Rearranged Immunoglobulin Heavy Chain Research Articles

SWIGH-SCORE: A translational light-weight approach in computational detection of rearranged immunoglobulin heavy chain to be used in monoclonal lymphoproliferative disorders

We present a lightweight tool for clonotyping and measurable residual disease (MRD) assessment in monoclonal lymphoproliferative disorders. It is a translational method that enables computational detection of rearranged immunoglobulin heavy chain gene sequences.•The swigh-score clonotyping tool emphasizes parallelization and applicability across sequencing platforms.•The algorithm is based on an adaptation of the Smith-Waterman algorithm for local alignment of reads generated by 2nd and 3rd generation of sequencers.For method validation, we demonstrate the targeted sequences of immunoglobulin heavy chain genes from diagnostic bone marrow using serial dilutions of CD138+ plasma cells from a patient with multiple myeloma. Sequencing libraries from diagnostic samples were prepared for the three sequencing platforms, Ion S5 (Thermo Fisher Scientific), MiSeq (Illumina), and MinION (Oxford Nanopore), using the LymphoTrack assay. Basic quality filtering was performed, and a Smith-Waterman-based swigh-score algorithm was developed in shell and C for clonotyping and MRD assessment using FASTQ data files. Performance is demonstrated across the three different sequencing platforms.

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

Significance of IgH Gene Rearrangement in Surveillance of Minimal Residual Disease after Autologous Hematopoietic Stem Cell Transplantation in Multiple Myeloma

To investigate the value of immunoglobulin heavy chain (IgH) gene rearrangement in monitoring minimal residual disease (MRD) in multiple myeloma (MM) received autologous hematopoietic stem cell transplantation(auto-HSCT). The clinical data of 26 MM patients who received auto-HSCT in the Department of Hematology, Wuhan First Hospital from 2018 to 2022 were collected. IgH rearrangement was detected by multiplex PCR combined with capillary electrophoresis fragment analysis to evaluate minimal residual disease (MRD), and the outcome of the disease was analyzed statistically. Among the 26 MM patients, 18 were males and 8 were females, with a median age of 59(41-70) years. The median follow-up time after transplantation was 33 (7-52) months. Compared with the IgH rearrangement negative group (n=17), the proportion of CR and sCR of patients with IgH rearrangement positive in bone marrow samples before auto-HSCT at 3 months after transplantation was lower （1/9 vs 14/17）, and the duration of remission (DOR) after transplantation was shorter（10.78±4.35 vs 15.88±5.22 months）, with statistically significant difference in DOR between the two groups(P < 0.05). Compared with IgH rearrangement negative group (n=21), the proportion of CR and sCR of patients with positive IgH rearrangement results from peripheral blood stem cell collection at 3 months after transplantation was lower（0/5 vs 15/21）, the duration of remission (DOR) after transplantation was shorter（9.60±4.83 vs 15.19±5.11 months）, and the difference in DOR between the two groups was statistically significant (P < 0.05). During the follow-up period, 5 patients (5/9) with positive IgH rearrangement results in bone marrow specimens died, and all patients with negative IgH rearrangement results survived. Four patients (4/5) with positive IgH rearrangement results by peripheral blood stem cell samples died, while one patient (1/21) with negative IgH rearrangement results died. In both bone marrow and peripheral blood stem cell samples, the survival time of IgH rearrangement-positive patients after transplantation was shorter than that of IgH rearrangement-negative patients(P < 0.05). Logistic regression analysis showed that gender, disease stage, the proportion of bone marrow smear plasma cells at initial diagnosis, stem cell mobilization plan, efficacy evaluation before transplantation (≥CR and <CR), and CD34+ cell count had no effect on IgH rearrangement results of stem cell collection (P >0.05). By detecting IgH rearrangement of MM patients receiving auto-HSCT, the depth of MRD can be further evaluated, which has a certain guiding significance for the efficacy and prognosis of the disease.

The Effect of Preemptive Rituximab Treatment in Patients with EBV Viremia after Solid Organ Transplantation on the Development of Post-Transplant Lymphoproliferative Disorder

The association between Epstein-Barr Virus (EBV) reactivation after solid organ transplantation (SOT) and Post-Transplant Lymphoproliferative Disorder (PTLD) is well described. There are currently no guidelines regarding the use of rituximab preemptively in patients with EBV viremia after SOT to reduce the risk of PTLD. We sought to determine whether rituximab in SOT patients with EBV viremia has benefit in preventing PTLD. This retrospective analysis of electronic medical record data identified SOT recipients who experienced EBV viremia post-transplant between January 2010 and February 2023 at Columbia University Irving Medical Center (CUIMC). Data queries were clinically reviewed. Logistic regression analysis was performed to investigate the relationship between predictor variables including rituximab administration, log-transformed EBV peak titer, duration of EBV viremia, and time from SOT to EBV reactivation, with the binary primary outcome of PTLD diagnosis status. The analysis encompassed both univariate and multivariate logistic regression. There were 2,168 patients who met the inclusion criteria of having EBV reactivation after SOT, out of 6,395 total SOT performed at CUIMC during this timeframe. Patient characteristics are described in table 1. The median peak EBV titer was 154 (range: not quantifiable - 5,335,816), the median duration of EBV viremia was 78 days (range: 1 - 6,980), and the median time from SOT to EBV reactivation was 688 days (range: 0-11103). Of the 2,168 patients, 185/2168 (8.5%) received rituximab for indications other than PTLD including EBV viremia, treatment of rejection, antibody desensitization therapy/immunosuppression peri-transplant, and other non-transplant related indications (e.g. lupus nephritis). Of the patients who received rituximab, 182/185 (98.4%) did not develop PTLD. The 3/185 (1.6%) patients who did develop PTLD were all ≤ 20 years-old at time of diagnosis, and had EBER(+), monomorphic PTLD, diffuse large B-cell lymphoma type, with a non-germinal center phenotype, involving the gastrointestinal tract. Two were CD20 positive, all 3 had partial weak CD30 positivity, and all 3 had clonal immunoglobulin heavy chain rearrangements. Among the patients who did not receive preemptive rituximab post-SOT, 195/1983 (9.8%) developed PTLD, whereas 1788/1983 (90.2%) did not. Prior rituximab therapy for any indication in patients with EBV viremia post-SOT was associated with lower risk of subsequent PTLD by univariate analysis (0.14% vs 8.39%, OR 0.15, p = 0.0013, 95% CI 0.048-0.48). Receipt of rituximab (OR 0.12, p=0.0002), log-transformed peak EBV titer (OR 1.10, p&amp;lt;0.0001), and duration of EBV viremia in months (OR 1.01, p&amp;lt;0.0001) were all independently associated with risk for PTLD by multivariate analysis (figure 1). The relationship between time from SOT to EBV viremia was not significantly associated with development of PTLD. This retrospective analysis suggests that administering preemptive rituximab in SOT patients with EBV viremia may lower the risk of developing PTLD. The fact that peak EBV titer and duration of EBV viremia remain associated with PTLD in multivariate analyses suggests that the benefit of rituximab may be independent of the degree or duration of EBV viremia. A prospective analysis of factors such as area under the curve of EBV exposure, HLA status, T cell clonotype diversity, organ type, and immunosuppression type, is needed to better understand individual risks for improved selection of those who would benefit from preemptive rituximab.

High-dose chemotherapy with transplantation of autologous hematopoietic stem cells in the first line of follicular lymphoma therapy

Introduction. The follicular lymphoma (FL) is the most common indolent lymphatic tumor with high sensitivity to immunochemotherapy un most cases. Although overall survival (OS) is generally long, the disease is characterized by multiple relapses. High-dose chemotherapy (HDCT) with transplantation of autologous hematopoietic stem cells (auto-HSCT) is used for recurrent FL. Aim: to evaluate the efficacy and safety of HDCT with aHSCT in the first line of FL therapy; identify risk factors for disease progression and refractoriness. Material and methods. A prospective single-center study (conducted from May 2015 to January 2023) included 35 patients aged 18–65 years (median 43) with PL 1–3A grade t(14;18)+ with stages III–IV or stage II with bulky, having at least one criterion for the need to start therapy (according to GELF). Patients were treated according to the FL-2015 protocol: 4 R-CHOP, 2 R-DHAP and BeEAM with auto-HSCT. The primary endpoint was the rate of overall response (OR) and/or complete remission (CR) at the end of chemotherapy. Secondary end points were 3-year survival rates: OS, relapse-free survival (RFS), progression-free survival (PFS), and event-free survival (EFS). Minimal residual disease (MRD) in blood and/or bone marrow was assessed by PCR based on immunoglobulin heavy chain (IGH) gene rearrangements and/or BCL2::IGH rearrangements. Statistical analysis (by intent to treat) was performed on January 12, 2023. Results. 86 % of patients had stage IV tumor and 79 % had 3–5 FLIPI factors. After the end of treatment, OR and PR were 90 % and 90 %, the incidence of POD24 was 3 %. After the end of induction (4 courses of R-CHOP), MRD-negativity was achieved in 77 % and 53 % of patients as determined by PCR-IGH and BCL2::IGH. After the full completion of the FL-2015 protocol, MRD was not detected in 96 % of patients (according to PCR-IGH). Three-year overall survival, disease-free survival, progression-free survival and event-free survival were respectively: 90 %, 90 %, 95 % and 85 % (with the same standarderror of 9 %) at a median follow-up (by inverse Kaplan-Meier estimate) of 19 months (range: from 1 to 91 months) There were no deaths due to early toxicity within 100 days of auto-HSCT. Prognostically unfavorable independent statistically significant (р &lt; 0.01; Wald test; hazard ratio &gt; 1) predictors of progression and refractoriness according to the results of multivariate analysis using the Fine-Grey competing risk model (р = 0.052 for the model) were: bone marrow disease, ECOG high risk, patient age &gt; 50 years, stage 4 disease, elevated serum lactate dehydrogenase and B-symptoms. Conclusion. The use of HDCT with auto-HSCT in the first line in patients with FL is highly effective and can significantly reduce the incidence of POD24 and early mortality from the tumor. The study is ongoing.

Presence of leukemic clone-specific immunoglobulin heavy chain rearrangements in neonatal blood spots of children with B-cell precursor acute lymphoblastic leukemia.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be traced back to birth using leukemic clone-specific immunoglobulin heavy chain (IGH) rearrangements, implying prenatal origin of this disease. We retrospectively analyzed neonatal blood spots (Guthrie cards) of 24 patients with childhood BCP-ALL aged 1-9.6 years (median 3.1 years) for the presence of clonotypic IGH rearrangements identified in diagnostic bone marrow samples. Based on the sequences of IGH rearrangements, 2 patient-specific primers were designed for each patient and used in semi-nested polymerase chain reaction for the detection of preleukemic clones at birth. Clonotypic IGH rearrangements were detected in neonatal blood spots of 54.2% of patients (13/24). In two cases with double IGH rearrangements detected at diagnosis, only one rearrangement was present at birth, while in the third case both leukemic rearrangements were detected in neonatal blood. Guthrie card-positive findings were significantly more frequent in children ≤5 years of age than in older children (p = 0.011). Regarding patients' characteristics at birth and at diagnosis, Guthrie card-positivity was not associated with sex, birth weight and mother's age, as well as with white blood cell count, percentage of bone marrow blasts, immunophenotype and the presence of ETV6/RUNX1 and TCF3/PBX1 fusion genes at diagnosis. Our study confirms that a large proportion of childhood BCP-ALL originates in utero, regardless of the molecular subtype defined by chromosomal aberrations. The observed trend toward younger age at diagnosis in Guthrie card-positive versus Guthrie card-negative patients implies that the age at diagnosis depends on the presence of preleukemic clone at birth, as well as on the timing of postnatal transforming genetic events.

A decade of CD4+ chimeric antigen receptor T-cell evolution in two chronic lymphocytic leukemia patients: were chronic lymphocytic leukemia cells present?

On Feb 2, 2022, Nature published the paper titled "Decade-long leukemia remissions with the persistence of CD4+ CAR T-cells" (Nature. 2022;602:503-9. doi: 10.1038/s41586-021-04390-6). According to the results presented, it could be argued that "chimeric antigen receptor (CAR) T-cells can actually cure patients with chronic lymphocytic leukemia (CLL)". CAR T-cells remained detectable more than ten years after infusion, and immunoglobulin heavy chain (IGH) rearrangement deep sequencing showed persistent deep molecular remission for both patients (no CLL clonotypes were detectable six months after CAR T-cell infusion and onwards). However, the existing actual disease status of both patients remained unclear, as it was unknown: (1) if CAR T-cells killed all leukemia cells during the initial anti-leukemic response phase, that is, soon after CAR T-cell infusion into both patients; (2) if few CLL cells survived, but persistent CAR T-cells had been able to destroy any leukemia cells before they reach detectable levels. In the first case, both patients could be considered definitely cured; in the second not and their decade-prolonged deep remission could be a consequence of the cytotoxic activity of the functionally active CD4+ CAR T-cells. The first version appears to be stronger and the supporting arguments have been included in a comprehensive commentary article. A new therapeutic intervention may emerge with the potential to fully improve the quality of life of both patients and in addition, ongoing research into CAR T-cells may turn in a new, more effective direction.

Characterization of CD3+/CD20+ canine large-cell lymphoma.

Immunophenotyping of canine large-cell lymphoma (LCL) for B-cell and T-cell surface antigens is commonly performed to better predict the clinical outcome. Expression of surface antigen CD3 is associated with T-cell malignancies; surface antigen CD20 is expressed on B cells. However, a small subset of canine LCLs expresses both CD3 and CD20 (CD3+/CD20+); this form of lymphoma remains poorly defined at the molecular level. In a retrospective study, we aimed to better characterize immunophenotypic properties and antigen receptor clonality of CD3+/CD20+ LCL. We selected formalin-fixed, paraffin-embedded tissues from 10 cases of CD3+/CD20+ LCL and breed-matched controls of peripheral large T-cell lymphoma (PTCL) and diffuse large B-cell lymphoma (DLBCL). Using PCR for antigen receptor rearrangement (PARR), we identified monoclonal T-cell receptor gamma (TCRγ) rearrangements in all CD3+/CD20+ cases. Three of 10 cases had monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of cross-lineage rearrangement. There was no significant difference in the frequency of antigen receptor rearrangement between CD3+/CD20+ and PTCL cases. In comparison with DLBCL, CD3+/CD20+ LCL had TCRγ rearrangement more frequently and IgH rearrangement less frequently, respectively. Immunolabeling of the B-cell marker PAX5 occurred less frequently in all CD3+/CD20+ LCL cases compared to the DLBCL controls. Immunolabeling for BCL-2 was robust, regardless of immunophenotype. Nuclear Ki67 positivity was variable in CD3+/CD20+ cases, indicating a heterogeneity in proliferation. Overall, cases of canine CD3+/CD20+ LCL had properties similar to PTCL, suggesting a similar histogenesis of these 2 subsets.

Characterization of B-cell receptor clonality and immunoglobulin gene usage at multiple time points during active SARS-CoV-2 infection.

Although monoclonal antibodies to the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are known, B-cell receptor repertoire and its change in patients during coronavirus disease-2019 (COVID-19) progression is underreported. We aimed to study this molecularly. We used immunoglobulin heavy chain (IGH) variable region (IGHV) spectratyping and next-generation sequencing of peripheral blood B-cell genomic DNA collected at multiple time points during disease evolution to study B-cell response to SARS-CoV-2 infection in 14 individuals with acute COVID-19. We found a broad distribution of responding B-cell clones. The IGH gene usage was not significantly skewed but frequencies of individual IGH genes changed repeatedly. We found predominant usage of unmutated and low mutation-loaded IGHV rearrangements characterizing naïve and extrafollicular B cells among the majority of expanded peripheral B-cell clonal lineages at most tested time points in most patients. IGH rearrangement usage showed no apparent relation to anti-SARS-CoV-2 antibody titers. Some patients demonstrated mono/oligoclonal populations carrying highly mutated IGHV rearrangements indicating antigen experience at some of the time points tested, including even before anti-SARS-CoV-2 antibodies were detected. We present evidence demonstrating that the B-cell response to SARS-CoV-2 is individual and includes different lineages of B cells at various time points during COVID-19 progression.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3' regulatory region (3'RR) (Sμ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3'RRrec counts per sample: Sμ-3'RRrecHigh cases (mostly unmutated CLL) and Sμ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3'RRrec appeared to be ongoing in Sμ-3'RRrecHigh CLL cells and comparison of Sμ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3'RRrec in Sμ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3'RRrec, even in the absence of AID for the latter.

Cell-free DNA for detection of clonal B cells in diffuse large B cell lymphoma by sequencing.

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in the western world. It is highly heterogeneous with a variable clinical course, but curable with chemo-immunotherapy in up to 70% of all cases. The lymphoma presents in lymph nodes and/or extranodal lymphoid tissue, and the diagnosis is based on invasive procedures for histopathologic evaluation. In this technical study, we evaluated cell-free DNA (cfDNA) from blood plasma to detect clonal B cells in patients with DLBCL using rearranged immunoglobulin heavy chain gene as targets by next-generation sequencing. Clonal B cell sequences and frequencies were determined from blood plasma cfDNA and cellular DNA from matched excised lymphoma tissues and mononuclear cells isolated from diagnostic bone marrow and blood samples from 15 patients. We showed that identical clonal rearrangements could be detected in blood plasma and excised lymphoma tissue and that plasma cfDNA was superior in detecting clonal rearrangements compared to blood or bone marrow-derived cellular DNA. These findings consolidate the role of blood plasma as a reliable and easily accessible source for detecting neoplastic cells in DLBCL.

Gastric Mucosa-associated Lymphoid Tissue Lymphoma Mimicking Signet Ring Cell Carcinoma

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is the most common type of extranodal non-Hodgkin lymphoma. Endoscopic findings are nonspecific and variable; therefore, differentiation of this malignancy from early gastric cancer is challenging during endoscopy. Although an endoscopic biopsy is the gold standard for diagnosis, a biopsy may not conclusively establish the diagnosis in all cases. Diagnostic confirmation requires interpretation of the biopsy specimen findings by an experienced histopathologist, and an additional immunoglobulin heavy chain (IgH) rearrangement test may aid with accurate diagnosis. We present a case of gastric MALT lymphoma that histopathologically mimicked signet ring cell carcinoma (SRCC) on evaluation of repeat endoscopic biopsies. Following endoscopic submucosal dissection (ESD), we confirmed the final diagnosis of gastric MALT lymphoma based on histopathological findings of prominent lymphoid infiltrates accompanied by lymphoepithelial lesions and results of the monoclonal IgH rearrangement test. Notably, a few carcinoma-like signet ring cells (SRCs) in the specimen were attributed to a reactive change. Clinicians should be mindful of possible SRCs in gastric MALT lymphoma specimens to avoid misdiagnosis of SRCC in patients with gastric MALT lymphoma. Confirmatory ESD may be useful for accurate diagnosis and appropriate management of such lesions.

HIGH RATE OF METABOLIC COMPLETE RESPONSE AFTER LOW DOSE RADIOTHERAPY AND OBINUTUZUMAB IN EARLY STAGE FOLLICULAR LYMPHOMA: INITIAL RESULTS OF THE GAZAI STUDY (GLA 2018‐3)

Introduction: The FORT trial demonstrated a higher complete remission (CR) rate using 12 × 2 Gy compared to 2 × 2 Gy in Follicular Lymphoma (FL) (67% vs. 47%). This benefit resulted also in an improved progression free survival (PFS). The TROG 99.03 trial and the MIR study showed an improved PFS with the addition of Rituximab to radiation therapy of 30–40 Gy in early stage FL. Preclinical data suggest that the anti-CD20 antibody may act synergistic with radiation therapy and therefore enhance the radiation effect of low dose radiotherapy. The prospective, multicentric phase II GAZAI study of the German Lymphoma Alliance (GLA) investigated the efficacy of low dose radiation therapy (2 × 2 Gy) in combination with the anti-CD20 antibody Obinutuzumab in early stage nodal FL. The primary endpoint was the metabolic complete remission at the end of therapy. Methods: Patients with early stage FL grade 1/2 were recruited in 11 German centers. Ann Arbor stage I or II was confirmed by FDG-PET/CT. Patients received 1000 mg Obinutuzumab flat dose intravenously in weeks 1, 2, 3, 4, 8, 12 and 16. An interim CT scan was performed in week 7 for response evaluation and subsequent treatment planning. Radiation therapy was applied to the initial involved sites (site of diagnostic surgical intervention and PET positive sites) in week 9. The radiation dose was 2 x 2 Gy on two succeeding days. A FDG-PET/CT was performed at the end of therapy in week 18 for evaluation of the metabolic and morphologic response. Minimal detectable/residual disease (MRD) in peripheral blood was monitored at baseline and at week 18 by allel-specific RQ PCR targeting t(14;18) translocations and clonal immunoglobulin heavy chain (IGH) rearrangements. Results: Of 89 patients included in the study, 54 entered the treatment phase showing FDG-PET-positive lymph nodes qualifying for the primary endpoint. At week 18, metabolic CR (Deauville score (DS) <3) was seen in 46/53 patients (87%; one patient only had a CT without FDG-PET). Partial metabolic remission with a DS 3 was seen in 3 patients (6%) and 3 patients showed a DS 4. Morphologic (CT based) CR/CRu according to Cheson 1999 criteria was seen in 21/54 patients (39%) at week 7 and in 49/54 patients (91%) at week 18. Morphologic PR was seen in 17 patients (32%) at week 7 and in 4 patients (7%) at week 18; one patient (2%) showed progressive disease (metabolic and morphologic) outside of the radiation field. 13/54 patients (24%) were initially MRD positive. All but one patient converted to MRD negativity at week 18. Conclusions: Low dose radiation therapy using 2 × 2 Gy in combination with Obinutuzumab is highly effective in early stage FL. Longer follow-up is necessary to investigate if this effectivity also results in a prolonged PFS. Currently, the FORTplus trial is looking for non-inferiority of this regimen in comparison to a conventional radiation dose (12 × 2 Gy) combined with Rituximab. The research was funded by: Roche Pharma Keywords: combination therapies, indolent non-Hodgkin lymphoma, radiation therapy Conflicts of interests pertinent to the abstract K. Herfarth Research funding: Roche Pharma Educational grants: Roche Pharma C. W. Scholz Consultant or advisory role: Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Educational grants: Roche Pharma K. Hübel Consultant or advisory role: Scientific advisory board Roche Pharma Honoraria: Speaker Honoraria Roche Pharma Research funding: Roche Pharma C. Buske Consultant or advisory role: Roche Pharma Honoraria: Roche Pharma Research funding: Roche Pharma J. Dürig Consultant or advisory role: Scientific advisory board Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Educational grants: Roche Pharma G. Lenz Consultant or advisory role: Advisory board Roche Pharma Honoraria: Speakers honoraria Roche Pharma Research funding: Roche Pharma C. Pott Research funding: Roche Pharma M. Dreyling Consultant or advisory role: Roche Pharma Honoraria: Speakers Honoraria Roche Pharma Research funding: Roche Pharma

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

The establishment of B cell-deficient Igh-J KO mouse model by gene editing and efficacy evaluation

Over the last few years, immunotherapy has made significant progress in treating various cancers with therapeutic antibodies. However, therapeutic antibodies have been validated for inducing an unintended immune response in human and animal models, which leads to the emergence of anti-drug antibodies (ADAs) and affects their effectiveness and safety. In preclinical research, ADAs production by B cells may accelerate antibody metabolism and result in missing potential candidate molecules. Thus, it is urgent to develop preclinical models that remove only B cells without affecting the function of T and NK cells. Rearrangement of immunoglobulin heavy chain J gene fragment (Igh-J) is the first link in B cell development, and immunotherapies are currently leaning toward combination treatments with PD-1/PD-L1 antibodies, here we created humanized PD-1, PD-L1 and Igh-J knockout (hPD-1/hPD-L1, Igh-J KO) mice and validated by using the reported high immunogenicity drug M7824 (a protein designed to simultaneously block PD-L1 and TGF-β pathways, poorly anti-tumor efficacy in immunocompetent mice). Phenotypic analysis revealed that human PD-1 and PD-L1 were detectable in hPD-1/hPD-L1, Igh-J KO mice, but not mouse IgM and IgD. Igh-J KO depleted B cells while increased the percentage of other immune cell types. Meanwhile, the humanization of PD-1/PD-L1 and Igh-J KO had neither effect on the overall development, differentiation, or distribution of T cell subtypes, nor on the activation of NK and T cells, indicating that mice can be used for T and NK-related immunotherapies. Furthermore, M7824 treatment of these B cell-deficient mice inhibited tumor growth significantly, with higher M7824 analog concentrations and lower ADA-positive rates. These findings demonstrate that Igh-J KO mice are an effective and stable preclinical model for testing drugs based on T and NK cells with high immunogenicity in vivo.

Clinical Significance of IGH Clonality Analysis with RNA Sequencing in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common pediatric malignancy. Using minimal residual disease (MRD) and genetic subtypes, risk stratification algorithms have considerably improved clinical outcomes. However, despite being in a better prognostic group, a non-negligible number of relapses occur, and prognostic markers for these patients are required for more effective treatment strategies. RNA sequencing (RNA-seq) is a useful tool for detecting patient-specific immunoglobulin heavy chain (IGH) junctional sequences (Li et al., Leukemia. 2020), which can be informative for evaluating the nature of leukemic clones. Here, we analyzed IGH clonality of pediatric BCP-ALL using RNA-seq data and revealed its validity for evaluating disease characteristics and impact on prognosis. Methods: We analyzed RNA-seq data of 136 pediatric BCP-ALL samples at diagnosis and 46 samples at relapse from the TARGET ALL Expansion Phase 2 cohort. Using MiXCR (Bolotin et al., Nat Methods 2015) and Vidjil (Duez et al., PLoS One 2016), we detected IGH complete VDJ and incomplete DJ rearrangements. Then, we estimated the expected number of IGH rearrangements corresponding to each read count (E-value) by power-law model. Finally, we identified leukemic "disease clones” with E-values less than 0.01 (Fig. 1a). Results: First, we detected a total of 55,518 IGH clones, of which 48,555 were complete rearrangements and 6,963 were incomplete. In total, 351 disease clones (median 2, range 0-8 per sample) were identified by power-law model: 193 clones were complete rearrangements, and 158 clones were incomplete. Interestingly, the numbers and compositions (complete or incomplete rearrangements) of disease clones varied significantly between samples. Thus, we defined three clonality patterns and classified each sample based on identified disease clones: "Undetectable disease clones (UDC)” if the sample had no disease clone (n=18); "Incomplete Disease Clones (IDC)” if there were one or more disease clones with incomplete rearrangements (n=72); and "Complete Disease Clones (CDC)” if all disease clones of that sample have complete rearrangements (n=46). Gene Set Enrichment Analysis (GSEA) indicated a strong enrichment for hematopoietic stem cell (HSC) and multipotent progenitor (MPP) signatures in UDC and pro-B cell signatures in IDC. Similarly, CIBERSORTx (Newman et al., Nat Biotechnol 2019) showed higher proportions of HSCs, MPPs, and pre-pro B cells in UDC and pro-B cells in IDC. Furthermore, there was a significant enrichment in pathways promoting cell cycle progression in IDC. Of note, in high hyperdiploid (HHD) cases, which are typically associated with a favorable prognosis, event-free survival (EFS) was significantly lower in UDC and IDC (p=0.01) (Fig. 1b). Moreover, even in MRD-negative HHD patients, those with UDC and IDC had significantly worse EFS (p=0.01) and overall survival (p=0.03) (Fig. 1b). Finally, we compared the primary and relapse clones in 46 patients. As a result, 37 patients had one or more disease clones at diagnosis, and 29 (78%) had these primary disease clones at relapse. On the other hand, 9 (24%) patients showed relapse-specific disease clones, which should lead to relapse in these cases. The other 9 primary patients with UDC had no disease clone also at relapse. Discussions: We identified three clonality patterns, UDC, IDC, and CDC based on RNA-seq analysis of the IGH rearrangement status. According to the findings of GSEA and CIBERSORTx, leukemic cells of UDC, IDC, and CDC patients have nature of progenitors to pre-proB cells, pro-B cells, and more mature cells, respectively. These findings are consistent with the knowledge that IGH rearrangement occurs during pro-B to pre-B maturation in normal B-cells. Patients with immature clonality status, UDC and IDC, were associated with poor prognosis in high hyperdiploid subtype and could be a potential prognostic marker in MRD-negative patients. In addition, we revealed that primary disease clones contributed to relapse in 78% of cases, while relapse-specific clones were implicated in relapse in 24% of cases. Additionally, UDC clonality pattern was carried over at relapse. These findings shed light on the mechanisms of leukemic clonal evolution and relapse, and suggest that the status of IGH rearrangement may serve as a novel prognostic marker in BCP-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The mammalian SKIV2L RNA exosome is essential for early B cell development.

The SKIV2L RNA exosome is an evolutionarily conserved RNA degradation complex in the eukaryotes. Mutations in the SKIV2L gene are associated with a severe inherited disorder, trichohepatoenteric syndrome (THES), with multisystem involvement but unknown disease mechanism. Here, we reported a THES patient with SKIV2L mutations showing severe primary B cell immunodeficiency, hypogammaglobulinemia, and kappa-restricted plasma cell dyscrasia but normal T cell and NK cell function. To corroborate these findings, we made B cell-specific Skiv2l knockout mice (Skiv2lfl/flCd79a-Cre), which lacked both conventional B-2 and innate-like B-1 B cells in the periphery and secondary lymphoid organs. This was linked to a requirement of SKIV2L RNA exosome activity in the bone marrow during early B cell development at the pro-B cell to large pre-B cell transition. Mechanistically, Skiv2l-deficient pro-B cells exhibited cell cycle arrest and DNA damage. Furthermore, loss of Skiv2l led to substantial out-of-frame V(D)J rearrangement of immunoglobulin heavy chain and severely reduced surface expression of μH, both of which are crucial for pre-BCR signaling and proliferative burst during early B cell development. Together, our data demonstrated a crucial role for SKIV2L RNA exosome in early B cell development in both human and mice by ensuring proper V(D)J recombination and Igh expression, which serves as the molecular basis for immunodeficiency associated with THES.

IgG kappa multiple myeloma with isolated central nervous system relapse.

A 76-year-old male presented with acute renal failure and cast nephropathy was diagnosed with IgG kappa multiple myeloma (MM). His paraprotein was 40 g/L, kappa-free light chain level was 17,058 mg/L, kappa: lambda light chain ratio was 1445. An automated full blood count showed hemoglobin 90 g/L, white blood cells 2.96 × 109/L, neutrophil 1.7 × 109/L, and platelet 179 × 109/L. Bone marrow trephine sample was hypercellular with nearly 100% plasma cells, which were positive for CD138, CD56, and cyclin D1, and showed kappa light chain restriction. Fluorescence in situ hybridization analysis with dual color/dual probes for immunoglobulin heavy chain (IGH) and MYC genes (Cytocell) and whole genome screening using 8 × 60K oligonucleotide arrays (Agilent) performed on the CD138 enriched cells (CD138 microbeads Miltenyi Biotech) from the liquid bone marrow sample showed no evidence of IGH rearrangement, MYC rearrangement, 1q gain, 1p loss, 17p loss, or any clinically significant imbalance. He was treated with eight cycles of bortezomib, cyclophosphamide, and dexamethasone (VCD) to very good partial response. He relapsed 30 months after diagnosis with paraprotein of 36 g/L, kappa light chains of 10,000 mg/L, and creatinine of 498 μmol/L. He was treated with eight cycles of bortezomib, thalidomide, and dexamethasone (VTD) to paraprotein undetectable and kappa light chain 29 mg/L. Seven months post completion of VTD (4 years after myeloma diagnosis), he presented with abnormal gait, falls, and a left-sided facial droop. His paraprotein was 2 g/L and serum-free kappa light chain was 81 mg/L. Magnetic resonance imaging (MRI) head and whole spine showed multifocal diseases in spinal canal and intracranially, involving the cranial nerves most prominent the fifth, seventh, eighth, and hypoglossal nerve on the left, anterior surface of the inferior pons medulla (Figure 1A). There were multiple lesions in the cervical spine as well as in the thoracic spine (Figure 1B). Computerized tomography (CT) chest abdomen pelvis did not show the evidence of other new diseases. Plasma cells were detected in cerebral spinal fluid (CSF) (Figure 1C, May–Grünwald–Giemsa stain x100 objective). He was treated with pomalidomide and dexamethasone. Weekly cytarabine intrathecal (IT) treatment was given until the clearance of plasma cells from CSF samples for three times. Four months later, he achieved the complete remission on MRI head and whole spine with undetectable paraprotein and normal light chain ratio. Serial MRI scans confirmed ongoing remission. A year later, he relapsed again with worsening mobility and reappearance of myeloma cells in CSF. By flow cytometry, these cells were positive for CD138, CD38, CD56, CD117, and negative for CD19. MRI head showed leptomeningeal disease with a new 2.2 cm frontal lobe lesion. His paraprotein remained undetectable and kappa light chain remained at below 100 mg/L. He continued systemic pomalidomide and dexamethasone chemotherapy and received palliative whole brain radiotherapy 20 Gy in 5 fractions for symptom control. His disease continued progressing and he passed away 3 months later (5.5 years after myeloma diagnosis). Here, we reported a myeloma patient with isolated CNS relapse, who achieved 1 year of remission with combination of intrathecal chemotherapy and systemic pomalidomide and dexamethasone treatment, but sadly passed away 4 months after second CNS relapse. CNS MM is a rare form of extramedullary disease, but carries a very poor prognosis. The optimal approach to treatment of CNS MM is not currently known. The current approach includes systemic therapy with agents known to cross the blood brain barrier, intrathecal therapy, and CNS irradiation. Innovative approach to treatment is urgently needed. The authors received no specific funding for this work. Ke Xu and Lucy Kamuriwo wrote the manuscript. Ke Xu, Lucy Kamuriwo, Claire Anderson and Parag Jasani critically revised the final version of the manuscript. The authors declare they have no conflicts of interest. No written or verbal consent obtained from patient. Patient has passed away.

Organization and Complexity of the Yak (Bos Grunniens) Immunoglobulin Loci.

As important livestock in Qinghai-Tibet Plateau, yak provides meat and other necessities for Tibetans living. Plateau yak has resistance to diseases and stress, yet is nearly unknown in the structure and expression mechanism of yak immunoglobulin loci. Based on the published immunoglobulin genes of bovids (cattle, sheep and goat), the genomic organization of the yak immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgL) were described. The assemblage diversity of IgH, Igλ and Igκ in yak was similar to that in bovids, and contributes little to the antibody lineage compared with that in humans and mice. Somatic hypermutation (SHM) had a greater effect on immunoglobulin diversity in yak than in goat and sheep, and in addition to the complementarity-determining region (CDR), some loci in the framework region (FR) also showed high frequency mutations. CDR3 diversity showed that immunological lineages in yak were overwhelmingly generated through linkage diversity in IgH rearrangements. The emergence of new high-throughput sequencing technologies and the yak whole genome (2019) publication have greatly improved our understanding of the immune response in yaks. We had a more comprehensive analysis of yak immunoglobulin expression diversity by PE300, which avoided the disadvantage of missing low-frequency recombination in traditional Sanger sequencing. In summary, we described the schematic structure of the genomic organization of yak IgH loci and IgL loci. The analysis of immunoglobulin expression diversity showed that yak made up for the deficiency of V(D)J recombinant diversity by junctional diversity and CDR3 diversity. In addition, yak, like cattle, also had the same ultra-long IgH CDR3 (CDR3H), which provided more contribution to the diverse expression of yak immunoglobulin. These findings might provide a theoretical basis for disease resistance breeding and vaccine development in yak.

Ezrin promotes antigen receptor diversity during B cell development by supporting immunoglobulin heavy chain variable gene recombination

Abstract Genome-level rearrangements of immunoglobulin genes during B cell development are critical for generation of a diverse repertoire of B cell antigen receptors (BCRs) that bind to a multitude of foreign antigens and some self-antigens. Bone marrow B cell development involves a variety of cell-cell interactions, cell migration and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins towards BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next generation sequencing to investigate if Ezrin plays a role in immunoglobulin heavy chain rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDR3s in the BCRs and reduced Shannon entropy. Ezrin-deficient pro-, pre- and immature B cells utilized similar number of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length, nucleotide additions and deletions, was also altered in Ezrin-deficient pro-, pre- and immature B cells. Mechanistically, Ezrin-deficient bone marrow B cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate a novel role for Ezrin in shaping the BCR repertoire through combinatorial and junctional diversification. Supported by grants from NIH (R01 AR067705) to N.G.