Immunoglobulin Heavy Chain Gene Research Articles

Clinical Utility of B- and T-Cell Gene Rearrangement Studies in Blood and Bone Marrow Samples

In the evaluation of bone marrow (BM) and peripheral blood (PB) for hematologic malignancy, positive immunoglobulin heavy chain (IG) or T-cell receptor (TCR) gene rearrangement results may be detected despite unrevealing results from morphologic, flow cytometric, immunohistochemical (IHC), and/or cytogenetic studies. The significance of positive rearrangement studies in the context of otherwise normal ancillary findings is unknown, and as such, we hypothesized that gene rearrangement studies may be predictive of an emerging B- or T-cell clone in the absence of other abnormal laboratory tests. Data from all patients who underwent IG or TCR gene rearrangement testing at the authors' affiliated VA Hospital between January 1, 2013 and July 6, 2018 were extracted from the electronic medical record. Date of testing; specimen source; and morphologic, flow cytometric, IHC, and cytogenetic characterization of the tissue source were recorded from pathology reports. Gene rearrangement results were categorized as true positive, false positive, false negative, or true negative. Lastly, patient records were reviewed for subsequent diagnosis of hematologic malignancy in patients with positive gene rearrangement results with negative ancillary testing. A total of 136 patients, who had 203 gene rearrangement studies (50 PB and 153 BM), were analyzed. In TCR studies, there were 2 false positives and 1 false negative in 47 PB assays, as well as 7 false positives and 1 false negative in 54 BM assays. Regarding IG studies, 3 false positives and 12 false negatives in 99 BM studies were identified. Sensitivity and specificity, respectively, were calculated for PB TCR studies (94% and 93%), BM IG studies (71% and 95%), and BM TCR studies (92% and 83%). Analysis of PB IG gene rearrangement studies was not performed due to the small number of tests (3; all true negative). None of the 12 patients with false positive IG/TCR gene rearrangement studies later developed a lymphoproliferative disorder, although two patients were later diagnosed with acute myeloid leukemia. Of the 14 false negatives, 10 (71%) were related to a diagnosis of plasma cell neoplasms. Results from the present study suggest that positive IG/TCR gene rearrangement studies are not predictive of lymphoproliferative disorders in the context of otherwise negative BM or PB findings. As such, when faced with equivocal pathology reports, clinicians can be practically advised that isolated positive IG/TCR gene rearrangement results may not indicate the need for closer surveillance. Disclosures No relevant conflicts of interest to declare.

R-m-NHL-BFM-90/R-EPOCH Protocol in the Treatment of Patients with Primary Mediastinal Lymphoma: First Results

Background. Response to the induction chemotherapy is an important predictor of favorable outcome in aggressive lymphomas. As it was shown recently (Kurtz et al.2018) combination of interim PET/CT (iPET/CT) negativity and early molecular response detected by cell-free DNA (cfDNA) analysis, robustly stratify EFS and OS in patients (pts) with diffuse large B-cell lymphoma. There is no consensus on the most optimal program for primary mediastinal lymphoma (PML) treatment. Both R-CHOP+\-RT and R-DA-EPOCH are highly effective frontline options for PML, but about 10-15% of pts develop relapse/progression mainly within 2 years from diagnosis. We hypothesize that intensification of induction with 2 courses of modified R-NHL-BFM-90 program (R-m-NHL-BFM-90), followed by 4 courses of R-EPOCH without dose escalation might improve treatment outcome in PML. Aim. To assess the efficacy of R-m-NHL-BFM-90/R-EPOCH regimen for untreated PML pts by means of PET/CT and cfDNA evaluation. Methods. Overall, 18 pts were enrolled in prospective study between 2015 and 2019 years; median age 34 (19-50); M/F=4/14; Ann Arbor stage >I in 18 (100%); adverse factors in 12 pts (bulky disease>10 cm in 11 pts, involvement of the soft tissues in 9 pts and breast in 4 pts, pleural effusion in 6 pts, elevated lactate dehydrogenase level in 10 pts). The treatment plan consisted of: (i) induction of remission (blocks A and B of R-mNHL-BFM-90 program); (ii) consolidation with 4 courses of R-EPOCH. PET/CT was performed in all pts before therapy, after 2 courses of induction and after the end of therapy. B-cell clonality was assessed detecting immunoglobulin heavy chain gene rearrangements IGH (VH-JH FR2) and immunoglobulin light kappa chain gene rearrangement (IGK), by the use of multiplex Biomed-2 primer system for PCR and Gene-Scan analysis. cfDNA was obtained before the start of the treatment, after the induction and after the end of therapy in 4 pts. DNA concentration was measured by real time PCR with beta-globin primers. Results. All 18 pts completed induction phase. After 2 courses of R-mNHL-BFM-90 PET/CT Deauville score (DS) were DS1-3 in 9 pts (50%) and DS4 in 9 pts (50%). 16 pts completed treatment; from these 12 pts received all 4 courses of R-EPOCH. 2 pts received 2 courses and 2 pts only 1 course due to hematologic toxicity grade 3-4 and infectious complications. After the end of therapy PET/CT were DS1-3 in 15 out of 16 (94%) pts. Only 1 patient response was assessed as DS4; after she received autologous HSCT PET/CT negativity was achieved. cfDNA assessment at all stages of treatment was available in 4 patients (Table 1). In all cases before treatment there were no clonal rearrangements in blood samples. Clonal rearrangements in cfDNA before treatment and in tumor samples were the same in all cases. After induction treatment in one case B-cell clonality persisted in cfDNA, but after the end of the treatment there was no amplification of cfDNA from all pts. Overall, with a median follow-up of 14 months (range 1-40) 16/16 pts (100%) are alive in complete remission. Of note, all 4 pts who received only 1-2 courses of R-EPOCH are in complete remission for 4, 13, 16 and 17 months. Conclusions. Our results suggest that R-m-NHL-BFM-90/R-EPOCH protocol is highly effective for treatment of patients with PML. Given our results, we consider the possibility of reducing the number of cycles of chemotherapy. We suggest that detection of the tumor markers in cfDNA could be used as a biomarker to adapt treatment strategies in the future. Disclosures No relevant conflicts of interest to declare.

Detection of Immunoglobulin Heavy Chain Gene Clonality By High-Throughput Sequencing for Minimal Residual Disease Monitoring in Chronic Lymphocytic Leukaemia

Introduction: The negative minimal residual disease (MRD) after treatment has been recently accepted as endpoint for Chronic Lymphocytic Leukaemia (CLL) clinical trials. Conventionally, MRD can be detected by using multi-color Flow Cytometry (FC) with high sensitivity. Determination of the clonal immunoglobulin gene rearrangement can be a useful monitoring marker in a broad range of B-cell lymphoproliferative neoplasms. Moreover, the mutational status of immunoglobulin heavy chain variable (IgHV) rearrangement is considered one of the most important prognostic factors in CLL. Therefore, the identification of the IgHV rearrangement can be a useful marker both at diagnostic and as monitoring marker for MRD. Nowadays, high-throughput sequencing (HTS) technologies has enabled highly sensitive cancer genomic testing in clinical laboratories. There are same initiatives based on HTS to use IgHV rearrangement as marker for MRD monitoring in Acute lymphoblastic leukemia or multiple myeloma, but it remains unharmonized for application on CLL in the clinical laboratory. Objective: We evaluated the performance and clinical applicability of HTS assay for IgHV rearrangement in CLL MRD monitoring in 69 samples from 19 CLL patients treated. Methods: The libraries including IGH locus were performed using the Sequencing Multiplex Kit on IGH consensus primers. To simplify and make automatic the analysis of the data obtained, we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Assessment of IgHV mutational status by Sseq, genomic DNA (gDNA; 50-100 ng), were used for IgHV analysis. gDNA was amplified using locus-specific primer sets for IgHV designed to allow for the amplification of all known alleles of the germline IgH sequence, as described previously. Inmunophenotypic studies were performed on erythrocyte-lysed whole PB samples according to Euroflow procedures. PB white blood cells (WBC) was systematically stained with the eight color combination panel recently proposed by the ERIC group for MRD detection (Rawstron AC et al. 2016). Data acquisition was performed on a FACSCanto II flow cytometer Becton-Dickinson Biociences using the FACSDiva software (V8.0; BD). For data analysis, the Infinicyt softwareTM (Cytognos SL, Salamanca, Spain) was used. The MRD levels were reported as fraction of CLL cells of all nucleated cells. MRD negativity was define as a fraction <10 -4 However, for treatments that preferentially clear the blood (for example monoclonal antibodies) the MRD was confirm in the bone marrow as has been recommended. Results: Patient demographics and the results of IGH clonality tests are summarized in Table 1. Interpretable results were obtained with higher sensitivity compared with Sseq at diagnosis stage (19/19 samples), and we are able to determine the same clone at subsequent samples. Among 50 follow-up samples, 44 MRD were positive in both techniques (HTS and FC); 3 follow-up samples, were negative in both determinations (HTS and FC); in contrast, 3 follow-up samples, were negative by FC but positive by HTS (8.3, 17.2, 18.2 samples). This is the case of Patient 8, in which the last follow-up sample, the detection of the primary clone is positive by HTS, while by FC is undetected (Figure 1). In addition, it was possible to detect IgHV clone in all Patient 7 samples. It was under long-term monitoring by HTS and FC, and 17 months after initial diagnosis and first line of treatment (7.5 sample), it was detected a loss of response (Figure 2.). Conclusions: We evaluated an HTS IgHV assay using initial and follow-up samples of 19 CLL patients. Using our pre-designed primer set in library preparation, and developed our specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation, we were able to demonstrate that our method was more sensitive than FC in detecting positive follow-up samples and could be able to propose it for MRD monitoring in CLL. However, the prognostic impact of these low-level MRD detected by HTS should be validated with further investigations. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Ortiz: GILEAD SCIENCES: Research Funding. Terol:Abbvie: Consultancy; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Astra Zeneca: Consultancy; Roche: Consultancy.

Comprehensive Profiling of Disease-Relevant Copy Number Aberrations Improves Risk Assessment and Unveils the Clonal Origin of Relapse in Pediatric Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with >20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Correlated dynamics of serum IGE and IGE+ clonotype count with allergen air level in seasonal allergic rhinitis

Mechanisms of maintenance of immunological memory in the chronic course of seasonal allergic rhinitis remain poorly understood. The detailed understanding of these mechanisms is required for design of new approaches for allergy treatment. It is known that the level of allergen-specific IgE antibodies (sIgE), which play a key role in the development of the disease, is increased in patients with seasonal allergic rhinitis during pollination season. This study aimed to investigate the dynamics of serum IgE levels and characteristics of the clonal repertoire of IgE-secreting lymphocytes depending on the intensity of the patient's contact with the allergen. For three patients, allergic to birch pollen (22, 22, and 28 y.o.), we measured total IgE and birch pollen specific IgE levels at 6 time points with 2 week interval during the birch pollination season. Immunoglobulin heavy chain gene (IGH) clonal repertoire data for several B-cell subpopulations at different time points were obtained for one patient. We observe growth of the sIgE level (91%, 37%, and 64% compared to the baseline) at the peak of pollination season in all three donors. Initial increase in sIgE and IgE levels coincides with the birch pollination initiation; sIgE and total IgE levels correlate with the birch pollen air level (sIgE: R2 = 0.98 at p < 0.05; total IgE: R2 = 0.95 at p < 0.05). We detected IgE clonotypes only in samples obtained during the birch pollination, which indicates an increase of IGE-expressing cells concentration during this period. The frequency of IgE clonotypes was extremely low compared to that of the clonotypes of other isotypes (IgE — 0.01%, IgM — 48.4%, IgD — 14%, IgG — 17.4%, IgA — 19.8%). Hypermutation and phylogenetic analysis of the sequences from the 13 detected IgE-containing clonal groups showed that these IgE clonotypes could originate from IgG as a result of sequential isotype-switching.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

IGHV mutational status and outcome for patients with chronic lymphocytic leukemia upon treatment: a Danish nationwide population-based study.

Patients with chronic lymphocytic leukemia and unmutated immunoglobulin heavy-chain variable region gene (IGHV) have inferior survival from time of treatment in clinical studies. We assessed real-world outcomes based on mutational status and treatment regimen in a nationwide population-based cohort, comprising all 4,135 patients from the Danish chronic lymphocytic leukemia registry diagnosed between 2008 and 2017. In total, 850 patients with known mutational status received treatment: 42% of patients received intensive chemoimmunotherapy consisting of fludarabine, cyclophosphamide plus rituximab, or bendamustine plus rituximab; 27% received chlorambucil in combination with anti-CD20 antibodies or as monotherapy, and 31% received other, less common treatments. No difference in overall survival from time of first treatment according to mutational status was observed, while treatment-free survival from start of first treatment was inferior for patients with unmutated IGHV. The median treatment-free survival was 2.5 years for patients treated with chlorambucil plus anti-CD20, and 1 year for those who received chlorambucil monotherapy. The 3-year treatment-free survival rates for patients treated with fludarabine, cyclophosphamide plus rituximab, and bendamustine plus rituximab were 90% and 91% for those with mutated IGHV, and 76% and 53% for those with unmutated IGHV, respectively, and the 3-year overall survival rates were similar for the two regimens (86-88%). Thus, it appears that, in the real-world setting, patients progressing after intensive chemoimmunotherapy as first-line therapy can be rescued by subsequent treatment, without jeopardizing their long overall survival. Intensive chemoimmunotherapy remains a legitimate option alongside targeted agents, and part of a personalized treatment landscape in chronic lymphocytic leukemia, while improved supportive care and treatment options are warranted for unfit patients.

A laboratory-based scoring system predicts early treatment in Rai 0 chronic lymphocytic leukemia.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x103/microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today’s chemofree era.

3142 Atypical Presentation of an Upper Gastrointestinal (GI) Bleed Manifesting as Extranodal Involvement of Diffuse Large B-Cell Lymphoma (DLBCL) Diagnosed in a Community Setting

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most common type of Non-Hodgkin's Lymphoma (NHL), with 5.4 new cases diagnosed per 100,000 persons as of 2016. There is a male predominance, with an average age of 64.2. One extranodal site involves the GI tract, predominantly the stomach. Commonly seen are mucosa associated lymphoid tissue lymphomas and DLBCL. The majority of cases present at stage III/IV, with 40% presenting as local involvement. Here we present an unusual case of extranodal DLBCL in a patient presenting with dysphagia and an upper GI bleed. CASE DESCRIPTION/METHODS: An 85 year-old female presented for evaluation of dysphonia, ongoing for several months. The patient complained of bilateral submandibular adenopathy, dysphagia, trouble breathing, and a 15 pound weight loss over two weeks. Computed tomography of the neck revealed a nasopharyngeal mass in the left (L) nasopharynx extending into the nasal cavity, right (R) nasopharynx, and eroding into the skull. Additionally, bilateral necrotic cervical lymph nodes and L supraclavicular adenopathy were also seen. Histologic sample was consistent with DLBCL, non-germinal center type. Fluorescence in-situ hybridization was negative for MYC rearrangement, t(8:14), t(4:18), and Epstein Barr Virus, but indeterminate for BCL6 rearrangement. She was staged IIA and underwent radiation therapy without chemotherapy. During treatment, the patient was admitted for evaluation of an upper GI bleed associated with dysphagia, fatigue, and progressive weakness. She underwent an esophagogastroduodenoscopy with findings showing Los Angeles class A esophagitis, large, inflammatory nodular tissue within the lesser curvature of the stomach with associated linear ulcerations, multiple punctate ulcers throughout the gastric body and antrum, duodenitis, and a small, clean-based ulcer in the duodenal sweep. Histology of the gastric mass revealed an atypical clonal lymphoid infiltrate involving the gastric mucosa, suggestive of DLBCL, with clonal immunoglobulin heavy chain gene rearrangement. DISCUSSION: This case represents an atypical presentation of an upper GI bleed that is the result of extranodal involvement of DLBCL. An atypical presentation like this should have lymphoma in the differential for those with a history. Treatment is dependent upon the extent of disease, usually starting with the regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone therapy. If left untreated, this disease can progress rapidly and prove fatal.

Clinical Utilization of Next-Generation Sequencing-Based Clonality Assays for Immunoglobulin Heavy Chain in Evaluating Suspected B-Cell Proliferation

Abstract Objectives B-cell clonality assays of the immunoglobulin heavy chain (IGH) and light chain (IGK) genes have been used widely in the workup of B-cell neoplasms. PCR is the current gold standard test for clonality. Recently, NGS has been suggested to both improve sensitivity and identify the specific V-(D-)J sequence required to track clones. Here we report our experience with using NGS for complementing our current procedures. Methods Our validated NGS test is performed by amplifying patients’ DNA using IGH-FR1 assay panel. Pooled amplicons get sequenced. The sequence data then get analyzed using in-house bioinformatics software, which sorts sequences and generates frequency distributions. Each sequence is ranked by its frequency. A clonal population is designated if the top sequence read is at least 1% of the total reads. For the standard PCR gene rearrangement test, we use a BIOMED-2 PCR-based B-cell assay. Results We retrospectively searched our database for all cases in which IGH and IGK clonality was tested by either standard PCR or NGS from 8/2016 to 7/2017. A total of 253 cases were tested by PCR while 21 cases were tested by NGS, which showed higher success rates. Of the 253 cases tested by conventional PCR, 39% (n = 98) were clonal (either IGH, IGK, or both), 34% (n = 86) were polyclonal, and the rest 28% (n = 69) showed a weak inconclusive signal or failed. Of the 21 cases analyzed by NGS, 71% (n = 15) were clonal, 24% (n = 5) were polyclonal, and just 5% (n = 1) failed. Conclusion Our experience indicates that NGS for IGH clonality, even more expensive, is an effective and more sensitive tool that produces higher informational content and yields higher success rates when compared to conventional PCR assay. NGS captures multidimensional data and provides a more precise determination of the clonal relationship between different lymphoid proliferations, which also is essential for the detection and measurement of minimal residual disease.

Evaluation of Positive T- and B-Cell Gene Rearrangement Studies Among Patients Without a Definitive Diagnosis by Other Assays

Abstract In the evaluation of bone marrow (BM) and peripheral blood (PB) for hematologic malignancy, positive immunoglobulin heavy chain (IG) or T-cell receptor (TCR) gene rearrangement results may be detected despite unrevealing results from morphologic, flow cytometric, immunohistochemical (IHC), and/or cytogenetic studies. The significance of positive rearrangement studies in the context of otherwise normal ancillary findings is unknown, and as such, we hypothesized that gene rearrangement studies may be predictive of an emerging B- or T-cell clone in the absence of other abnormal laboratory tests. Data from all patients who underwent IG or TCR gene rearrangement testing at the authors’ affiliated VA hospital between January 1, 2013, and July 6, 2018, were extracted from the electronic medical record. Date of testing; specimen source; and morphologic, flow cytometric, IHC, and cytogenetic characterization of the tissue source were recorded from pathology reports. Gene rearrangement results were categorized as true positive, false positive, false negative, or true negative. Lastly, patient records were reviewed for subsequent diagnosis of hematologic malignancy in patients with positive gene rearrangement results with negative ancillary testing. A total of 136 patients, who had 203 gene rearrangement studies (50 PB and 153 BM), were analyzed. In TCR studies, there were 2 false positives and 1 false negative in 47 PB assays, as well as 7 false positives and 1 false negative in 54 BM assays. Regarding IG studies, 3 false positives and 12 false negatives in 99 BM studies were identified. Sensitivity and specificity, respectively, were calculated for PB TCR studies (94% and 93%), BM IG studies (71% and 95%), and BM TCR studies (92% and 83%). Analysis of PB IG gene rearrangement studies was not performed due to the small number of tests (3; all true negative). None of the 12 patients with false-positive IG/TCR gene rearrangement studies later developed a lymphoproliferative disorder, although 2 patients were later diagnosed with acute myeloid leukemia. Of the 14 false negatives, 10 (71%) were related to a diagnosis of plasma cell neoplasms. Results from the present study suggest that positive IG/TCR gene rearrangement studies are not predictive of lymphoproliferative disorders in the context of otherwise negative BM or PB findings. As such, when faced with equivocal pathology reports, clinicians can be practically advised that isolated positive IG/TCR gene rearrangement results may not indicate the need for closer surveillance.

Minimal Residual Disease in Chronic Lymphocytic Leukemia: A New Goal?

In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with TP53 deletion/mutation or unmutated IGHV genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.

Clinical significance of DNA methylation in chronic lymphocytic leukemia patients: results from 3 UK clinical trials.

Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.

Molecular diagnosis angioimmunoblastic T-cell lymphoma

to determine molecular diagnostics routine for different tissue samples in angioimmunoblastic T-cell lymphoma. Molecular studies were performed for 84 primary AITL patients. The median age was 61 year (29-81); the male to female ratio was 48/36. T-cell and B-cell clonality was assessed by GeneScan analysis of rearranged T-cell receptor (TCRG, TCRB) and immunoglobulin heavy chain genes. For the quantitative determination of cells with RHOA G17V mutation real - time polymerase chain reaction (PCR) with allele - specific LNA modified primers was used. In lymph nodes rearrangements of T-cell receptor genes were determined in 76 (90.5%) of 84 patients and were absent in 8 (9.5%) cases. Identification of the same clonal products of the TCRG and TCRB genes in the lymph node and in peripheral blood and/or bone marrow indicated the prevalence of the tumor process and was observed in 64.7% of patients. Clonal products in peripheral blood and/or bone marrow different from those in the lymph node indicated reactive cytotoxic lymphocyte population and were noted in 58.8% of AITL cases. Simultaneous detection of T- and B-cell clonality in the lymph node was observed in 20 (24.7%) of 81 patients. Cells with RHOA G17V mutation were detected in lymph node in 45 (54.9%) of 82 patients. The use of allele - specific PCR with LNA modified primers revealed presence of the tumor cells in peripheral blood in 100% and in bone marrow in 93.9% of patients with G17V RHOA mutation in the lymph nodes. The validity of different molecular assays performed on certain tissue samples for the diagnosis of angioimmunoblastic T-cell lymphoma has been evaluated. Quantitative allele - specific PCR assay for RHOA G17V mutation based on LNA modified primers possesses sufficient sensitivity for tumor process prevalence evaluation and minimal residual disease monitoring.

Elevated Lactate Dehydrogenase Has Prognostic Relevance in Treatment-Naïve Patients Affected by Chronic Lymphocytic Leukemia with Trisomy 12.

Chronic Lymphocytic Leukemia (CLL) patients with +12 have been reported to have specific clinical and biologic features. We performed an analysis of the association between demographic; clinical; laboratory; biologic features and outcome in CLL patients with +12 to identify parameters predictive of disease progression; time to treatment; and survival. The study included 487 treatment-naive CLL patients with +12 from 15 academic centers; diagnosed between January 2000 and July 2016; and 816 treatment-naïve patients with absence of Fluorescence In Situ Hybridization (FISH) abnormalities. A cohort of 250 patients with +12 CLL followed at a single US institution was used for external validation. In patients with +12; parameters associated with worse prognosis in the multivariate model were high Lactate DeHydrogenase (LDH) and β-2-microglobulin and unmutated immunoglobulin heavy-chain variable region gene (IGHV). CLL patients with +12 and high LDH levels showed a shorter Progression-Free-Survival (PFS) (30 months vs. 65 months; p < 0.001), Treatment-Free-Survival (TFS) (33 months vs. 69 months; p < 0.001), Overall Survival (OS) (131 months vs. 181 months; p < 0.001) and greater CLL-related mortality (29% vs. 11% at 10 years; p < 0.001) when compared with +12 CLL patients with normal LDH levels. The same differences were observed in the validation cohort. These data suggest that serum LDH levels can predict PFS; TFS; OS and CLL-specific survival in CLL patients with +12.

Prognostic markers for immunodeficiency-associated primary central nervous system lymphoma

Immunodeficiency is a major risk factor for primary central nervous system lymphoma (PCNSL), but data on the disease in immunocompromised hosts are scarce. We aimed to define clinical and imaging features and determine prognostic factors for immunodeficiency-associated PCNSL. All PCNSL cases seen at Yale-New Haven Hospital between 2002 and 2017 were retrospectively screened for immunodeficiency. For patients with immunosuppression, biopsies were evaluated and clinical data were collected. Predictors of survival were identified using Kaplan-Meier survival analysis and log-rank test. p values < 0.05 were considered significant. 23 patients with immunodeficiencies were identified: eleven on immunosuppressants after solid organ transplantation, seven with human immunodeficiency virus infection, and five on immunosuppressive treatment due to various autoimmune disorders. PCNSL cases were largely Epstein-Barr-Virus positive (78%), histologically classified as diffuse large B cell lymphomas (87%), and showed peripheral contrast enhancement (81%) and corresponding heterogeneous diffusion-weighted imaging patterns (DWI) on magnetic resonance imaging (MRI) (71%). Median overall survival was 31months. Age > 60years at diagnosis (p < 0.01), peripheral enhancement of the mass on MRI (p = 0.04), heterogeneous DWI patterns (p = 0.04), and clonal immunoglobulin heavy chain gene rearrangement (IgHR) (p = 0.03) were found to be negative prognostic markers. Immunodeficiency-associated PCNSL presents with similar clinical, pathological and imaging features. Age > 60years, clonal IgHR, heterogeneous DWI pattern and peripheral enhancement on MRI may serve as predictors of less favorable outcome.

Venetoclax and Obinutuzumab in Patients with CLL and Coexisting Conditions

BackgroundThe BCL2 inhibitor venetoclax has shown activity in patients with chronic lymphocytic leukemia (CLL), but its efficacy in combination with other agents in patients with CLL and coexisting conditions is not known.MethodsIn this open-label, phase 3 trial, we investigated fixed-duration treatment with venetoclax and obinutuzumab in patients with previously untreated CLL and coexisting conditions. Patients with a score of greater than 6 on the Cumulative Illness Rating Scale (scores range from 0 to 56, with higher scores indicating more impaired function of organ systems) or a calculated creatinine clearance of less than 70 ml per minute were randomly assigned to receive venetoclax–obinutuzumab or chlorambucil–obinutuzumab. The primary end point was investigator-assessed progression-free survival. The safety of each regimen was also evaluated.ResultsIn total, 432 patients (median age, 72 years; median Cumulative Illness Rating Scale score, 8; median creatinine clearance, 66.4 ml per minute) underwent randomization, with 216 assigned to each group. After a median follow-up of 28.1 months, 30 primary end-point events (disease progression or death) had occurred in the venetoclax–obinutuzumab group and 77 had occurred in the chlorambucil–obinutuzumab group (hazard ratio, 0.35; 95% confidence interval [CI], 0.23 to 0.53; P<0.001). The Kaplan–Meier estimate of the percentage of patients with progression-free survival at 24 months was significantly higher in the venetoclax–obinutuzumab group than in the chlorambucil–obinutuzumab group: 88.2% (95% CI, 83.7 to 92.6) as compared with 64.1% (95% CI, 57.4 to 70.8). This benefit was also observed in patients with TP53 deletion, mutation, or both and in patients with unmutated immunoglobulin heavy-chain genes. Grade 3 or 4 neutropenia occurred in 52.8% of patients in the venetoclax–obinutuzumab group and in 48.1% of patients in the chlorambucil–obinutuzumab group, and grade 3 or 4 infections occurred in 17.5% and 15.0%, respectively. All-cause mortality was 9.3% in the venetoclax–obinutuzumab group and 7.9% in the chlorambucil–obinutuzumab group. These differences were not significant.ConclusionsAmong patients with untreated CLL and coexisting conditions, venetoclax–obinutuzumab was associated with longer progression-free survival than chlorambucil–obinutuzumab. (Funded by F. Hoffmann–La Roche and AbbVie; ClinicalTrials.gov number, NCT02242942.)

Unveiling the Diversity of Immunoglobulin Heavy Constant Gamma (IGHG) Gene Segments in Brazilian Populations Reveals 28 Novel Alleles and Evidence of Gene Conversion and Natural Selection.

Even though immunoglobulins are critical for immune responses and human survival, the diversity of the immunoglobulin heavy chain gene (IGH) is poorly known and mostly characterized only by serological methods. Moreover, this genomic region is not well-covered in genomic databases and genome-wide association studies due to particularities that impose technical difficulties for its analysis. Therefore, the IGH gene has never been systematically sequenced across populations. Here, we deliver an unprecedented and comprehensive characterization of the diversity of the IGHG1, IGHG2, and IGHG3 gene segments, which encode the constant region of the most abundant circulating immunoglobulins: IgG1, IgG2, and IgG3, respectively. We used Sanger sequencing to analyze 357 individuals from seven different Brazilian populations, including five Amerindian, one Japanese-descendant and one Euro-descendant population samples. We discovered 28 novel IGHG alleles and provided evidence that some of them may have been originated by gene conversion between common alleles of different gene segments. The rate of synonymous substitutions was significantly higher than the rate of the non-synonymous substitutions for IGHG1 and IGHG2 (p = 0.01 and 0.03, respectively), consistent with purifying selection. Fay and Wu's test showed significant negative values for most populations (p < 0.001), which indicates that positive selection in an adjacent position may be shaping IGHG variation by hitchhiking of variants in the vicinity, possibly the regions that encode the Ig variable regions. This study shows that the variation in the IGH gene is largely underestimated. Therefore, exploring its nucleotide diversity in populations may provide valuable information for comprehension of its evolution, its impact on diseases and vaccine research.

PS1131 HIGH‐THROUGHPUT B‐CELL IMMUNOPROFILING AT DIAGNOSIS AND RELAPSE OFFERS FURTHER EVIDENCE OF FUNCTIONAL SELECTION THROUGHOUT THE NATURAL HISTORY OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is divided into two broad prognostic categories, namely mutated (M) and unmutated (U) CLL, according to the somatic hypermutation (SHM) status of the clonotypic heavy chain immunoglobulin (IGHV) gene. This is perceived to remain stable over time, as evidenced by low‐throughput studies, which however precluded investigation of subclonal architecture and evolution overtime.Aims:Here, we sought to comprehensively assess the B cell receptor (BcR) IG gene repertoire at CLL diagnosis and 1st relapse after chemoimmunotherapy (FCR) by next‐generation sequencing (NGS).Methods:We studied 5 patients, of whom 1 each expressed BcR IG typical of stereotyped subset #1 (U‐CLL), subset #4 (M‐CLL) or subset #6 (U‐CLL), whereas the remaining 2 concerned non‐stereotyped U‐CLL. Genomic DNA was amplified by multiplex PCR and products were subjected to paired‐end NGS. Sequence data were processed by a validated bioinformatics pipeline performing strict quality filtering. Rearrangements with identical IGHV gene and CDR3 amino acid (aa) sequence were defined as clonotypes. The dominant clonotype was defined as major, and clonotypes with the same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3 were considered as its satellite subclones.Results:In total, we analyzed 1,682,728 filtered‐in, productive IGHV‐IGHD‐IGHJ gene rearrangements (median 195,460 rearrangements/sample). In all cases, the major clonotype (IGHV and CDR3 aa sequence) was identical to that determined by Sanger sequencing and remained the same at diagnosis and relapse. However, it consisted of multiple distinct nucleotide (nt) sequences; while most nt sequences displayed the same SHM load as with Sanger (median 73.9% of all nt sequences of the major clonotype), there were other nt sequences with higher or lower SHM load. In all cases, nt sequences with discordant SHM status were practically negligible (median frequency 0.01%). Notably, the relative frequency of the dominant nt sequence increased at relapse for all cases (median increase by 3.7%, p &lt; 0.01). A particular note for subset #4, which by definition displays high SHM, was the emergence of major clonotype nt sequences with lower or even absent SHM at relapse. The median frequency of the major clonotype at diagnosis was 92.5% (range 86.1–93.3%) and remained stable at relapse (92.6%, range 89.9–93.8%). In all cases, the major clonotype came along with numerous satellite subclones (same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3) at both diagnosis and relapse (median n = 685 vs n = 604 respectively, p&gt;0.05). When considering both the major clonotype and its satellite subclones, the respective cumulative frequency corresponded to nearly the entire B cell repertoire/sample (median 99.6%, range 97.6–99.8%). Subset #4 satellite sublones displayed recurrent SHM patterns within the CDR3 at relapse, which actually diverged from the major clonotype and converged towards the consensus CDR3 sequence for this subset as defined by 176 different subset #4 patients.Summary/Conclusion:Overall, our study provides insight into the evolving architecture of the BcR IG repertoire of relapsing CLL, revealing that, similar to genomic subclones, there is a universe of BcR IG subclones as well. While the possibility of technical error cannot be excluded, the identification of recurrent SHM patterns and trends at relapse strongly points towards functional selection.

PB2110 MRD ANALYSIS IN MULTIPLE MYELOMA: IMMUNOGLOBULIN NGS VERSUS ASO‐PCR

Background:MRD monitoring is a valuable tool for treatment guidance and prognostication in hematological malignancies. The real‐time quantitative allele specific oligonucleotide PCR (qASO‐PCR) is a highly standardized technique across Europe and beyond for molecular MRD detection. For multiple myeloma (MM) patients, MRD monitoring becomes also more and more important and although the qASO‐PCR is suitable in this disease, it has a lower applicability due to the lower number of targets suitable for ASO design and the presence of somatic hypermutations in the immunoglobulin (IG)‐genes. Modern techniques such as next‐generation sequencing (NGS) overcome certain limitations of this qASO‐PCR.Aims:The purpose of this study is to compare the IG‐NGS technique with the qASO‐PCR for MRD detection in MM.MethodsPCR‐based NGS of the IG heavy chain (IGH) and IG kappa (IGK) genes was performed by the LymphoTrack® Dx IGH FR1/IGK Assay Panel ‐ MiSeq kits from Invivoscribe. For MRD quantification by NGS, a spike‐in of three different reference molecules (gBlocks) for each receptor was used as calibrator. The MRD data obtained by NGS were compared to the MRD data previously obtained by qASO‐PCR.A cohort of 25 bone marrow samples of 14 MM patients was used. The samples originated from the MYVAC2‐study, a pilot study in newly‐diagnosed symptomatic MM patients ≤ 65 years achieving at least a very good partial response (VGPR) after first‐line autologous stem cell transplantation (ASCT) and for whom therapy consists of a combination of lenalidomide maintenance and 4 autologous dendritic cell vaccines against MAGE‐A3/C1. Several time points after ASCT where tested for MRD.Results:The analytical sensitivity of the NGS assay was comparable with the qASO‐PCR and reached 1E‐05 when 500 ng DNA was used. Dilution experiments showed a very good linearity over a 5 log range (r = 0.96).The NGS‐MRD levels correlated well with the qASO‐PCR MRD levels in the total MM cohort (r = 0.85). The correlation was even better when the ‘positive non quantifiable’ MRD data (positive but below 1E‐04) by qASO‐PCR where eliminated (r = 0.96).MRD monitoring by NGS was applicable for all MM patients while MRD monitoring performed by qASO‐PCR was suitable for only 8/14 patients, indicating the higher applicability of the NGS technique for MRD detection in MM. A more detailed overview of the MRD results by NGS and qASO‐PCR is given in table 1. Overall, 24% off the samples (6/25) were negative by NGS, compared to 32% (8/25) by qASO‐PCR. In the positive non‐quantifiable group by qASO‐PCR, 100% of the samples (6/6) were positive by NGS. These findings indicate that an additional 32% (8/25) of the samples could be quantitated by NGS, reflecting already the higher sensitivity/specificity of the NGS approach.MRD negativity did result into a prolonged progression free survival benefit in the studied patient population, which validates its use as a surrogate endpoint.Summary/Conclusion:The PCR‐based NGS assay for MRD detection shows a good correlation with the patient‐specific qASO‐PCR. Moreover, the NGS assay is applicable in almost all MM patients while the qASO‐PCR is not, due to ASO design failure. The sensitivity of the assay is comparable with the qASO‐PCR and can only be augmented when the amount of DNA input is increased.image