Clonal Immunoglobulin Gene Rearrangements Research Articles

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

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

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B‐cell neoplasms

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Rearrangements of Immunoglobulin Genes in Tumor Cells of Patients with Primary Mediastinal (Thymic) Large B-Cell Lymphoma

Background. Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is a malignant tumor with large atypical lymphoid cells expressing post-germinal differentiation markers. Rearrangements of immunoglobulin genes in PMBCL are revealed in 30-65 % of cases. Immunoglobulin molecules, however, are expressed neither on the surface, nor in cytoplasm of tumor cells. Aim. To assess cell clonality rate on the basis of rearrangements of immunoglobulin heavy/light chain genes; to determine rearrangement stability at the time of relapse development; to study the range of rearrangements and clonal relationship with primary tumor in metachronous development of mediastinal gray zone lymphoma. Materials & Methods. The assessment of rearrangements of immunoglobulin heavy/light chain genes was based on molecular analysis of 29 primary tumor biopsies and 4 tissue samples with histologically and immunohistochemically verified relapses or metachronous lymphoma development. Results. In 16 (55.2 %) out of 29 cases a rearrangement of immunoglobulin heavy chain genes was reported, in 7 (24.1 %) cases a rearrangement of light chain genes was identified, in 6 (20.7 %) cases no rearrangements of immunoglobulin heavy/light chain genes were found. On the basis of immunoglobulin gene analysis in 2 patients with early relapse a tumor clone was detected that was identical with the one identified at the onset of the disease. In 2 patients with complete remission a metachronous development of mediastinal gray zone lymphoma was reported, whereas molecular genetic analysis revealed a change/disappearance of initial clonal rearrangements of immunoglobulin genes. Conclusion. Total detection rate of B-cell clonality in PMB-CL was 79.3 %. Molecular genetic analysis confirmed that initial clonal rearrangements of immunoglobulin genes were preserved in early relapses, and invalidated tumor clonal relationship in a metachronous development of mediastinal gray zone lymphoma.

Expanding the Spectrum of EBV-positive Marginal Zone Lymphomas: A Lesion Associated With Diverse Immunodeficiency Settings.

Traditionally low-grade B-cell lymphomas have been excluded from the category of monomorphic posttransplant lymphoproliferative disorders. However, recent reports identified Epstein-Barr virus-positive (EBV) extranodal marginal zone lymphomas (MZL), almost exclusively seen in the posttransplant setting. Some reported cases responded to reduced immunosuppression, suggesting that they should be considered as a form of posttransplant lymphoproliferative disorders. We identified 10 cases of EBV MZL, 9 in extranodal sites and 1 presenting in lymph node. Two cases arose following solid organ transplantation, but other settings included iatrogenic immunosuppression for rheumatoid arthritis (2); prior chemotherapy (2); congenital immune deficiency (1); and increased age (3), as the only potential cause of immune dysfunction. There were 4 males and 6 females; age range 18 to 86. The atypical plasmacytoid and/or monocytoid B cells were positive for EBV in all cases, with either latency I or II in all cases tested. Monotypic light chain expression was shown in all with 6 cases positive for IgG, and 2 for IgM, undetermined in 2. Clonal immunoglobulin gene rearrangement was positive in all cases with successful amplification. MYD88 L265P was wild type in the 6 cases tested. We show that EBV MZLs can arise in a variety of clinical settings, and are most often extranodal. Treatment varied, but most patients had clinically indolent disease with response to reduction of immune suppression, or immunochemotherapy.

Aggressive B-cell lymphomas in patients with myelofibrosis receiving JAK1/2 inhibitor therapy

AbstractInhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1−/− mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

Detection and application of bcl-2/IgH gene translocation and immunoglobulin gene rearrangement in follicular lymphoma

Objective: To evaluate the application of FISH testing of bcl-2/IgH gene translocation and IgH/L gene rearrangement in different stages of follicular lymphoma. Methods: In 32 follicular lymphoma cases, which were collected at Guangdong General Hospital from September 2014 to December 2016, the bcl-2/IgH gene ectopic state was detected by FISH while the IgH/L gene rearrangement was tested using PCR-GeneScan to analyze the relationship between bcl-2/IgH gene translocation, different stages of follicular lymphoma and clonal immunoglobulin (IgH/L) gene rearrangements. Results: From the paraffin sections of all 32 follicular lymphomas, 17 cases showed bcl-2/IgH gene translocation, and the percentages of FL1, FL2 and FL3 translocation were 12/13, 3/5 and 2/14, respectively. Among the 24 cases of IgH/L gene arrangements identified from the total sample, the occurrence rates of FL1, FL2 and FL3 gene arrangement were 7/13, 4/5 and 13/14, respectively. Spearman's rank correlation analysis and χ(2) analysis showed that bcl-2/IgH gene translocation was negatively correlated with follicular lymphoma stage and the association was statistically significant. In more advanced stages of follicular lymphoma, the occurrence of bcl-2/IgH gene translocation tended to decrease with distinct FL1, FL2 and Fl3 gene expression (P<0.05). As IgH/L gene rearrangement in FL3 was higher than that in FL1 and FL2, its detection may be complimentary to FISH test for bcl-2/IgH gene translocation in diagnosing follicular lymphoma. Conclusions: The combined use of FISH and PCR-GeneScan increases the positive rate of follicular lymphoma diagnosis, and this combination is more sensitive than FISH or clonal analysis only to detect the chromosomal abnormality or the gene rearrangement.

Persistent indolent pancolonic marginal zone lymphoma of MALT-type with plasmacytic differentiation – A rare post-transplant lymphoma?

Marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is associated with chronic inflammatory disorders. We present an indolent pancolonic MALT lymphoma occurring in a 39-year-old female with history of autoimmune hepatitis requiring liver transplant in 1997 and ulcerative colitis diagnosed in 2004. Random biopsies from a grossly unremarkable surveillance colonoscopy in 2015 revealed a dense monomorphic plasmacytoid infiltrate causing expansion of lamina propria without significant crypt infiltration or destruction. These cells were positive for CD79a and CD138 and showed lambda restriction; however, CD20, CD43, CD56, HHV8, and EBER were negative. A similar pancolonic infiltrate was identified in all prior colorectal biopsies from 2010 and 2012 upon retrospective review. Subsequent computed tomography of the abdomen revealed no bowel wall thickening nor enlarged lymph nodes. Bone marrow revealed involvement consistent with stage IV disease. Biopsies from 2010 and 2015 demonstrated clonal immunoglobulin gene rearrangement. MYD88 mutation was not detected. The overall features were indicative of MALT lymphoma. Although low-grade B-cell lymphomas are not considered part of the post-transplant lymphoproliferative disorder spectrum, such cases have been reported, and are typically EBV-negative. Patient underwent treatment with pentostatin for her MALT lymphoma reaching a sustained remission despite additional immunosuppression for resurgent hepatic dysfunction. To our knowledge, this is the first reported case of EBV-negative pancolonic MALT lymphoma with plasmacytic differentiation post liver transplant presenting in an indolent, asymptomatic fashion with persistence for greater than five years successfully managed without compromising the patient's liver transplant.

Is Next-Generation Sequencing the way to go for Residual Disease Monitoring in Acute Lymphoblastic Leukemia?

Minimal residual disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL). Since it has been implemented into in treatment stratification strategies, cure rates have improved significantly for all age groups. Real time quantitative (RQ)-PCR of clonal immunoglobulin and T-cell receptor gene rearrangements using allele-specific primers is currently regarded as the gold standard for MRD analysis in ALL, as it is not only highly sensitive and specific but also provides accurate MRD quantification. Following recent advances in next-generation sequencing (NGS), much attention has been devoted to the development of NGS-based MRD assays. This new technique can enhance sensitivity provided that sufficient numbers of cells are analyzed. Recent reports have shown that NGS-MRD also tends to be more specific for relapse prediction than RQ-PCR. In addition, NGS provides information on the physiological B- and T-cell repertoire during and after treatment, which has been shown to be prognostically relevant. However, before implementation of NGS-MRD detection in clinical practice, several issues must be addressed and the whole workflow needs to be standardized, including not only the analytical phase (spike-in calibrators, quality controls) but also the pre-analytical (e.g. sample preparation) and the post-analytical phases (e.g. bioinformatics pipeline, guidelines for correct data interpretation). These topics are currently addressed by a European network, the EuroClonality-NGS Consortium. In conclusion, NGS is a promising tool for MRD detection with the potential to overcome most of the limitations of RQ-PCR and to become the new gold standard for MRD detection in ALL.

International Laboratory Comparison of Methodologies for Determining Minimal Residual Disease (MRD) in Childhood Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

MRD assessment is used for risk stratification, prognosis, and treatment decisions (eg, hematopoietic stem cell transplant (HSCT) recommendation) in adult and childhood ALL. In Ph+ ALL, three distinct assay methods may be used to detect MRD: polymerase chain reaction (PCR) for clonal immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, quantitative RT-PCR (RQ-PCR) measurement of BCR-ABL1 transcripts, and flow cytometry (Flow). If results vary by assay method, different treatment decisions might be made. To assess this possibility, we examined the concordance of MRD levels and corresponding HSCT recommendations across the three methods for measuring MRD in patients (pts) enrolled to CA180372, a phase 2 study of dasatinib added to standard of care chemotherapy in pediatric pts with newly diagnosed Ph+ ALL.CA180372 (Children's Oncology Group (COG) AALL1122) was a collaboration between the COG, EsPhALL (European Intergroup Study on Post Induction Treatment of Ph+ ALL), and Bristol-Myers Squibb. Prior to study initiation, validation and proficiency testing were completed for standardization of methods used to detect MRD and for interpretation of IG/TR and BCR-ABL1 in accordance with guidelines developed by the EuroMRD consortium. All Flow analyses were performed at Johns Hopkins University. All pts received continuous treatment with dasatinib (60 mg/m2/d starting at day 15 of induction therapy) added to successive blocks of the EsPhALL multiagent chemotherapy regimen for a maximum of 2 y. MRD was analyzed 4 times during therapy (induction IA/baseline, end induction IA, start high risk block 1 (HR1), and end HR block 3 (HR3)). Pts were recommended for HSCT if:· Start of HR1 ≥0.05% by IG/TR PCR (or by Flow), less than 3-log reduction in MRD as measured by RQ-PCR for BCR-ABL1 OR· Start of HR1 0.005-0.05% by IG/TR PCR (or by Flow) or any positivity by BCR-ABL1 AND MRD remains positive at any detectable level (providing the assay limit is at least 0.1%) at the end of HR3Clinical decisions were based on a single MRDmethodology, with the hierarchy of IG/TR > BCR-ABL1 > Flow. In this report we evaluated the concordance in HSCT recommendation among all three MRD methods.A total of 106 pts >1 y and <18 y were treated. HSCT eligibility could be determined for 89 (84%) pts by IG/TR, 41 (39%) by BCR-ABL1, and 104 (98%) by Flow. HSCT eligibility could not be determined for 17 (16%) by IG/TR due to technical challenges in assay development, 65 (61%) by BCR-ABL1 due to assay-specific requirements not being met, and 2 (2%) by Flow due to data missing from the database.These data were used to conduct inter-assay HSCT recommendation comparisons (Table). Rates of concordance in HSCT recommendations varied from 79.5-89.7% across methods. Among the discordant HSCT recommendations observed between each pair-wise comparison, 6/9 (IG/TR vs Flow), 2/4 (IG/TR vs BCR-ABL1), and 4/8 (BCR-ABL1 vs Flow) numerical MRD results were within 1-log, but fell on opposite sides of the assay cutoff point; assay-specific protocol criteria resulted in discordant HSCT recommendation in those cases.All pts enrolled to CA180372 were followed by at least one MRD method. HSCT recommendations could be made for most (98%) pts by Flow, while HSCT recommendation by IG/TR could be made for 84% of pts. Only 39% of pts could be recommended for HSCT based on BCR-ABL1 results. The main reason that BCR-ABL1 results were available for fewer pts was the requirement for at least 10,000 copies at baseline in order to assess if a 3-log reduction was achieved. Re-evaluation of assay-specific requirements is warranted. The overall concordance in HSCT recommendation was approx 80-90% across all three MRD methods. Overall, HSCT recommendation would have differed in 14 pts. Discordance between assays may be attributable (in part) to protocol-specific criteria, assay-specific threshold cut points, and/or differences in MRD results over different marrow pulls. Outcome data are not mature enough at this time to know whether pts recommended against HSCT by one method have increased risk for relapse when found to be eligible for HSCT by another method. [Display omitted] DisclosuresBorowitz:Becton Dickinson Biosciences, Medimmune: Research Funding. Geese:BMS: Employment. Healey:Bristol-Myers Squibb: Employment. Gastier-Foster:Bristol-Myers Squibb: Research Funding. Hunger:Bristol-Myers Squibb: Employment; Jazz Pharmaceuticals; Sigma Tau Pharmaceuticals; Erytech: Honoraria.

Clinical and Pathologic Features of Intravascular Large B Cell Lymphoma: A Single Institution Experience

BackgroundIntravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal large B-cell lymphoma characterized by the selective growth of large lymphoma cells within smaller vessel lumina. With less than 500 reported cases, little is understood about this extranodal B-cell lymphoma. This entity is considered widely underdiagnosed due to its nonspecific clinical presentation, often not detected until autopsy. Cytogenetic and immunophenotypic analyses of IVLBCL cases have revealed the presence of numerous chromosomal aberrations and the non-germinal center (post-germinal center) activated B-cell phenotype. Chromosomal abnormalities and clonal immunoglobulin rearrangements have been reported, however, very few cases have been studied.MethodsWe performed an IRB-approved retrospective review of the electronic medical records in our institution from January 2007 to January 2015 and identified five cases of IVLBCL. Morphologic evaluation, immunohistochemical (IHC) stains and PCR analysis for clonality was performed using primers specific for the immunoglobulin heavy (IgH) and light chains (IgK and IgL). IHC was used to characterize the neoplastic cells into germinal center B cell (GCB) and non-GCB phenotypes according to the Hans criteria.ResultsWe identified five patients with the pathologic diagnosis of IVLBCL, three of whom were autopsy cases and two that were living patients referred for consultation. The clinical and pathologic features of these cases are summarized in Table 1. All three autopsy cases had multiorgan involvement by IVLBCL evidenced by CD20 positivity (see Image 1) and with the diagnosis made for the first time post-mortem. The two living patients had an initial presentation of rash and skin involvement. Brain involvement was present in two cases; in the first, which was an autopsy case, it presented in a perivascular morphologic pattern, and in the second case, it presented six months following the intradermal diagnosis of IVLBCL and subsequent R-CHOP treatment, as an extravascular 4.5 cm mass-forming lesion. This patient has been disease-free since receiving autologous stem cell transplant. Of the four cases with available material for IHC, the neoplastic cells were of non-GCB phenotype for three cases and GCB phenotype for one case. Clonal IgH rearrangement was identified in one of the five cases. Clonal light chain rearrangements were identified in two cases.ConclusionsIVLBCL is an elusive diagnosis and more than half of our cases were diagnosed first time post-mortem. Similar to literature described, this is a highly aggressive lymphoma. The two patients in our series with mostly skin involvement appear to have a better prognosis. We also observed IVLBCL may not always present intravascularly, particularly in the brain, where it may present as perivascular or even form extravascular mass lesions. PCR analysis could detect clonal immunoglobulin gene rearrangements. However, assessing heavy chain gene rearrangement alone is not sensitive. PCR of the immunoglobulin light chain, particularly IgK, is a useful technique to increase the sensitivity for detection of clonality for IVLBCL. However, PCR analysis for clonality is inderminate in two cases and this could be attributed to DNA degradation as well as low tumor volume. Future directions that will be explored include performing next generation sequencing on VDJ junctions for these cases.Table 1CaseAgeSexClinical FeaturesOrgan Involvement by HistologyGCB vs. non-GCBPCR181MRespiratory failure, splenomegaly, thrombocytopenia, coagulopathy.MultiorganNon-GCBIgH: Indeterminate IgL: Indeterminate IgK: Indeterminate276FDeceased; presented with encephalopathy. Bone marrow involvement present.MultiorganNon-GCBIgH: Indeterminate IgL: Indeterminate IgK: Clonal361F6 month of progressive rash, weakness, SOB, fever. Presented 6 month later with 4.5 cm left frontal brain lymphoma. Currently in remission.Skin initially. Subsequently brain.GCBIgH: Indeterminate IgL: Indeterminate IgK: Indeterminate456FRemote history of non-Hodgkins lymphoma who subsequently presented with rash.N/AN/AIgH: Indeterminate IgL: Clonal IgK: Clonal584MPresented with 3 months of fevers, chills, new thrombocytopenia.MultiorganNon-GCBIgH: Clonal IgL: Clonal IgK: Clonal [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Predictive Power of Early, Sequential MRD Monitoring in Peripheral Blood and Bone Marrow in Patients with Mantle Cell Lymphoma Following Autologous Stem Cell Transplantation with or without Rituximab Maintenance; Interim Results from the LyMa-MRD Project, Conducted on Behalf of the Lysa Group

INTRODUCTION : Minimal residual disease (MRD) is emerging as an important predictor of clinical outcome in patients with mantle cell lymphoma (MCL). However, its utility in everyday clinical practice remains uncertain since standardized MRD monitoring strategies and response criteria are not yet formally established. To address this question, we conducted the LyMa-MRD project as an ancillary biology study in a prospective phase III trial in MCL (NCI NCT00921414; LyMa Trial).METHODS : The present MRD analysis was performed in a subgroup of randomized patients (n=178) of the 299 MCL patients (<66yrs) enrolled in the LyMa trial (Sept 2008 to Aug 2012). Briefly, all patients were previously untreated and received 4 courses of R-DHAP followed by ASCT using an R-BEAM conditioning regimen (n=257). After ASCT, patients were randomized between observation (obs) (n=120) versus Rituximab maintenance (RM) (n=119). The first planned interim-analysis, with a median follow-up of 40.6 months was presented at ASH 2014 and indicated superior PFS in the RM versus Obs arms (Le Gouill et al. ASH 2014). Sequential MRD monitoring was a predefined secondary objective and was performed throughout. At interim analysis the aim was to determine if peripheral blood (PB) and/or bone marrow (BM) MRD status, pre- and post ASCT, can predict patient outcome in terms of PFS according to RM or Obs. MRD was quantitatively assessed in PB and / or BM after induction, after ASCT by using gold standard EURO-MRD RQ-PCR assays and interpretation guidelines, targeted to clonal immunoglobulin gene rearrangements, in national reference laboratories. MRD data for survival analysis was generated from assays with a minimal sensitivity of at least 10-4. MRD status was assigned according to EURO-MRD interpretation guidelines. MRD negativity at a given time point was defined as absence of RQ-PCR amplification product in a given follow-up sample (minimal assay sensitivity of 10-4).RESULTS: Among the 299 patients enrolled in the LyMa trial, MRD data in PB and / or BM was successfully generated pre- and/or post-ASCT phases, for a total of 178 randomized patients. Of the 61 out of 239 randomized patients without MRD data, 9 are ongoing and other reasons were no MRD target or MRD assay failure. Pre-ASCT MRD in BM and PB was negative in 66% (n=98) and 80% (n=120), of samples, respectively. Post-ASCT, MRD in BM and PB was found negative in 82% (n=122) and 95% (n=162), respectively. MRD status pre-ASCT in either BM or PB was predictive of longer PFS (respectively; p= 0.0451, p= 0.0016). In contrast, PB MRD status, post-ASCT failed to predict PFS while there was a trend for a better PFS for patients achieving MRD negativity in the BM (median PFS: NR vs 43.4 months ; p= 0.07 ). We next investigated patient outcome according to MRD status pre-ASCT and randomization arms. In BM and PB, respectively, 72% and 79% of patients in the obs arm were MRD negative compared to 59% and 80% in the RM arm, respectively. The estimated 3y-PFS for MRD pos/obs, neg/obs, pos/RM, neg/RM patients, according to BM and PB MRD status were: 61,6% (IC95%, 35.4-79.8) vs 83.9% (IC95%, 70.1-91.7) vs 86.2% (IC95%, 67.3-94.6) vs 91.8% (IC95%, 76.3-97.3) (p=0.0110) and 51.8% (IC95%, 24.4-73.6) vs 86.5% (IC95%, 73.5-93.4) vs 80% (IC95%, 50-93.1) vs 92.8% (IC95%, 81.6-97.3) ( p=0.0027), respectively.CONCLUSION : Pre-ASCT MRD status in both BM and PB is an early predictor of PFS in younger MCL patients receiving ASCT. RM provides longer PFS regardless of MRD status pre-ASCT. Early sequential MRD monitoring at the pre-ASCT treatment phase thus offers strong potential for early clinical outcome prediction and MRD-guided, risk-adapted treatment in future MCL trials. Our preliminary results also suggest continued, clinically relevant, direct or indirect Rituximab-mediated anti-tumor activity, in rare residual circulating or 'tissue-resident' MCL cells. DisclosuresThieblemont:St. Louis Hospital, Paris, France: Employment. Casasnovas:Roche: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Consultancy. Ribrag:Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Cutaneous plasmacytosis: a clinicopathologic study of 12 cases in Taiwan revealing heterogeneous underlying causes.

Cutaneous plasmacytosis is clinically characterized by multiple pigmented papules and plaques that occur mainly on the trunk and many plasma cells in the lesional skin. Most of the cases reported have occurred in Japan. Whether cutaneous plasmacytosis is a unique reactive disease or a neoplastic condition has not been established. We collected 12 cases fulfilling the criteria for a diagnosis of cutaneous plasmacytosis at our institution from 2001 to 2013. We analyzed clinicopathologic features and performed immunohistochemical and immunoglobulin gene rearrangement studies. All 12 patients presented with characteristic cutaneous lesions mainly on the trunk and proximal extremities. Many plasma cells were observed in the lesional skin, and the basal layer was hyperpigmented. Four patients had numerous plasma cells in lymph nodes, and one patient also had increased plasma cells in bone marrow. One patient fulfilled the criteria for immunoglobulin G4 (IgG4)-related lymphadenopathy. Elevated serum IgG and IgG4 concentrations were found in seven patients. Monoclonal immunoglobulin gene rearrangements were detected in four patients. The median duration of follow-up was four years. One patient was lost from follow-up. Partial remission of the cutaneous lesions was observed in five patients, and progressive disease was noted in five patients. One patient died of respiratory failure. Our study suggests that the so-called "cutaneous plasmacytosis" has heterogeneous underlying causes with or without systemic involvement and may be associated with clonal immunoglobulin gene rearrangements and IgG4-related lymphadenopathy. No effective treatment is available for this condition.

BLAST: A Confirmatory, Single-Arm, Phase 2 Study of Blinatumomab, a Bispecific T-Cell Engager (BiTE®) Antibody Construct, in Patients with Minimal Residual Disease B-Precursor Acute Lymphoblastic Leukemia (ALL)

▪Introduction: In ALL, minimal residual disease (MRD) is defined as the detection of leukemic cells in bone marrow by PCR or flow cytometry in the presence of hematological complete remission (CR). Patients with persistent/recurrent MRD after induction therapy have a higher risk of relapse than those with no detectable MRD. Effective treatment of patients with MRD aims to avoid hematologic relapse, reduce MRD load, and provide a bridge to subsequent HSCT.Blinatumomab, an investigational BiTE® antibody construct, redirects CD3+ T cells to CD19+ target cells, resulting in serial lysis of CD19+ B cells. In a phase 2 study of blinatumomab in 21 patients with MRD+ ALL in first-line treatment, 80% of evaluable patients achieved a complete MRD response. BLAST, a confirmatory single-arm, phase 2 study evaluated efficacy, safety, and tolerability of blinatumomab in patients with MRD+ ALL in a larger population.Methods: Adults (≥18 years) with B-precursor ALL in hematologic CR (<5% blasts in bone marrow) after ≥3 intensive chemotherapy treatments and with MRD ≥10-3 were eligible. Patients with current CNS pathology or extramedullary disease, previous allogeneic HSCT, or Ph+ ALL eligible for treatment with tyrosine kinase inhibitors were excluded. Blinatumomab 15 µg/m²/day was given by continuous IV infusion for 4 weeks, followed by a 2-week treatment-free period (1 cycle). Responders could receive ≤4 cycles of treatment or undergo HSCT after ≥1 cycle. Patients with hematologic relapse discontinued treatment. Rate of complete MRD response (absence of MRD confirmed with a sensitivity of at least 10-4 after 1 cycle) was the primary endpoint. MRD was assessed by allele-specific quantitative real-time PCR for clonal rearrangements of immunoglobulin or T-cell receptor genes by a central laboratory. Overall survival (OS), relapse-free survival (RFS), duration of complete MRD response, and incidence and severity of adverse events (AEs) were secondary endpoints. Per protocol, OS and RFS will be analyzed after 18 months minimum follow-up.Results: 116 patients enrolled and received blinatumomab. Median (range) age was 45 (18–76) years; 15 (13%) patients were ≥65 years of age. 35% of the patients were treated in second or later remission (Table). As of February 2014, 106 patients had ended treatment: 74 had completed treatment (4 cycles or 1 cycle followed by HSCT) and 32 had discontinued treatment due to AEs, disease relapse, or investigators’ decision; 79 patients were still alive and being followed. Three patients were excluded from the efficacy analysis: one had no central lab assay and two had assays with a sensitivity of 5×10-4. Eighty-eight patients (78% [95% CI, 69%–85%]) had a complete MRD response after 1 cycle of treatment. The lower CI bound exceeded 44% (the null hypothesis response rate), confirming that the study met its primary objective. Two additional patients had a complete MRD response after >1 cycle of blinatumomab; across all cycles the complete MRD response rate was 80%. The rate of complete MRD response did not differ significantly across baseline age, sex, line of treatment, and MRD burden categories (Table). All patients experienced ≥1 AE. AEs occurring in ≥20% of patients included pyrexia (88%), headache (38%), tremor (29%), chills (25%), fatigue (24%), nausea (22%) and vomiting (22%). Serious AEs (SAEs) occurred in 60% of patients; 59% and 27% of patients had grade ≥3 and grade ≥4 AEs, respectively. SAEs occurring in ≥5% of patients were pyrexia (15%), tremor (7%), aphasia (5%), encephalopathy (5%) and overdose (5%). Two fatal AEs occurred on treatment: subdural hemorrhage and atypical pneumonia (the latter was deemed treatment-related).Conclusion: This is the largest prospective trial with an experimental compound in MRD+ ALL.Blinatumomab treatment resulted in complete MRD response across multiple patient demographics including patients in second-line treatment and those with high MRD burden. With a complete MRD response rate of 78%, the study met its primary objective. Among patients with a complete MRD response, 98% had a response within the first treatment cycle. In patients with MRD+ ALL following intensive therapy, rapid MRD response induced by blinatumomab has the potential to improve patient outcomes. [Display omitted] DisclosuresGoekbuget:Amgen Inc.: Consultancy, Honoraria, Research Funding. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Dombret:Amgen Inc.: Honoraria, Research Funding. Bonifacio:Amgen Inc.: Consultancy. Graux:Amgen Inc.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Buss:Amgen Inc.: Reimbursement for participation in clinical studies, Reimbursement for participation in clinical studies Other; BMS: Travel support, Travel support Other; Novartis: Travel support Other; Pfizer: Reimbursements for participation in clinical studies, Reimbursements for participation in clinical studies Other. Bruggemann:Amgen Inc.: Consultancy, Research Funding. Stieglmaier:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Wessels:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Haddad:Amgen Ltd.: Employment. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment, Equity Ownership, Related to blinatumomab Patents & Royalties. Bargou:Amgen Inc.: Consultancy, Honoraria.

Clonal origins of ETV6-RUNX1⁺ acute lymphoblastic leukemia: studies in monozygotic twins.

Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage.

Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma

AimsWe aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL).MethodsClonal rearrangements were...

Extracavitary primary effusion lymphoma: clinical, morphological, phenotypic and cytogenetic characterization using nuclei enrichment technique.

Primary effusion lymphoma (PEL) is a rare form of aggressive B-cell lymphoma, which typically manifests as malignant effusion in the body cavities. However, extracavitary solid variants are also described. The aim of this study was to investigate copy number aberrations in two cases of solid PEL at their first occurrences and relapse by applying a newly developed methodology of tumour nuclei enrichment. Using histological and genetic techniques, a novel protocol for tumour nuclei enrichment by flow sorting and array-comparative genomic hybridization, we characterized two cases of extracavitary PEL, one of which later relapsed as effusion. Both primary tumours were positive for HHV8 and EBV, confined to lymph nodes, and aberrantly expressed CD3, yet displaying clonal immunoglobulin gene rearrangements indicating B-cell origin. Cytogenetic characterization of primary tumours revealed modest number of aberrations, partially overlapping with previously reported affected loci. The effusional relapse in case 1 was cytogenetically related to the primary tumour but showed dramatic increase of chromosomal instability. We for the first time demonstrate a cytogenetic relationship between solid and effusional presentations of PEL. Moreover, we provide an indirect evidence of multiple malignant clones, which gave rise to clonally-related, yet karyotypically different relapsing lymphoma manifestations.

Prognostic Value Of Deep Sequencing Approach For Minimal Residual Disease (MRD) Detection In Multiple Myeloma Patients

Background and AimMost multiple myeloma (MM) patients will experience relapse due to the persistence of residual tumor cells, or MRD. We compared the prognostic value of traditional response criteria with MRD measured by three different methods: a sequencing-based method, termed the LymphoSIGHT™ platform, multiparameter flow cytometry (MFC) and ASO-PCR of Ig genes in a cohort of 133 uniformly-treated MM patients from the Spanish Myeloma Group trials. MethodsBone marrow samples were obtained from 133 patients at diagnostic and post-treatment time points on GEM clinical trials (GEM00 and GEM05). All 133 patients were either in CR or VGPR at the post-treatment time point. Using the sequencing assay, we identified clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, and IGK) genes in diagnostic samples. We then assessed MRD in follow-up samples, analyzing concordance between: sequencing, MFC and ASO-PCR of Ig genes MRD results, and comparing the prognostic value of each method with traditional response criteria. ResultsThe sequencing assay detected the presence of a myeloma-specific gene rearrangement in diagnostic samples from 121 of 133 (91%) patients. We tested MRD in follow-up time points in 109 of the 121 patients.Of the 109 patients, 79 were positive by sequencing at MRD levels of 10-5 or higher and 30 were MRD negative. The Time to Tumor Progression (TTP) and Overall Survival (OS) were significantly longer in the MRD negative group compared with the MRD positive group by sequencing (TTP, median 80 vs. 31 months, p<0.0001; and OS, median not reached vs. 80 months p=0.02) (Figures 1A and 1B). All patients in VGPR, with the exception of 4 cases, were MRD positive by sequencing (92%). Three of the 4 cases with MRD negative and positive immunofixation became immunofixation negative after maintenance treatment. When restricting the analysis to the 61 patients that were in conventional CR (negative immunofixation), 35 of 60 patients were positive by sequencing at MRD levels at 10-5 and higher and 26 were MRD negative. There was a significantly improved TTP in the MRD negative group compared with the MRD positive group (median 131 vs. 35 months, p=0.001) (Figure 1C).A high correlation between MFC and sequencing MRD results was observed (r2=0.87); as well as between ASO-PCR of Ig genes and sequencing (r2=0.89). Discordant results between FCM and sequencing (SEQ) included: 14 cases who were positive by SEQ and negative by FCM, and 5 cases negative by SEQ but positive by FCM; while the remaining cases were concordant: 63 cases were positive by both methods and 16 were negative by both methods. Correlation between ASO-PCR and SEQ was possible in 45 patients: 10 were discordant (9PCR-SEQ+ and 1 PCR+SEQ-) while 35 were concordant (20 PCR+SEQ+, 15 PCR-SEQ-). [Display omitted] ConclusionsThe sequencing applicability for MRD studies in MM is 91%, which is essentially equivalent with that of MFC (96% Rawstron AC et al, JCO 2013). There is a high correlation between MRD levels by sequencing, ASO-PCR and MFC. Based on these results, MRD assessment by sequencing is a useful method for patient risk stratification and can be used to determine molecular CR in MM. Disclosures:Pepin:Sequenta, Inc.: Employment, Equity Ownership. Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Herpesvirus-Associated Central Nervous System Diseases after Allogeneic Hematopoietic Stem Cell Transplantation

Herpesvirus infections of the central nervous system (CNS) are associated with encephalitis/myelitis and lymphoproliferative diseases in immunocompromised individuals. As of now, data of herpesvirus-associated CNS diseases in transplant recipients is limited. Hence, in this prospective study, we investigated the incidence of herpesvirus-associated CNS diseases and explored the diagnosis of these diseases in 281 allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Herpesvirus-DNA and cerebrospinal fluid (CSF) cells were sampled from 58 recipients with herpesvirus-associated diseases or with unexplainable CNS manifestations. Results showed that 23 patients were diagnosed as herpesvirus-associated CNS diseases, including 15 Epstein-Barr virus (EBV)-associated diseases (4 encephalitis and 11 lymphoproliferative diseases), 5 herpes simplex virus type 1 encephalitis, 2 cytomegalovirus encephalitis/myelitis and 1 varicella zoster virus encephalitis. The median time of diseases onset was 65 (range 22-542) days post-transplantation. The 3-year cumulative incidence of herpesvirus-associated encephalitis/myelitis and post-transplant lymphoproliferative disorder (PTLD) was 6.3% ±1.9% and 4.1% ±1.2%, respectively. Of the evaluable cases, CSF cells mainly consisted of CD19+CD20+ B cells (7/11) and had clonal rearrangement of immunoglobulin genes (3/11) in patients with CNS-PTLD. On the contrary, in patients with encephalitis/myelitis, CSF cells were comprised of different cell populations and none of the gene rearrangement was detected. Herpesvirus-associated CNS diseases are common in the early stages of allo-HSCT, wherein EBV is the most frequent causative virus. The immunophenotypic and clonal analysis of CSF cells might be helpful in the differential diagnosis between encephalitis and lymphoproliferative diseases.

Peripheral T-cell Lymphomas of Follicular T-Helper Cell Derivation With Hodgkin/Reed-Sternberg Cells of B-cell Lineage

Peripheral T-cell lymphomas (PTCLs) are functionally and morphologically complex. Epstein-Barr virus (EBV)-positive B cells have been reported in angioimmunoblastic T-cell lymphoma (AITL) and other PTCLs and may mimic Hodgkin/Reed-Sternberg (HRS) cells, but EBV-negative HRS-like B cells have not been described. We wished to assess the nature of the PTCL associated with HRS-like cells and to determine whether EBV-negative HRS-like cells may be seen. We identified 57 PTCL cases reported as containing HRS-like cells. These included 32 AITL, 19 PTCL, not otherwise specified (NOS), 3 PTCL-NOS, follicular variant, 1 PTCL-NOS, T-zone variant, and 2 adult T-cell leukemia/lymphoma cases. All patients were adults with a median age of 63 and presented with lymphadenopathy. The male:female ratio was 31:26 (1.2:1). Clonal TRG rearrangement was detected in 46/53 cases. Six of 38 cases had a concomitant clonal immunoglobulin gene rearrangement. In 52/57 cases the HRS cells were positive for EBV. Five cases, 3 classified as AITL and 2 as PTCL-NOS, follicular variant, contained HRS-like cells negative for EBV. All PTCLs with EBV-negative HRS cells had a T follicular helper cell immunophenotype. The neoplastic T cells expressed CD3, CD4, and PD-1 and formed rosettes around the HRS-like cells. The HRS-like cells were positive for CD20 (variable intensity), PAX5, CD30, and CD15 (4/5). We conclude that both EBV-positive and EBV-negative HRS-like B cells may occur in the background of PTCL; caution is needed to avoid misdiagnosis as classical Hodgkin lymphoma. The close interaction between the HRS-like cells and the rosetting PD-1-positive T cells suggests a possible pathogenetic role in this phenomenon and provides new insights into the abnormal B-cell proliferations that occur in the context of TFH malignancies.

Prognostic value of deep sequencing method for minimal residual disease (MRD) detection in multiple myeloma.

8511 Background: Most multiple myeloma (MM) patients will relapse due to persistence of residual tumor cells, or MRD. We compared the prognostic value of traditional response criteria and MRD measurement by a sequencing-based method, LymphoSIGHT, and multiparameter flow cytometry (MFC) in a cohort of 68 uniformly-treated MM patients from the Spanish Myeloma Group trials. Methods: Bone marrow samples were obtained from 68 patients at diagnostic and post-treatment time points on GEM clinical trials (GEM00 and GEM05). All patients were in CR or VGPR at the post-treatment time point. Using sequencing, we identified clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, and IGK) genes in diagnostic samples. We assessed MRD in follow-up samples, analyzed concordance between sequencing and MFC MRD results, and compared the prognostic value of each method with traditional response criteria. Results: The sequencing assay detected a myeloma-specific gene rearrangement in diagnostic samples from 59 of 68 (87%) patients. We tested MRD in follow-up time points in 56 of the 59 patients. We observed high correlation between MFC and sequencing MRD results (r2=0.86), with MFC underestimating the myeloma burden (Slope=0.4). Of the 56 patients, 45 were positive by sequencing at MRD levels of 10-5 or higher and 11 were MRD negative. There was significantly improved overall survival (OS) in the MRD negative group versus the MRD positive group (median not reached vs. 86 mos, p=0.026). Similar differences were found in progression free survival. When limiting the analysis to the 35 patients in conventional CR, 24 of 35 patients were positive by sequencing at MRD levels at 10-5 and higher and 10 were MRD negative. There was significantly improved OS in the MRD negative group versus the MRD positive group (median not reached vs. 80.92 mos, p=0.041). Conclusions: Our data shows high correlation between MFC and sequencing MRD levels in MM patients. For patients in CR by traditional response criteria, the presence or absence of MRD by sequencing delineated 2 groups of patients with significantly different OS. MRD negativity by sequencing may be a better prognostic indicator than CR by traditional response criteria.