granulocytes, B lymphocytes/NK cells). Principal component analyses demonstrated a high similarity and low variability of transcription profiles within the replicates of HPC committed to the same cell lineage. In addition, distinct genome-wide shifts and differential gene expression profiles were detected in HPC committed to each specific cell differentiation pathway. Specifically, the HPC commit to CD8+ T cells utilized a diverse repertoire of modulators promoting development of T cell maturation, specific immune response regulation, memory T cells, cytotoxicity and interferon induction, which were significantly higher than shown in HPC that differentiate to other cell lineages. In parallel, specific repression genes were identified in the HPC commit to CD8+ T cells, which develop TGF-β receptor, rearrangement of Ig heavy chain genes and inhibitory receptors. The T cells differentiated were mainly CD45RO+ memory CTL and fully rejuvenated without immune checkpoints expression and regulatory T cells and with high anti-MM activities. Conclusions: These findings identify genetic and epigenetic mechanisms and regulatory elements, which play key roles during lineage specific commitment of HPC developed in iPSC into CD8+ CTL and help to further design a next generation of regenerative medicine that provide the appropriate signals for T cell lineage commitment from progenitor cells.

Development of blood components samples collection as a source for new biomarkers search in multiple myeloma.

e15009 Background: Multiple myeloma (MM) is a B-cell malignancy resulting from the abnormal proliferation of neoplastic plasma cells that produce monoclonal immunoglobulin (Ig). The high variability of the course of this disease, its genetic clonal heterogeneity, is due to chromosomal deletions, chromosomal hyperploidy involving an odd number of chromosomes, as well as genetic aberrations, such as rearrangement of the Ig heavy chain gene loci. Since the available biomarkers do not take into account this feature of MM, there is a need to develop more advanced biomarkers that will more accurately predict the course of the disease and response to treatment. Methods: The collection of MM samples included biological samples obtained from patients over 18 years of age with a diagnosis of MM, who received treatment in the National Medical Research Center of Oncology since 2019. Only patients who signed an informed consent for the use of their biomaterial for scientific purposes were included in the project. The material was collected according to the developed algorithm of clinical information and biological material collection, sample preparation, quality control and storage in the cryostorage of the National Medical Research Centre for Oncology of the Ministry of Health of Russia (Rostov-on-Don). Results: As of December 23, 2021, collection consists of 387 samples of whole blood, serum, plasma and mononuclear cells obtained from 42 MM patients of both sexes, whose average age was 59.7 ± 1.49 (±SD) years. Each patient was assigned a unique identification number. Freezing of the obtained samples occurred in accordance with the low-temperature storage protocol. Registration, accounting and certification of the material were carried out in a specialized database for recording and storing information about biological samples. Conclusions: The identification of MM biomarkers is important for increasing the sensitivity of molecular monitoring, which makes it possible to stratify patients into risk groups for early relapses and treatment resistance development. Thanks to the accumulated experience, the Biobank of the National Medical Research Centre for Oncology of the Ministry of Health of Russia serves as a valuable resource for providing research in the development of new predictive molecular markers.