IgH Gene Research Articles

Composite angioimmunoblastic T-cell and diffuse large B-cell lymphoma

A58-year-old female smoker with a history of hypertension presented to the emergency department with a recurrent episode of worsening shortness of breath over five days. Her exam was significant for sinus tachycardia, cervical lymphadenopathy, and a firm left supraclavicular node. Laboratory values demonstrated a normal white cell count and differential, normocytic anemia, thrombocytopenia, and a lactate dehydrogenase of 803 IU/L. She was HIV negative. Computerized tomography (CT) scan of the chest/abdomen/pelvis demonstrated extensive bilateral cervical, supraclavicular and axillary adenopathy, mediastinal and hilar lymphadenopathy with infiltrate and consolidation of the right lower and posterior upper lobe and encasement of the right pulmonary artery, as well as splenomegaly with paraaortic and retroperitoneal adenopathy. An echocardiogram revealed a normal left ventricle and a severe pericardial effusion. She underwent a pericardial window and an excisional supraclavicular lymph node biopsy, which revealed T-cells with features of angioimmunoblastic T-cell lymphoma (AITL) and large atypical B-cells consistent with diffuse large Bcell lymphoma (DLBCL) (Figure 1A). Destruction of the lymph node architecture was noted, with many medium-sized atypical lymphocytes with irregular nuclei and moderate amounts of pale cytoplasm. In addition, scattered and focally increased large atypical lymphocytes with prominent nucleoli were seen. Blood vessels were increased. Immunostains demonstrated atypical T-cells expressing CD3 (Figure 1B), CD4 (Figure 1C), CD5, CD2, CD7, CD4, CD10, PD-1 (Figure 1D), as well as clustered large B-cells positive for expression of CD20, Bcl-6 and BOB-1 (Figure 1E). Polymerase chain reaction (PCR) testing revealed a clonal TCR gamma gene rearrangement, as well as a clonal IgH gene rearrangement. Epstein-Barr virus-encoded RNA (EBER) was negative by in situ hybridization (ISH). Bone marrow biopsy and CSF analysis were negative for lymphoid or plasma cell infiltrates. The patient was initiated on R-CHOEP (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone) and completed three cycles of chemotherapy. Her CT scan after three cycles was consistent with a partial response, with significantly diminished bilateral axillary lymphadenopathy, improved mediastinal, perihilar and right lower lobe infiltrates, decreased splenomegaly, and upper abdominal and retroperitoneal adenopathy. The patient eventually had progression of her disease during the course of subsequent R-CHOEP cycles. She then progressed through two cycles of gemcitabine-carboplatin and one course of romidepsin. Her performance status deteriorated, not enabling any further treatment and the patient finally expired, approximately 24 months after initial diagnosis. This case illustrates a rare hematologic malignancy presenting with a dual primary lymphoma population. The treatment of composite lymphoma with simultaneous Band T-cell components is challenging and the less favorable prognosis component determines

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known.To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against.We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.

The evolution and development of the antibody repertoire.

The evolution and development of the antibody repertoire.

Epstein-Barr virus-positive diffuse large B-cell lymphoma in children: a disease reminiscent of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly.

Pediatric Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV+ DLBCL) is a rare disease in nonimmunocompromised hosts. In a review of 231 cases of malignant lymphoma (87 Hodgkin lymphoma and 144 non-Hodgkin lymphoma) occurring in Iraqi children, 7 cases (5% of NHLs) were classified as EBV+ DLBCL. Six children presented with nodal disease, and 1 presented with extranodal localization (bone). In all cases, the disease was at an advanced clinical stage (III/IV). Evidence of immunodeficiency (Evans syndrome and selective IgA deficiency) was observed in a single case. Two cases were "monomorphic" with immunoblastic histology, and 5 cases were "polymorphic" with histologic aspects reminiscent of nodular lymphocyte-predominant Hodgkin lymphoma (2 cases) and of CD30+ classical Hodgkin lymphoma (3 cases). In all cases, tumor cells were EBV infected (EBER+/LMP-1+), were medium-large B-cells (CD20+/CD79a+/PAX-5+/BOB-1+/OCT-2+) of non-germinal center (non-GC) origin (CD10-/MUM-1+), and had high proliferative activity (50%-70%). Chromosomal translocations involving BCL2, MYC, and IGH genes were not observed. IGH monoclonality could be demonstrated in 3 of 3 investigated cases. Six cases of EBV-negative DLBCL (4% of NHL) were present in the same series. All had monomorphic histology with centroblastic/immunoblastic morphology; 3 cases were of GC type and 3 of non-GC type. Our findings indicate that in Iraq, DLBCLs are 9% of NHLs. Moreover, 2 different types of the disease do exist; the EBV-positive cases, with strong histologic and immunohistochemical resemblance with EBV+ DLBCL of the elderly, and the EBV-negative cases, which are similar to the pediatric DLBCL usually observed in Western populations.

Primary cutaneous diffuse large B-cell lymphoma, leg type: a study of clinicopathology, immunophenotype and gene rearrangement

To study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL). Seven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study. All the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up. PCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.

Characterization of the torafugu (Takifugu rubripes) immunoglobulin heavy chain gene locus.

In this study, we investigated the immunoglobulin heavy (IGH) gene locus of torafugu (Takifugu rubripes) from publicly available assembly sequences and presented an annotated locus map, including the IGHV genes, pseudogenes, and IGHC genes. Three new IGHV gene families (IGHV3-IGHV5) were discovered. We observed the interspersion of IGHV1 and IGHV2 family members and that they often intermingled with each other, while other family members were further interspersed. Conservation of the promoter and recombination signal sequences (RSS) was observed in a family-specific manner. In addition to known variable region genes present on chromosome 5 (current torafugu genome assembly), we found 34 additional IGHV genes on scaffold 287 and three novel potentially functional IGHD genes on scaffold 483. In total, the variable region of the torafugu IGH locus consists of at least 48 IGHV genes, seven IGHD genes, and six IGHJ genes. IGHC genes have also been mapped in this study, with three genes encoding immunoglobulin classes: IgT, IgM, and IgD. We confirmed the expression of newly identified IGHV3 family sequences in the spleen and kidney of adult torafugu and found a favorable IGHV segment usage by IgM and IgT. Possible structural variation in the IGHδ locus was observed based on the current torafugu assembly. The complete characterization of the torafugu IGH locus will facilitate detailed studies of large-scale mechanisms associated with the recombination of the variable region genes and will offer insights into the genetic basis of the potential diversity in the antibody response observed in torafugu.

Significance of detecting IgH and TCRγ gene rearrangements in patients with hemopoietic maligancies by real-time quantitative PCR.

The aim of our study was to investigate the association of IgH and TCRγ gene rearrangements in hematological malignancies with the disease and clinical application. IgH and TCRγ gene rearrangements were determined in 69 paraffin and bone marrow specimens with SYBR Green I fluorescent dye and RQ-PCR method, including 21 paraffin-embedded tissues of the onset cases and 48 bone marrow samples, representing 15 ALL and 25 AML cases. After chemotherapy, 8 cases were NHL; the 10 cases of the negative control group were healthy people. Among the ALL cases, the IgH rearrangement occurred in 80.0%, the TCRγ rearrangement in 46.7%, and both gene rearrangements in 46.7%. Among the AML cases, the IgH rearrangement occurred in 72.0%, the TCRγ rearrangement in 68.0%, and both gene rearrangements in 60.0%. In the lymphoma cases, the IgH rearrangement occurred in 93.1%, the TCRγ rearrangement in 51.7%, and both gene rearrangements in 44.8%. In the negative control group, the 10 cases were all negative. There was the phenomenon of "sequence-non-fidelity" in the hematologic malignancies; the detection rate of both genes was much higher than that of the single gene. The application of the RQ-PCR method in the detection of IgH and TCRγ gene rearrangements in hematologic malignancies has important clinical significance in MRD monitoring.

CDKN2 Gene Deletion as Poor Prognosis Predictor Involved in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients.

Deletion of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is well known in many hematologic malignancies, but only few reports have investigated this deletion effect on clinical prognosis. This study performed analysis of the CDKN2 deletion in 215 adult B- lineage acute lymphoblastic leukemia (B-ALL) patients, and related cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; Mixed Lineage Leukemia (MLL) rearrangement; MYC, Immunoglobulin heavy locus (IGH) translocation). The prevalence of CDKN2 deletions in all study populations was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells (WBC) count, BM blast percentage, extra infiltration and induction complete remission (CR) rate. Analysis in relapse patients revealed that the distribution of CDKN2 deletion is higher in relapse patients (44.6%) than all patients (28.4%, P=0.006). Deletion of CDKN2 was significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free-survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.002); Also, CDKN2 deletion was strongly associated with IGH translocation (P=0.021); and had an adverse effect on patients with BCR-ABL fusion gene or with MLL rearrangement. Patients with CDKN2 gene deletion benefited from allogenic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was poor prognostic marker in long-term outcomes.

IgG-related disease in a patient with prior pancreatic cancer and splenopancreatectomy

IgG4-related disease is a newly recognised fibro-inflammatory condition characterised by a tendency to form tumefactive lesions in multiple sites; characteristic histopathological appearance with dense lymphoplasmacytic infiltrate, storiform fibrosis and obliterative phlebitis; and, often but not always, elevated serum IgG4 concentrations. We present a case of 69-year-old male with a past history of treated, recurrent mucinous carcinoma of the pancreas who presented with retroperitoneal, axillary and mediastinal lymphadenopathy and a renal mass. Axillary node excisional biopsy showed reactive changes with marked interfollicular increase in IgG4+ plasma cells, along with scattered non-necrotising granulomata. Core biopsies of the renal mass showed polymorphous infiltrate of lymphocytes and predominant plasma cells that stained IgG4. IgH and TCR gene rearrangements were polyclonal. Full blood count was normal other than eosinophilia. Serum IgG4 and IgE were significantly elevated at 11.70 g/L and 258 IU/mL respectively; CRP, ESR, IL-2 and IL-6 were normal. An infectious screen was also negative. The patient was diagnosed with IgG4-related disease and treated, with an excellent response, with prednisolone 0.6 mg/kg. IgG4-related disease is a newly recognised fibro-inflammatory condition characterised by a tendency to form tumefactive lesions in multiple sites; characteristic histopathological appearance with dense lymphoplasmacytic infiltrate, storiform fibrosis and obliterative phlebitis; and, often but not always, elevated serum IgG4 concentrations. We present a case of 69-year-old male with a past history of treated, recurrent mucinous carcinoma of the pancreas who presented with retroperitoneal, axillary and mediastinal lymphadenopathy and a renal mass. Axillary node excisional biopsy showed reactive changes with marked interfollicular increase in IgG4+ plasma cells, along with scattered non-necrotising granulomata. Core biopsies of the renal mass showed polymorphous infiltrate of lymphocytes and predominant plasma cells that stained IgG4. IgH and TCR gene rearrangements were polyclonal. Full blood count was normal other than eosinophilia. Serum IgG4 and IgE were significantly elevated at 11.70 g/L and 258 IU/mL respectively; CRP, ESR, IL-2 and IL-6 were normal. An infectious screen was also negative. The patient was diagnosed with IgG4-related disease and treated, with an excellent response, with prednisolone 0.6 mg/kg.

Peripheral T Cell Non-Hodgkin's Lymphoma following Treatment of Hodgkin's Lymphoma.

Previous reports have suggested that non-Hodgkin's lymphoma (NHL) is more likely to develop in patients with Hodgkin lymphoma (HL) compared to the general population. These two can occur synchronously or metachronously. We report here on a case of nodular sclerosis classical HL and T cell NHL that occurred in a patient metachronously. Peripheral T cell lymphoma (PTCL) of the patient was found about 2 years after treatment of classical HL. When the patient was diagnosed with HL, biopsy revealed typical RS cells, presenting positive for CD30 and CD15 and negative for CD79a and CD3 in immunohistochemistry. And PCR analysis showed IgH gene rearrangement; however, T cell receptor gene rearrangement and Epstein-Barr virus (EBV) were not detected on PCR analysis. After 2 years of treatment of HL, colonoscopic biopsy and lymph node biopsy showed CD3 positive atypical cells intermixed with small reactive lymphoid cells and plasma cells, indicating T cell lymphoma. PCR analysis demonstrated T cell receptor gene rearrangement and did not detect EBV. Although it is rare, synchronous or metachronous HL and NHL may occur. Therefore, we may need to ensure pathological confirmation, especially in case of lymphoma that did not respond to chemotherapy.

Submicroscopic deletions of immunoglobulin heavy chain gene (IGH) in precursor B lymphoblastic leukemia with IGH rearrangements.

Translocations leading to fusions between the immunoglobulin heavy chain gene (IGH) and various partner genes have been reported in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, submicroscopic deletions within IGH in B-ALL have not been rigorously assessed. In this study, we investigated characteristics of IGH submicroscopic deletions, by FISH, in B-ALL with IGH rearrangements. FISH was performed by using commercially available IGH dual-color break-apart rearrangement probes (Abbott/Vysis, Downers Grove, IL, USA; Kreatech, Amsterdam, Netherlands). The study group included seven B-ALL patients with IGH rearrangements, observed by FISH. Among them, two exhibited deletion of the 5' variable region of IGH by FISH. The B-ALL in these two patients included two kinds of abnormal cells; one had an IGH rearrangement without any IGH submicroscopic deletion, while the other had an IGH submicroscopic deletion, which showed that one normal fusion signal and one 3' IGH signal were detected. Thus, submicroscopic deletion of the IGH 5' variable region may have occurred in either the native or rearranged chromosome 14. These findings indicate that B-ALL with IGH rearrangements may be accompanied by submicroscopic deletions of the IGH 5' variable region, which can be detected by FISH. The clinical significance of such deletions is unclear, but the loss of part of the IGH gene in B-ALL warrants further study.

A Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Background:Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM).Methods:To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure.Results:The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time.Conclusions:IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Favorable Outcomes for Older Adolescents and Young Adults (AYA) with Acute Lymphoblastic Leukemia (ALL): Early Results of U.S. Intergroup Trial C10403

▪Background: Retrospective analyses have demonstrated significantly improved survival for AYA ALL patients (pts) aged 16-21 years (yrs) when treated on pediatric versus adult U.S. NCI Cooperative group regimens where 2-yr event-free survivals (EFS) have been only 35-40%. The purpose of C10403, a large prospective US intergroup trial, was to evaluate the feasibility and effectiveness [with EFS as a primary endpoint), of treating AYA ALL pts (ages 16-39 yrs) using the standard arm of the successful Children’s Oncology Group regimen (COG AALL0232) .Methods: Newly diagnosed AYA patients with B-precursor (B-ALL) or T-precursor (T-ALL) ALL were eligible to enroll on C10403. Burkitt type and Ph+ ALL were excluded. The regimen was identical to the Capizzi methotrexate arm of COG AALL0232 (Larsen E. JCO 2011; 29 suppl:3) and consisted of four intensive courses: remission induction, remission consolidation, interim maintenance, delayed intensification, and prolonged maintenance therapy. Pts with M2 marrow response (>5% but < 25% lymphoblasts) after remission induction received an extended remission induction course of therapy. Events were defined as induction failure (M3 [³25% blasts] day 29 of induction or M2 day 43 of extended induction), death, relapse, or second malignancy. Key correlative science studies in a subset of the total accrued population included assessment of Minimal Residual Disease (MRD) using quantitative real-time PCR of clonal IgH or TCR gene rearrangements as well as Low Density Microarray (LDA) assays designed to detect a previously validated gene expression profile (Harvey, R; ASH 2013, abstract 826) which can prospectively identify ALL pts with Ph-like (BCR-ABL1-like) ALL.Results: 318 pts were enrolled on C10403 from 11/2007 to 8/2012; 22 withdrew prior to therapy. Of 296 evaluable pts, the median age at diagnosis was 24 yrs (range: 17 to 39): 25% were 17-20 yrs, 53% were 21-29 yrs, and 22% were 30-39 yrs. The majority had B-ALL (76%) and were male ( 61%). Approximately 25% were non-Caucasian and 15% were Hispanic or Latino. 32% of pts were obese (BMI³30). There were 5 (2%) treatment-related deaths during protocol therapy: liver failure (n=2, both during induction), infection (1 in induction, 1 in consolidation), and ventricular arrhythmia (1 in induction). Overall, treatment toxicities were similar to those reported in the standard arm of COG AALL0232, with an increased thrombosis and early hyperbilirubinemia for C10403 pts, as reported previously (Advani A, ASH 2013, abstract 303). To date, 70 deaths have been reported and 87 pts remain on protocol therapy. With a median follow-up of 28 months for surviving pts, 105 events have been observed. The median EFS overall is 59.4 months (95% CI: 38.4 to NR) and the 2-yr EFS rate overall is 66% (95% CI: 60 – 72%) [Fig 1a] with similar 2-yr EFS rates for B and T- ALL pts (65%, and 68%). The 2-yr OS rate is 78% (95% CI: 72 – 83%)[Fig 1b], which is similar for B (78%, 95% CI: 72 – 84%) and T-ALL, (80%, 95% CI: 70 – 91%). These results allow rejection of the null hypothesis of this Phase II trial that the true median EFS is, at most, 32 months. In multivariable analysis of presenting clinical features, age > 20 years and initial WBC count ³ 30k/microliter were significantly associated with worse EFS and OS. Presence of MRD at day 28 following initiation of induction therapy and presence of a Ph-like gene expression signature were significantly associated with both worse EFS and OS. Notably, absence of detectable MRD noted in 22/58 [38%] evaluable pts at day 28 of induction was associated with 100% EFS (p=0.0006). The Ph-like like signature was detected in 28% of pts tested on C10403 and the 2 yr EFS for these patients was only 52%, compared to 81% for those without Ph-like disease (p = 0.04).Conclusions: This large prospective US adult intergroup trial (C10403) for pts 16-39 years old employing an intensive pediatric regimen demonstrates a significant improvement (compared to historical controls) in AYA EFS and OS and validates this approach for treatment of AYA with ALL by adult hematologists. The improved clinical outcomes and the predictive value of the correlative studies in this trial lay the foundation for the design of future trials, where incorporation of novel agents to eradicate MRD, and/or use of tyrosine kinase inhibitors to target the frequently detected Ph-like ALL in AYA pts may further improve survival for young adults with ALL. [Display omitted] [Display omitted] DisclosuresStock:Sigma-Tau: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Advani:Sigma Tau: Membership on an entity’s Board of Directors or advisory committees; Jazz: Membership on an entity’s Board of Directors or advisory committees. Liedtke:Onyx: Membership on an entity’s Board of Directors or advisory committees. Larson:Novartis: Consultancy, Research Funding.

Detection of B-Cell Clonality in Bone Marrow Is Independent Predictor of Outcome in De Novo Diffuse Large B-Cell Lymphoma Patients Treated with High-Dose Chemotherapy

Introduction: Detection of bone marrow involvement (BM) is a factor of poor prognosis for diffuse large B-cell lymphoma (DLBCL), since DLBCL BM is characterized by aggressive course and low response level to standard chemotherapy (CT), and 5-year overall survival (OS) rate for this group is less than 30% after ÑHOP-like CT. Intensification of therapy in this group of patients can improve the results, except patients with bone marrow involvement at diagnosis. Bone marrow involvement is a poor prognostic factor for patients treated with high-dose chemotherapy.Bone marrow involvement in DLBCL by histology is detected in 10-30% patients. The importance of B-cell clonality examination in bone marrow as prognostic and staging factor has not been described in the literature.Aim: To evaluate the significance of the immunoglobulin heavy chain gene rearrangement analysis performed by PCR for identification of the bone marrow involvement frequency and its value for staging and prognosis in patients with de novo DLBCL treated with high-dose chemotherapy (HDC).Patients and methods: We performed a pilot prospective trial, including 175 consecutive adult patients (median age 45 years, range 18–67) with newly diagnosed DLBCL who were enrolled in HDC protocol (mNHL-BFM-90 program or scheme R-EPOCH/R-HMA) since June 2007 till July 2014. Their clinical characteristics included such factors as IPI and phenotype DLBCL by immunohistochemical study. Out of the 175 patients, 85 had a GCB phenotype and 90 - non-GCB; 36 patients (20%) had low IPI risk, 40 patients (23%) had low-intermediate, 38 patients (22%) had high-intermediate, 61 patients (35%) had high risk.B-cell clonality was evaluated using PCR amplification by IGH (FR1, FR2, FR3) and IGK (Vκ-Jκ, Vκ/intron-Kde) gene rearrangements with multiplex BIOMED-2 primer sets and subsequent fragment analysis using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).In 105 of 175 patients the study by BIOMED-2 multiplex polymerase chain reaction protocol of B-cell clonality of bone marrow was fulfilled. Statistical analysis was done using JMP ver. 10.0 (SAS, Cary, NC).Results: Histological and immunohistochemical studies validated bone marrow involvement in 19 patients (10.8%), including: concordant in - 12 patients (63%), and discordant - in 7 patients (37%). In 14 of these patients the B-cell clonality detection was performed and confirmed immunoglobulin (IG) heavy chain gene rearrangement in all cases. In 26 out of 105 patients, bone marrow involvement (24.7%) was revealed: in 14 patients bone marrow involvement was identified by histological examination and in 12 patients only by clonality of bone marrow. 12 patients with only B-cell clonality in bone marrow were classified by IPI: 4 (33%) patients had high IPI, 6 (50%) patients - high-intermediate IPI and low-intermediate IPI - 2 (17%) patients. So, in 17% patients had to change the risk group according to bone marrow involvement. Thus, 31 patients with bone marrow involvement signs were identified.Univariate analysis of the entire cohort (n = 175) revealed that both IPI and phenotype were not of prognostic significance (p > 0.05). In multivariate analysis, detection of B-cell clonality in bone marrow was an independent predictor of OS and relapse-free survival (RFS) (p < 0.005). (Fig.1-2). OS, RFS in patients with histological detection bone marrow involvement and with only B-cell clonality in the bone marrow did not differ.Conclusion:Detection of B-cell clonality in the bone marrow seems to be an independent predictor of outcome in de novo DLBCL pts, treated with high-dose chemotherapy, while IPI and phenotype DLBCL cannot be considered as risk factors for this group of pts. It seems to be reasonable to include the detection of B-cell clonality in the bone marrow in primary diagnostics of the DLBCL for staging and predicting response in HDC. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia Patients Harbor Very Low Frequency Stereotypic Immunoglobulin CDR3 Sequences

Introduction : Recent studies have shown that leukemic clones in approximately 10-20% of CLL patients have stereotypic IgH gene sequences (Stamatopoulos et al., Blood 2007). These stereotypic IgH sequences are characterized by closely homologous complementarity-determining regions 3 (CDR3) sequences. The presence of these sequences supports the model of antigen-mediated disease pathogenesis in CLL. While previous studies have focused on the dominant CLL clone present at diagnosis, recent advances in next-generation sequencing enable analysis of the full IgH repertoire. Here we assessed whether stereotypic IgH sequences are present at very low frequency among the total IgH repertoire in diagnostic and follow-up samples from 36 CLL patients.Methods:We utilized the LymphoSIGHT method to sequence the IgH repertoire in 36 CLL patients and 185 patients with non-CLL lymphoid neoplasms. Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments in diagnostic and follow-up samples. Amplified products were sequenced, and CLL-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. We selected a set of 289 stereotypic IgH CDR3 sequences that were previously curated (Stamatopoulos et al., Blood 2007; Messmer et al., Leukemia Res 2008). We assessed whether the stereotypic CDR3 regions were present in the IgH repertoire of both CLL and non-CLL patients using sequence alignment tools.Results: 154,318 IgH clonotypes were observed in the 36 CLL patients, while we identified 8,248,032 IgH clonotypes from 185 patients with non-CLL lymphoid neoplasms. Specifically, we tested 14 mantle cell lymphoma (MCL), 19 hodgkin disease (HD), and 28 acute lymphoblastic leukemia (ALL) patients as well as three cohorts of multiple myeloma (MM) with 26, 30, and 68 patients. We assessed whether each of the 289 CDR3 stereotypes were present in the IgH repertoire of the CLL or non-CLL patients. Twenty two of the stereotypic IgH sequences were observed in any patient: 2 in CLL patients and 20 in the non-CLL patients. This is not surprising given that non-CLL patients have over 50 times the total number of clones compared to the CLL group. One of the stereotypic IgH clonotypes was detected in one CLL patient (p-value= 0.018; not significant with Bonferroni correction). The other stereotypic IgH clonotype was present in two CLL patients but not in any of the non-CLL lymphoid neoplasm cohorts (p=0.00034; 0.0074 Bonferroni corrected). In contrast, we tested for association between each of the 20 stereotypic sequences and each of the 6 control cohorts (3 MM, 1 MCL, 1 HD, and 1 ALL), and only two significant associations were observed (p=0.045 and p=0.01; neither were significant with Bonferroni correction). This underscores that the only significant association was seen in the tested CLL cohort. Of note, both of these stereotypic IgH clonotypes were present at very low frequency in the IgH repertoire (<10-4) (Figure 1). The two stereotypic IgH sequences identified in CLL patients were unmutated and match the previously published V, D, and J segments. Specifically, the stereotypic sequence ARHRLGYCSSTSCYYYYYGMDVWGQGTT present in one CLL patient has IGHV4-39, IGHJ6, and IGHD2-2 and the stereotypic sequence CARDSPLVVPAAIFYYYYGMDVW present in two CLL patients has IGHV3-48, IGHJ6, IGHD2-2. In contrast the vast majority of the hits in the non-CLL samples have V, D, and J sequences that are different from the published segments for the specific stereotypic sequence.Conclusion : Our results demonstrate that CLL patients harbor stereotypic IgH sequences. These sequences were not the dominant, high-frequency clonotypes identified at diagnosis in the CLL patients and were absent in a much larger set of clones derived from a cohort of non-CLL cancer patients. These results suggest that a subset of CLL patients may possess an antigen environment that selects for multiple B cells with stereotypic IgH sequences at low frequencies. This selection process may predispose patients to CLL that develops later via accumulation of multiple genetic hits in a pre-leukemic cell containing the stereotypic IgH sequence driving it to high-frequency. [Display omitted] DisclosuresKlinger:Sequenta, Inc.: Employment, Equity Ownership. Hervold:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

High-Throughput Molecular Profiling of Multiple Myeloma (MM) Clonotypic CD19+ B Cells Highlights Pathways Potentially Involved in the Disease Endurance

Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics.Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence.Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively.Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells.By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed.SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A).Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (<-2FC>2; FDR=0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed in every analyzed cell compartment; on the contrary, Hedgehog pathway was overall down regulated.Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells.Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply.Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Late-Relapse Diffuse Large B-Cell Lymphoma Frequently Represents Recurrence of the Original Disease, and Demonstrates Evidence of Superimposed Clonal Heterogeneity and Clonal Evolution

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. While most relapses occur within 2 years, a small proportion of patients present with late relapse (LR) after 5 years. As there are very few studies addressing the pathobiology of LR-DLBCL, the aim of this study is to further characterize the clinical, pathologic and molecular features of these neoplasms.METHODS: A retrospective analysis of all patients with DLBCL treated at Johns Hopkins Hospital between 1984 and 2013 was performed. Patients with low-grade lymphoma at any time-point were excluded. Disease-free intervals (DFI) of 5 years or greater were designated as LR. Five paired diagnostic (D) and relapse (R) samples were available for further studies. DNA was extracted from formalin fixed paraffin embedded tissue. IGH gene rearrangement status was determined by PCR. SNP microarray was performed, and copy number variations (CNV) were defined as loss or gain of signal over at least 2 megabases. Targeted next generation sequencing (NGS) using a cancer hotspot panel was also performed. Variant calls were generated using Torrent variant caller and a laboratory-developed analysis pipeline.RESULTS: One hundred thirty-three patients with relapsed DLBCL were identified. Forty-three (32.3%) patients were diagnosed in the pre-rituximab era. One hundred fourteen (85.7%) patients had early relapse (ER) with 99 (74.4%) patients recurring within 2 years. Nineteen (14.3%) patients had LR (mean 7.9 years; median 7.3 years; up to 15.6 years). There were no significant differences in age at diagnosis, race, staging marrow status, or overall survival (OS) in ER versus LR patients. Extra-nodal presentation at diagnosis (89.5% vs. 65.8%; p = 0.04) and extra-nodal-only disease over time (73.7% vs. 48.2%; p = 0.04) were more common in LR cases. Both groups had similar rates of recurring at a different site from the original disease (79.3% vs. 89.5%; p = 0.30).Table 1Molecular profile of paired D and R DLBCLPatientIGH clonality comparison (D vs. R)Clonal Heterogeneity (D / R)Total CNVs (D / R)Shared CNVsUnique CNVs (% of D / % of R)1Same+ / +24 / 211537.5 / 28.62Same+ / +15 / 321126.7 / 65.63Same+ / +32 / 15778.1 / 53.342 in D / 1 persists in R+ / +8 / 20187.5 / 95.05Different- / +4 / 50100 / 100The average DFI was 7.1 years in the 5 LR patients selected for additional studies. IGH gene rearrangement analysis demonstrated identical D and R IGH clones in 3 cases (Table 1). Patient 4 showed 2 rearranged alleles at D with only 1 persisting at R. Patient 5 had lymphomas with unique IGH rearrangements. SNP microarray data demonstrated the presence of clonal heterogeneity in all but 1 sample (4 of 5 at D; 5 of 5 at R). Among the 4 patients with clonally related IGH gene rearrangements, there was only partial overlap in CNVs (approximately 40% on average) between the D and R lymphomas. The average CNVs was similar in the D and R samples (16.6 vs. 18.6 respectively; p = 0.75). Chromosomes 2, 3, 6, 9, and 17 were frequently altered, and CNVs involving the BCL-6, CDKN2A, TP53, and MYC loci were also commonly seen; but there was no systematic difference between the CNVs identified at D and R.NGS showed a variety of mutations, but no consistent pattern of mutations acquired at R. There was a nonsense mutation in exon 2 of CDKN2A in the R sample in patient 1, and both D and R samples showed the same copy-neutral loss-of-heterozygosity of 9p encompassing the CDKN2A gene. In addition, missense mutations of TP53 were detected in patients 4 (only at R) and 5 (only at D).CONCLUSIONS: This study demonstrates that LR-DLBCL is an uncommon phenomenon with most cases representing recurrence of the original disease. LR patients have similar OS as ER patients, and the only clinical factors segregating LR from ER are higher rates of extra-nodal presentation and extra-nodal-only sites of disease. Although most paired D and R cases share IGH clones, there is clear evidence of clonal heterogeneity with clonal evolution over time. This suggests that DLBCL may contain minor subclones not susceptible to chemotherapy, which persist subclinically acquiring additional mutations over time eventually generating clinically-evident relapse. In rare cases, the late “relapse” may occur as an unrelated lymphoma that arises spontaneously or secondary to the mutagenic effects of chemotherapy. The precise mechanism of this long latency is yet unclear, and requires further investigation. DisclosuresBorowitz:Becton Dickinson Biosciences: Research Funding.

Spontaneous Mutations of Bcor and Jak1/2 genes Lead to an Aggressive Leukemia of B-1 Progenitor B Cells

NUP98 gene fusions, generated by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies and have been shown to alter normal hematopoietic stem and progenitor cell (HSPC) gene expression programs. A recurrent t(11;17)(p15;p13) translocation in patients with AML leads to the production of a NUP98–PHF23 (NP23) fusion gene. The consequent NP23 fusion protein retains the PHD domain, known to bind H3K4me3, and is thought to have aberrant chromatin regulation properties. We have generated a transgenic mouse model of the NUP98-PHF23 gene fusion which develops a range of hematologic malignancies, most commonly pre-T LBL and AML. However, approximately 10% of NP23 mice develop an aggressive B-1 progenitor acute lymphoblastic leukemia (pro B-1 ALL).B-1 and B-2 lymphocytes have distinct developmental pathways and are thought to represent arms of the innate and adaptive immune systems, respectively. Mature B-2 lymphocytes predominate in the peripheral circulation, and are characterized by expression of B220; whereas B-1 lymphocytes are more prevalent in the pleural and peritoneal cavities, and do not express B220. Murine B cell malignancies typically stain positive for B220, and represent transformed B-2 cells. In the present study, NP23 progenitor ALLs displayed an immunophenotype (Lin-B220- CD19+ AA4.1+) that was identical to that of the recently described B-1 progenitor cell. All B-1 progenitor ALLs exhibited clonal rearrangements of the IgH gene locus. Specifically, these rearrangements involve favored usage of 3’ VH regions, similar to observations with fetal B-1 progenitor cells, further supporting the notion that these are leukemias of B-1 progenitors. Using whole exome sequencing, we found acquired mutations in the BCL6 interacting corepressor (Bcor) gene in 5 out of 7 B-1 progenitor leukemias. The mutations were all frame shift or nonsense mutations, and were located within a 9 bp “hot spot” in Bcor exon 8. In addition, 4 of 7 cases had somatic mutations of Janus kinase 1 (Jak1) or 2 (Jak2), and 7/7 cases showed hyperphosphorylation of Stat3 or Stat5, consistent with the contention that the Jak1/2 mutations are activating mutations, and leading to a hypothesis that the NP23 pro B-1 ALLs which do not harbor Jak1/2 mutations may have acquired an unidentified mutation in the Jak-Stat pathway. Of note, Jak1/2 mutations have previously been identified in a subset of high-risk pediatric B-cell precursor ALL patients. The striking correlation between Bcor and Jak1/2 mutations, occurring specifically in a subset of NP23 leukemias, implies that these three mutations (NP23, Bcor, and Jak1/2) collaborate and provide the oncogenic setting for B-1 progenitor transformation. DisclosuresNo relevant conflicts of interest to declare.

Frequent Igh Fusion Transcripts with Clinical Impact in Multiple Myeloma

Background: Significant heterogeneity has been described in Multiple Myloma (MM), especially at the genomic level. Frequent gains and losses of DNA along with various mutations have been observed, and differential allelic expression is being characterized. Fusion proteins are common and maybe associated with cell transformation, growth and lethality of tumor cells. However, unlike in leukemia, no consistent fusion gene product has been consistently identified. As IgH-related translocations have an important role in myeloma, we have investigated fusion genes involving IgH, to understand their biology and explore a possible effect on survival in MM.Methods: We performed deep RNA-Seq on purified MM cells from 430 newly-diagnosed MM patients and analyzed gene expression profiles, isoform signatures and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. We also correlated genomic data with patient data including cytogenetics and FISH, as well as survival.Results: We primarily focused on fusions involving the IGH gene (chromosome 14) and found that about one fourth of the patients (57 out of 430) presented an IGH fusion gene (97 patients according to TopHat, 303 according to Mapsplice, 57 according to both). These included the well described t(4,14) fusion involving the MMSET gene (found in 47 patients by both algorithms, 49 by Tophat, 54 by Mapsplice). Additionally we observed fusions involving chromosomes 14 and chromosomes 1, 4, 11, 12, and 16. The counterpart genes involved in the IgH fusions included PDE3A (chromosome 12 - 4 patients); HFM1 (chromosome 1, 2 patients); NFKB1, FGFR3, CIITA, WWOX and MRPL21 (chromosomes 4, 16, and 11, 1 patient). As RNA-Seq data allows the precise localization of the breakpoints, we were able to identify that out of the 47 t(4,14) patients, 62% were MB4-1, 9.5% were MB4-2 and 28.5% were MB4-3. Interestingly, we did not see fusion products involving IgH and other known parts on Chromosomes 8 and 20. We studied event-free survival in a subset of 265 patients with available survival data and found that, as predicted, patients with an IgH-MMSET fusion had significantly lower survival than others. However, patients with a fusion gene involving IGH and any other partner have a significantly better prognosis as a group. Moreover, the poor prognosis of IgH-MMSET fusion appears to be driven by MB4-3 patients. Importantly, the fusions identified using RNA-seq were also validated by FISH analysis. All t(4,14) fusions were characterized by a very high MMSET expression (FPKM greater than 20) while patients with other fusions presented a lower MMSET expression (FPKM lower than 10).Conclusion: Our study suggests that IgH-related translocations in myeloma may impact tumor biology by a number of mechanism, one of which is the generation of fusion proteins with functional consequences. It also highlights a possible clinical impact that requires validation in larger cohorts. DisclosuresNo relevant conflicts of interest to declare.

P1-19-1 - Detection of Minimal Residual Disease in Autografts by Aso-Pcr for Patients with Multiple Myeloma

Background: The high-dose melphalan followed by autologous blood stem cell transplantation (ASCT) is a standard treatment for younger patients with multiple myeloma (MM). Recent reports have shown that achieving molecular complete response is associated with longer progression-free survival. However, the impact of minimal residual disease (MRD) in peripheral blood cell autografts on outcome remains unknown. We report the clinical and molecular follow-up of 6 MM patients who underwent ASCT.Methods: Six patients in whom the treatment was performed at Iwate Medical University Hospital and MRD in autografts was evaluable were included in the study. All patients had achieved at least a partial response after ASCT. Fresh bone marrow cells at diagnosis as well as autografts were obtained for DNA extraction. The CDR III coding region of the IgH gene was studied by direct sequencing of PCR product from bone marrow samples at diagnosis, and then MRD in each autograft was measured by ASO-PCR.Results: Four of 6 patients are alive: 2 who had no MRD in their autografts maintain a stringent complete response (CR) for 40 and 8 months after ASCT, while 2 who had MRD in them have serum M protein assessable by immunofixation for 20 and 10 months after ASCT. Two patients died from disease progression, both of which had MRD in their autografts and had have serum M protein after ASCT.Conclusion: These observations suggest that no MRD in peripheral blood cell autografts may be associated with a durable CR after ASCT. In addition, the post-ASCT treatment such as consolidation or maintenance therapy should be considered for MM patients who have MRD in autografts. Larger study and serial follow-up should be recommended to establish the clinical impact of MRD measurement in autografts in MM patients. As of writing, follow-up MRD analyses for total 13 MM patients are ongoing. Updated results will be presented.