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IF 15.132 (2017); International audience; The introduction of novel agents has led to major improvements in clinical outcomes for patients with multiple myeloma. To shorten evaluation times for new treatments, health agencies are currently examining minimal residual disease (MRD) as a surrogate end point in clinical trials. We assessed the prognostic value of MRD, measured during maintenance therapy by next-generation sequencing (NGS). MRD negativity was defined as the absence of tumor plasma cell within 1 000 000 bone marrow cells (

We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma(MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allelespecific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD- by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P, This research project was supported by grants PS09/01370, PS09/1450, PI12/01761, and PI12/023121 from the Fondo de Investigaciones Sanitarias; RD12/10 Red de Cáncer (Cancer Network of Excellence) from the Instituto de Salud Carlos III, Spain, and from the Fondo de Investigaciones Sanitarias, Asociación Española Contra el Cáncer (AECC, GCB120981SAN) and the CRIS foundation. This study was supported in part by research funding from Sequenta, Inc. (F.P., L.W., M.M., M.F.).

Introduction Achieving complete remission (CR) improves outcomes in multiple myeloma (MM). We have shown that 50% of patients enrolled in Total Therapy (TT) trials achieve CR within one year of enrollment irrespective of risk as defined by the GEP70 risk signature. Nevertheless, the majority of patients with high risk (HR) MM show an early relapse with grim prognosis, while most standard risk (SR) patients tend to relapse late, most often years after completion of maintenance therapy. Evaluation of minimal residual disease (MRD) has been shown to be of prognostic relevance in patients in CR with MRD negativity being associated with better outcomes. Here we report the impact of next generation sequencing (NGS)-based MRD assessment in TT patients who have achieved at least a very good partial response (VGPR) at two different time points during their treatment protocol: first after auto stem cell transplant (ASCT at 4-8 months of enrollment) and secondly during maintenance therapy (12-24 months). Materials and methods In brief, our TT protocols incorporate induction therapy consisting of Velcade and Thalidomide in conjunction with chemotherapy (Cisplatin, Doxorubicin, Cytoxan, Etoposide) followed by ASCT, consolidation and three years of maintenance with Velcade, Revlimid and Dexamethasone. HR and SR were assigned using the GEP70 risk signature. For MRD testing, we included 119 patients who were treated on our TT3b -TT6 protocols and who achieved at least VGPR and had bone marrow samples available at 4-8 months post ASCT and 12-24 months into maintenance. Thirty-eight patients had HR MM (32%), 75 patients had SR MM (63%) and no GEP70 data was available for 6 patients (5%). For NGS-based MRD assessment (Adaptive Biotechnologies Corp), genomic DNA was amplified using locus-specific primer sets for immunoglobulin heavy-chain complete (IGH-VDJH) and incomplete (IGH-DJ) as well as for immunoglobulin κ locus (IGκ). The amplified products underwent sequencing and clonal gene rearrangements were analyzed. MRD levels were calculated at a sensitivity level of 1 x 10-5. PET-CT and/or MRI were obtained at same time point as MRD assessment to evaluate presence of focal lesions. Results Using MRD status post-ASCT and during maintenance as a predictor, there were significant differences for PFS and OS in HR and SR MM. In HR disease, MRD positivity post ASCT was associated with significantly worse clinical outcomes with 2 year PFS/OS at 33% and 52% for MRD positive patients compared to 82%and 83% in MRD negative patients. Further 5 year PFS and OS for the same patient group were 11% and 24% for MRD positivity compared to 45% and 75% for HR patients that had achieved MRD negativity. In contrast to the HR group, there was no significant difference between MRD positivity and negativity in SR patients at the time point post ASCT we have investigated. In this latter patient group a significant difference only became obvious later during maintenance with 5 year PFS and OS at 55% and 63% for MRD positive patients compared to 83% and 93% for MRD negative patients. Of the 54 SR patients that were MRD positive post ASCT, 25 (46%) became negative later during maintenance, while in HR disease only 4 of 21 MRD positive patients post ASCT became negative during maintenance (19%). Importantly, a stratified analysis of our MRD data showed that even during maintenance the majority of cases in the favorable CD-2 subgroup were MRD positive, indicating that MRD results should further be interpreted in the context of patients' molecular subgroup. Conclusions Our data suggest that HR patients that do not achieve MRD negativity after their first ASCT have a very high likelihood of disease progression and death within 24 months. In contrast, SR patients that are still MRD positive post ASCT tend to have continuing response to treatment and MRD testing only becomes prognostic during maintenance. Of interest is that a high proportion of HR patients that achieve MRD negativity post ASCT and a smaller proportion of MRD negative SR patients still relapse within 5 years of enrollment suggesting that remaining residual MM cells are not detected by molecular MRD testing. Imaging studies with PET-CT and MRI could further stratify these patients as they detect any residual focal lesions that are not assessed by molecular MRD testing. We have combined PET-CT/MRI data for all of the 119 patients that were included in this study and analyzed data will be presented at ASH 2016. Disclosures Carlton: Adaptive Biotechnologies: Employment, Equity Ownership. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies: Employment. Barlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

Neoantigens arising through somatic mutations are increasingly recognized as key tumor antigens driving clinical immune responses. We sought to identify human lymphoma neoantigens through a genomic and proteomic characterization of peptide ligands of major histocompatibility complex class I (MHC-I) and class II (MHC-II) of a cohort of patients with untreated mantle cell lymphoma. Peptide identification was performed through analysis of MHC-I and MHC-II peptide ligands using liquid chromatography and tandem mass spectrometry (LC/MS-MS), and augmented by generating patient-specific proteome databases through tumor:normal whole exome sequencing and targeted immunoglobulin gene sequencing in 17 patients. A total of 66 neoantigen peptides from 9 patients were found to be presented by MHC, and surprisingly all neoantigenic peptides were derived from either the lymphoma immunoglobulin heavy or light chain genes. There was no detectable MHC presentation of hundreds of candidate somatic neoantigenic peptides from other genes despite widespread antigen presentation of the proteome including proteins encoded by germline heterozygous single nucleotide polymorphisms. Clonotypic lymphoma immunoglobulin sequences were among the most commonly presented proteins by both MHC-I and MHC-II, yet MHC-I presentation of variable regions was nearly absent and included only a single neoantigen derived from IGVH. In contrast, the variable region was frequently presented by MHC-II and included neoantigen peptides created through somatic hypermutation or VDJ recombination. Autologous circulating T-cells specific for immunoglobulin-derived neoantigens could be detected using MHC-II tetramers and their frequency dynamically changed with vaccination and therapy. These findings suggest that immunoglobulin derived neoantigens are frequently presented by lymphoma, and represent potential targets for CD4, but not CD8, T-cell mediated therapies. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Disclosures Newman: Roche: Consultancy. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies: Employment. Faham:Adaptive Biotechnologies Corp: Employment.

Background: Autologous stem cell transplantation (ASCT) in conjunction with therapeutic drugs such as bortezomib, thalidomide, and lenalidomide can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Here we utilized a next-generation sequencing (NGS) approach for MRD assessment, which offers at least 1 to 2 logs greater sensitivity (10-6) compared to allele-specific oligonucleotide PCR (ASO-PCR) and flow cytometry, respectively (Faham et al Blood 2012). Previous studies have shown that NGS-based MRD assessment 90 days post-ASCT has prognostic value (Martinez-Lopez et al Blood 2014). In this study, we compared the prognostic value of MRD assessment in autografts and bone marrow (BM) samples from MM patients in the ASCT setting. Methods: One hundred and twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients received ASCT and were followed between June 15, 2004 and April 25, 2015. All patients had achieved a partial response (PR) or better after ASCT. Analyzed samples included: (1) BM slides from 96 MM patients at diagnosis, (2) fresh/frozen BM cells from 27 MM patients at diagnosis, (3) autografts and/or (4) post-ASCT BM cells obtained at the time of best response based on serum and urine tests. IGH-based ASO-PCR was performed as described previously (Methods Mol Biol 2009). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014). Results: We compared MRD results in 51 samples assessed by ASO-PCR and NGS. We observed a high correlation between NGS and ASO-PCR results at MRD levels of 10-5 or higher (r=0.86, P Conclusions: In this study, we show the prognostic value of NGS-based MRD assessment in autografts of patients with MM. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Importantly, this retrospective study suggests that therapeutic intervention based on NGS-based MRD positivity has a significant effect on patient outcome in the post-ASCT setting. Disclosures Zheng: Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches. Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014). Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. Disclosures Zheng: Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Background: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Chronic lymphocytic leukemia (CLL) is typically considered a malignancy of mature B-cells and is thought to originate from the oncogenic transformation of a single pre- or post-germinal B-cell. Activation-induced deaminase (AID), an enzyme that induces somatic hypermutation (SHM) at the heavy and light chain Ig loci, has been shown to be active in CLL cells in vitro (Patten et al., Blood 2012). Previous studies suggest that multiple CLL-specific Ig clonotypes related by SHM may be present in patients (pts) with dominant CLL clones possessing somatically mutated or unmutated Ig loci (Logan et al., PNAS 2011; Campbell et al., PNAS 2008). To our knowledge, evolution of the dominant CLL-specific Ig clonotype over the course of treatment has not been demonstrated. Here we utilized the LymphoSIGHT™ method, a next-generation sequencing-based method for lymphocyte characterization and quantification, to quantify clonal evolution at the Ig heavy and kappa chain (IGH and IGK) loci in 63 pts with CLL. Methods: Samples were collected at Stanford University and the Dana-Farber Cancer Institute. Peripheral blood mononuclear cells were isolated, and genomic DNA was extracted. Using unbiased universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al., Blood 2012). CLL-specific clonotypes were identified for each patient based on their high frequency (>5%) within the B-cell repertoire of a diagnostic (dx) sample. The highest frequency CLL clonotype identified in a dx sample is termed the “index clonotype”. Dx and post-treatment peripheral blood samples were assessed for evidence of evolved CLL clonotypes using LymphoSIGHT. A clonotype was considered “evolved” based on CDR3 sequence homology to the dx “index clonotype.” Results: CLL clonotypes were identified in dx samples from 63 pts (51 unmutated IGHV; 12 mutated), and we assessed post-treatment samples for the presence of CLL clonotype-associated oligoclonality. Two of 63 pts exhibited clonal evolution in post-treatment samples. One patient with unmutated CLL was MRD negative for over 7 years following allogeneic hematopoietic cell transplant (HCT), and subsequently became MRD positive with the evolved clonotype (differing by 1 nucleotide from the index clonotype) leading to clinical relapse 9 months after MRD positivity, while the original index clone remained undetectable. The patient was treated with ibrutinib upon clinical relapse and continues to have detectable MRD with the same evolved CLL clonotype (Fig 1A). In a second patient with mutated IGHV, we observed several evolved clonotypes in the dx sample. Multiple evolved clonotypes, including 5 that exhibited a significant increase in their frequency relative to the index clonotype, were present in the follow-up sample after treatment with fludarabine and rituximab (Fig 1B). These evolved clonotypes differed from the index clonotype by 1-4 nucleotides, but otherwise shared CDR3 identity, excluding independently arisen B cell clonotypes. Conclusions: We observed evidence of clonal evolution at Ig loci in a small subset (3.2%) of pts with CLL undergoing treatment. The presence of evolution in pts with CLL indicates that either the SHM mechanism, including the AID enzyme, remains active after neoplastic transformation, or the evolved clonotypes arose through a mechanism distinct from SHM. These evolved CLL clonotypes may have a selective advantage, and may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Additional cases are under investigation and updated results will be presented. Figure 1. CLL clonal evolution during therapy. MRD levels of two related Ig clonotypes, expressed as leukemia molecules per million leukocytes in peripheral blood, are shown at multiple time points following allogeneic HCT (A). In another patient undergoing conventional treatment, the level of each individual evolved clonotype as a fraction of the total CLL molecules is plotted at dx and post treatment time points. The index clone, evolved clones with increasing levels post-treatment, and evolved clones with decreasing levels post-treatment are shown in red, blue, and white, respectively (B). Figure 1A. Figure 1A. Figure 1B. Figure 1B. Disclosures Moorhead: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

*Equally Contributed Introduction: Recent studies show better progression-free (PFS) and overall survival (OS) for newly diagnosed multiple myeloma (NDMM) pts achieving MRD negativity by multicolor flow cytometry (MFC) or next-generation sequencing (NGS). Here, we report on the comprehensive assessment of MRD in a uniformly treated cohort of 45 MM patients (Korde et al. ASH 2013). Methods: 45 NDMM pts were treated with 8 cycles of combination therapy (carfilzomib, lenalidomide and dexamethasone) followed by 2 years of maintenance lenalidomide. Median potential follow-up was 17.3 mos. All patients were evaluated by NGS by LymphoSIGHT™ method. Briefly, using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining (VDJ) gene segments from genomic DNA obtained from CD138+ BM cell lysate or cell free bone marrow (BM) aspirate at baseline. A MM clonotype was defined as an immunoglobulin rearrangement identified by NGS at a frequency >=5%. MRD assessment by NGS, MFC and PET was repeated when patients achieved a complete response (CR) or completed 8 cycles of therapy. In a subset of patients, we performed NGS in peripheral blood (plasma) at baseline and after 2 cycles of treatment. Results: 40/45 (89%) of pts achieved VGPR or better after combination therapy. At least one clonal rearrangement was identified in 31/34 (91%) of BM CD138+ cell samples and in 34/45 (76%) of cell free BM aspirates; overall clonal rearrangement was detected in 37/45 (82%) bone marrow aspirates at baseline. Repeat MRD assessment at CR or the completion of 8 cycles in 32 pts show residual disease in cell free BM aspirates by NGS in 18 (56% of pts tested and 40% of the total study population). Estimated 12-mo and 18-mo PFS for MRD neg vs. pos by NGS was 100% vs 94% and 100% vs 84%, respectively (p=0.025). MFC testing for MRD was feasible in 43/44 pts (98%). PFS probabilities at 12-mo and 18-mo for flow neg vs pos was 100% vs 79% and 100% vs 63%, respectively (p=0.0022). Among pts assessed by both MRD methods (n=31), 23 samples were concordant (9 pos and 14 neg); among 8 discordant cases, all were positive by sequencing and negative by flow (p=0.0078). Abnormal PET scans were noted in 38/45 (84%) of pts at baseline. 24/43 (56%) pts at CR or after 8 cycles of CRd had a neg/dec PET response and 19/43 (44%) pts had a pos/partial PET response. At 12-mo and 18-mo, PFS by a neg/dec PET response vs pos/partial PET response was 100% vs 89% and 92% vs 89%, respectively (p=0.54). Furthermore, in 14 pts, we performed NGS in peripheral blood samples collected at baseline. At least one MM clonotype identified in baseline BM was detectable in corresponding plasma sample in 13/14 pts. Number of myeloma-specific molecules per million diploid genomes in the plasma was 3-log fold lower than in the BM (median 252 vs 730,950 MM specific clonal molecules per million diploid genomes). After 2 cycles of CRd treatment, 12/13 pts were still pos by serum electrophoresis and/or immunofixation while only 1 had detectable myeloma clonotypes in the plasma. Conclusions: This prospective evaluation of MRD testing in MM has several key findings: 1. Detection of myeloma-specific clonotypes by NGS of the Immunoglobulin VDJ segments in the BM is feasible in majority of pts with NDMM. 2. MRD detection by NGS compares favorably to MFC since all pts with residual disease by MFC are also MRD positive by sequencing; an additional 8 pts who were MRD negative by flow MFC were MRD positive by sequencing. 3. MRD negativity by MFC and NGS are both associated with significantly better PFS. 4. Detection of myeloma-specific clonotypes by NGS of the immunoglobulin VDJ segments (i.e. cell free DNA) in the peripheral blood plasma is feasible in NDMM pts at diagnosis; however, since tumor load in the plasma is >2000-fold lower than in the BM; using standard volumes of peripheral blood (plasma), the levels of myeloma-specific clonotypes were too low to be quantified already after 2 cycles of combination therapy. This was true despite presence of positive serum electrophoresis and/or immunofixation. Additional studies to understand the dynamics of the myeloma clonotype level in peripheral blood plasma are necessary to determine optimal MRD testing regimen. Disclosures Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Moorhead:Sequenta, Inc.: Employment.

Background: Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual, which is laborious and time-consuming. We utilized the LymphoSIGHTTM platform, which employs consensus primers and next-generation sequencing (NGS) to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone, to assess mCR. This technique has been shown to have 1-2 logs greater sensitivity compared to ASO-PCR and flow cytometry, respectively (Faham et al, Blood 2012). Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts and bone marrow (BM) in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Methods: One hundred and nine Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or better after ASCT. BM slides from 84 MM patients and fresh/frozen BM cells from 25 MM patients at diagnosis, as well as autografts/post-ASCT BM cells from each patient, were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. Results : Myeloma clonotypes could be identified in autografts/post-ASCT BM cells in 98 of 109 patients (90%) and by ASO-qPCR in 63 of 101 patients (62%). MRD by NGS was assessed in autografts of 89 patients. 70 of 89 patients (79%) were positive by NGS; 28 of 62 patients (45%) were positive by ASO-qPCR. Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, the sensitivity of ASO-PCR was 10-4-10-5, whereas that of NGS was 10-6 or lower when a sufficient amount of DNA was available for analysis. Eight cases where MRD was not detected in the autograft by NGS (MRDNGS(-)) and 38 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide, while 11 MRDNGS(-) cases and 32 MRDNGS(+) cases were followed without post-ASCT therapy. The MRDNGS(-) cases without post-ASCT therapy showed significantly better progression-free survival (PFS) than the MRDNGS(+) cases without post-ASCT therapy (P = 0.012) (Figure 1A) although overall survival rates were comparable between these groups. To investigate the value of sensitive detection by NGS, we compared PFS in 11 MRDNGS(-) cases (Group 1) with the 12 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 showed significantly better PFS than Group 2 (P = 0.027) (Figure 1B). Furthermore, 9 MRDNGS(-) in post-ASCT BM cases tended to show a better PFS than 18 MRDNGS(+) in post-ASCT BM cases (P = 0.075) (Figure 1C). In a multivariate analysis, post-ASCT therapy using novel agents (P Conclusions: In this study, we showed the prognostic value of MRD detection using the NGS-based LymphoSIGHT platform in autografts of patients with MM who received and in those who did not receive post-ASCT therapy with novel agents. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Patients with low level MRD detected by NGS but not by ASO-qPCR have worse prognosis compared to patients who are MRD negative by sequencing, which underscores the need for sensitive detection. Figure 1 Figure 1. Disclosures Zheng: Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Multiple myeloma (MM) is a malignancy of the plasma cells, which are at the terminal stage of B cell development. MM is a clonal disease originating from the transformation process of a single plasma cell and, thus, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. Based on preliminary evidence of oligoclonality, we utilized the LymphoSIGHT™ platform, a high-throughput sequencing method, to detect evidence of oligoclonality at the Ig heavy and kappa chain (IGH and IGK) loci. The sequencing approach can be used to examine two general models for oligoclonality. In the first model, two unrelated clonal Ig sequences are observed indicating the presence of two independent myelomas. Alternatively, in the second model, two related myeloma clonal Ig sequences are observed indicating that both myeloma clones are derived from a common ancestor that arose after the pro B cell stage when VDJ recombination is completed. The common ancestor can be a cell with premalignant lesion or after the MM has developed. Using the sequencing platform, we looked for evidence of these two models of oligoclonality in 193 MM patients. Methods Two cohorts of newly diagnosed MM patients were included in this analysis (N=125, N=68). Using universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments from genomic DNA or RNA from bone marrow collected at initial diagnosis. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al, Blood 2012). In the first cohort (N=125), we assessed gene rearrangement at the IGH-VDJ and IGK loci in 120 patients using RNA only and in 5 patients, we used both DNA and RNA to assess the IGH-VDJ, IGH-DJ and IGK loci. In the second cohort (N=68), we analyzed gene rearrangement at the IGH-VDJ, IGH-DJ and IGK loci using genomic DNA. Myeloma-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic sample. To identify clonotypes that are present in more than one cell we looked for patterns that are not consistent with having a maximum of one functional and one non-functional clonotype in a cell. Results We observed oligoclonality in 23 of 193 (12%) MM patients. Unrelated Ig sequences, which are consistent with the first model of oligoclonality, were present in 8 of the 193 (4%) patients. Fifteen of 193 (8%) patients exhibited related Ig clones, which is consistent with the second model of oligoclonality. In 4 of the 15 patients clones were related to each other via a somatic hypermutation process and differed by only a few bases (Figure 1), while in other 11 patients, the same VDJ sequence was associated with two distinct isotypes (IgA and IgG). Interestingly, in cases with both RNA and DNA sequencing and oligoclonality, we observed differential expression levels compared to clonal content at the DNA level, suggesting that a low frequency clone could be contributing as a predominant secretory clone. Conclusions This study demonstrates frequent oligoclonality in MM patients and suggests that this phenomenon does occur due to two distinct processes, either as unrelated sequences consistent with independent clones or as related sequences consistent with evolution after the MM malignant lesions occur. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. Currently, this analysis was limited to high frequency clones, using a threshold of 5% for identification of the myeloma-specific clones. Additional analysis is being performed to assess the presence of lower frequency clones, and data will be presented. Disclosures: Carlton: Sequenta, Inc. : Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Background Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We utilized a sequencing method, termed the LymphoSIGHT™ platform, which employs consensus primers and high-throughput sequencing to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. The sequencing method is quantitative at frequencies above 10-5 and the lower limit of detection is below 10-6. Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. Methods We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Because myeloma cells exist patchily in bone marrow (BM), myeloma cells in PBSC autografts may reflect the whole amount of tumor in vivo. Thirty-six Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or complete response (CR) after ASCT. BM slides from 28 MM patients and fresh BM cells from 8 MM patients at diagnosis as well as autografts were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using next-generation sequencing (NGS). Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples (Faham et al, Blood 2012). Results MRD in autografts could be assessed 36 of 36 (100%) by NGS and 30 of 36 (83%) by ASO-qPCR. MRD in autografts was detected in 27 of 36 (75%) by NGS and 11 of 30 (37%) by ASO-qPCR (Figure 1A). Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, ASO-qPCR could not detect myeloma cells at MRD levels of 10-5 or lower. Two cases where MRD was not detected by NGS (MRDNGS(-)) and 14 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide while 7 MRDNGS(-) cases and 13 MRDNGS(+) cases were followed without post-ASCT therapy. The best post-ASCT responses were as follows: 6 (67%) mCR, 1 (11%) sCR and 2 (22%) VGPR in 9 MRDNGS(-) cases; 2 (14%) mCR, 2 (14%) sCR, 2 (14%) CR, 8 (58%) VGPR in 14 MRDNGS(+) cases with post-ASCT therapy; 2 (15%) sCR, 10 (78%) VGPR and 1 (7%) PR in 13 MRDNGS(+) cases without post-ASCT therapy. The MRDNGS(-) cases tended to show a better PFS than the MRDNGS(+) cases with post-ASCT therapy (P = 0.400) and showed a significantly better PFS than those without post-ASCT therapy (P = 0.032) (Figure 1B) although overall survival rates were comparable among the three groups. To investigate the value of sensitive detection by NGS, we compared PFS in 7 MRDNGS(-) cases (Group 1) with the 6 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 tended to show a better PFS than Group 2 (P = 0.091) (Figure 1C). This underscores the value of sensitive detection of MRD in MM. Conclusions A high correlation between NGS and PCR MRD results was observed, and MRD-negativity in PBSC autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR. Disclosures: Zheng: Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment.

Background Multiple myeloma (MM) therapies are becoming increasingly effective with deeper responses. Indeed, recent prospective clinical trials based on carfilzomib, lenalidomide and dexamethasone (CRd) show complete response (CR)/near (n)-CR rates of over 75%. Consequently, newer techniques are needed to detect minimal residual disease (MRD). We are conducting a prospective Phase II clinical trial studying response to CRd in newly diagnosed MM patients. We assessed treatment responses obtained by traditional IMWG response criteria, MRD measurement by 8-color multiparameter flow cytometry (MFC) based on a minimum of 3 million events (maximum detection rate: 0.0005%), and MRD measurement by high throughput sequencing in 14 newly diagnosed MM patients who achieved very good partial response (VGPR)/CR/nCR. Methods Bone marrow (BM) and/or plasma samples were obtained from 14 newly diagnosed MM patients receiving CRd therapy. Samples were subjected to deep sequencing using the LymphoSIGHT™ platform, which has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood (Faham et al, Blood 2012). Briefly, using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA obtained from CD138+ BM cell lysate at baseline, as well as post-treatment plasma samples. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency within the B-cell repertoire in the CD138+ cell lysate BM sample. The presence of the myeloma-specific clonotype was then quantified in plasma samples obtained post-treatment. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. For patients who achieved VGPR/CR/nCR, we compared the results from MFC of the BM with those obtained by sequencing the cell free DNA in plasma samples. Results We detected a high frequency myeloma-specific gene rearrangement in 13 of 14 (93%) CD138+ BM cell lysate samples obtained at diagnosis. We assessed for MRD by flow cytometry and deep sequencing for the seven patients who were in VGPR/CR/nCR based on traditional protein response criteria. One patient was MRD positive by flow cytometry of the marrow and negative by deep sequencing in blood. Another patient was MRD positive by deep sequencing but negative by flow cytometry. Additionally, in the diagnostic BM sample of one patient, we observed two distinct high-frequency IGH clones that were related through a process of somatic hypermutation. The specific mutation pattern observed is consistent with a branched model of evolution where the two observed clones did not evolve from each other but rather from a common ancestor. We are currently analyzing samples collected at baseline and post-treatment in an additional 23 MM patients enrolled on the CRd trial, and results will be presented. Conclusions The development of sensitive, non-invasive MRD assays is becoming increasingly important to assess the impact of modern anti-myeloma therapies. One novel approach for MRD detection, termed the LymphoSIGHT™ platform, relies on high-throughput sequencing of VDJ rearrangements at the immunoglobulin locus. Based on our CRd clinical trial for newly diagnosed MM patients, we found sequencing of cell free tumor DNA in bone marrow aspirates and peripheral blood (plasma) to be technically feasible. Among 6 patients who obtained VGPR/CR/nCR and were found to be MRD negative by MFC of the BM, we found one patient to be MRD positive by sequencing in the peripheral blood (plasma compartment). One patient was MRD positive by flow cytometry but negative by deep sequencing. These preliminary results suggest that a sequencing-based MRD blood test may be more sensitive than standard protein response criteria and complementary to MFC-based BM tests. Analysis of additional samples will be presented. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Weng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Research Funding.

Introduction Allogeneic hematopoietic cell transplantation (allo-HCT) provides long-term immunologic disease control for a substantial portion of patients with hematologic malignancies. Chronic lymphocytic leukemia (CLL) is sensitive to graft-versus-leukemia (GVL) effects as evidenced by responses to reduced-intensity conditioning (RIC) allo-HCT, and to donor lymphocyte infusions (DLI) for post-HCT relapse. To identify potential alloreactive (AR)/GVL T cells, we performed in vitro mixed lymphocyte reactions (MLRs) between recipient CLL cells and donor T cells derived from blood apheresis products acquired for DLI. Responder and non-responder T cell populations from MLRs and recipient post-HCT blood samples underwent T cell receptor beta (TCRB) high-throughput sequencing (HTS). The prevalence of candidate AR/GVL TCRB clonotypes at various times following HCT was quantified and correlated with CLL disease burden and graft-versus-host disease (GVHD). Methods CLL cells isolated from cryopreserved PBMC aliquots of 7 patients who experienced post-HCT relapse were pre-stimulated in vitro for 72 hours with CpG oligodeoxynucleotides in X-VIVO 15 supplemented with IL-4, IL-7, BAFF, and GM-CSF. Upregulation of CD80, CD86, CD40L, MHCI, and/or MHCII was confirmed by flow cytometry. Donor T cells were isolated from cryopreserved DLI using pan-T cell isolation beads (Miltenyi), labeled with CFSE, and incubated with CpG-stimulated CLL cells for 7 days. Upon the conclusion of the MLR incubation, T cell populations were sorted into rapid responders (RR; CFSEdim), slow responders (SR; CFSEbrightCD69pos) and non-responders (NR; CFSEbrightCD69neg) and RNA was isolated from each cell population. RNA was then amplified and TCRB sequenced using the LymphoSIGHT platform (Sequenta). PBMC samples collected and cryopreserved pre-HCT and regularly following HCT and DLI were also subjected to TCRB-HTS. Results RR cells comprised 11.5 +/- 9.2% of MLR T cells, whereas SR were 4.2 +/- 3.5% and NR were 84.3 +/- 10.1% (Fig 1A). RR, SR, and NR populations demonstrated clonotypic exclusion with a mean 4.4% +/- 5.5% coincidence between populations (Fig 1B). TCRB diversity in the RR population was more restricted compared with diversity in the SR and NR populations, with the mean number of clonotypes comprising the top 50th percentile of total TCRB reads being 11.8 +/- 6.5%, 17.7 +/- 8.5%, and 20.2 +/- 1.8% of unique reads, respectively (p Conclusions In vitro MLR between donor T cells and CpG-stimulated CLL cells selects clonotypically distinct T cell populations with an oligoclonal RR population. Persistence of adoptively transferred candidate AR/GVL clones identified by MLR appears to correlate with likelihood of maintaining clinical remission beyond 2 years in CLL patients undergoing RIC allo-HCT. Failure to adoptively transfer AR/GVL clonotypes may be associated with early treatment failure. Disclosures: Klinger: Sequenta, Inc.: Employment, Research Funding. Moorhead:Sequenta, Inc.: Employment, Research Funding. Zheng:Sequenta, Inc.: Employment, Research Funding. Faham:Sequenta Inc.: Employment, Stockholder Other.

Abstract 2512 Background: Tremendous progress has been made in the management of acute lymphoblastic leukemia (ALL) in children, in part, through the wide use of minimal residual disease (MRD) monitoring to guide therapeutic intensification before and after allotransplantation. Unfortunately, poor accessibility and the high costs of MRD testing have limited its use in the management of adult ALL. A universally applicable MRD quantification method has the potential to revolutionize the management of ALL in adults. Most B- and T-cell ALL patients exhibit clonal rearrangements of one or more immunoglobulin (heavy chain, IGH; light chain, IGK/IGL) or T-cell receptor (beta, B; delta, D; gamma, G) genes. Such rearrangements may be quantified in a mixture of polyclonal B or T cells by massively parallel high-throughput sequencing (HTS), enabling highly sensitive MRD quantification. Methods: Thirty-six allografted ALL patients were selected for this retrospective study based on availability of a diagnostic sample containing leukemic cells, which was necessary for validating the amplification and sequencing method with each disease clonotype. Using Sequenta's LymphoSIGHT platform, we amplified and sequenced rearranged immunoreceptor (IR) loci from genomic DNA extracted from peripheral blood (PB) or bone marrow aspirates (BM) using V and J segment consensus primers for each IR gene (IGH, TCRB, TCRD, and TCRG) and, in some cases, D segment primers for incomplete IGH-DJ rearrangements. Sequences were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high prevalence in a PB or BM sample at a time of high disease burden. MRD levels were then determined in serial samples of PB or BM and quantified using spiked-in reference sequences. A total of 227 samples with a median 442,672 input genomes (range 8,038 – 7,162,715) were evaluated by IR-HTS. Results: A clonal IGH sequence was identified in 17/36 (47%) ALL patients. Amongst patients who did not have a detectable IGH clonotype, 14/19 (74%) had clonal sequences at one or more other loci, including partial IGH-DJ rearrangements (5/14; 36%), TCRB (4/14; 29%), TCRD (3/14; 21%), or TCRG (6/14; 43%). In total, 31 of 36 (86%) ALL patients had a clonal IR sequence suitable for MRD quantification. In 4 of 5 cases without an identified clonal sequence, only PB, but no BM samples, were available. Twenty patients achieved MRD negativity following HCT and 11 did not. In the MRD negative group, 12/20 patients (60%) ultimately relapsed with a median time to clinical progression of 231 days (range 77–889), whereas 11/11 patients (100%) in the MRD positive group relapsed with a median time to clinical progression of 139 days (range 60–304). The 12 patients who relapsed after achieving MRD negativity showed a median time to molecular disease progression of 93 days (range 59–689 days). Of the 8 patients who maintained MRD negativity following HCT, 7 remain alive at a median 1542 days (range 1133–2557 days). All 22 patients with MRD detected following HCT relapsed and 21 (95%) died (median survival 387 days; range 85–1991 days) (Figure 1). The lead time between molecular disease detection by IR-HTS and clinical relapse was a median 69 days (range 0–207 days) with significant likelihood of MRD detection in a PB sample at least one, three, and six months (each p Conclusions: We demonstrate a method for quantifying acute lymphoblastic leukemia MRD in the majority of patients using high-throughput sequencing of multiple immunoreceptor genes. IR-HTS MRD quantification provides valuable lead-time prior to clinical relapse that could provide an opportunity to apply additional therapeutic maneuvers while disease burden is low. Because MRD sensitivity in BM is significantly higher than in PB, marrow assessment may further improve the lead-time for predicting disease recurrence using IR-HTS. The dismal outcome of patients with ALL, particularly when disease burden persists after allogeneic transplant, demands further clinical study of both post-transplant MRD monitoring and novel methods for treating ALL relapse in evolution. Disclosures: Faham: Sequentia Inc.: Employment. Carlton:Sequenta, Inc.: Employment. Zheng:Sequenta: Employment. Moorhead:Sequenta: Employment. Klinger:Sequenta, Inc.: Employment. Willis:Sequenta, Inc.: Employment.

1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient's reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results: We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients ([Table 1][1]). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples ([Table 1][1]). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient's clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions: We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing. | Sample | Number of input cells | Number of input leukemic clone molecules | Sequencing MRD (%) | Flow cytometry MRD (%) | Clinical outcome | |:------:| --------------------- | ---------------------------------------- | ------------------ | ---------------------- | ---------------- | | 1 | 162,945 | 12 | 0.008 | 0.1 | Relapse | | 4 | 291,352 | 48 | 0.02 | 0.01-0.1 | CR | | 5 | 1,901,076 | | | 0.01-0.1 | CR | | 6 | 1,968,511 | 5,855 | 0.3 | >0.1 | CR | Table 1. Comparison of Day 29 (end of induction) MRD results using sequencing and FC methods Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. [1]: #T1

2532 [][1] Background: The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain >1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children's Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results: With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by > 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions: Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing. | Patient | Number of input cells | Number of input leukemic clone molecules | Sequencing MRD (%) | ASO-PCR MRD (%) (High MRD ≥ 0.1%) | Clinical outcome | |:-------:| --------------------- | ---------------------------------------- | ------------------ | --------------------------------- | ----------------- | | 1 | 163,333 | 17,170 | 5.3 | 0.008 | Relapse | | 2 | 2,043,860 | 9,958,326 | ∼100 | 18.2 | Induction failure | | 3 | 2,052,651 | 2,464 | 0.06 | 6.5 | Alive and well | | 4 | 1,298,291 | 67 | 0.001 | 0.001 | Alive and well | | 5 | 984,918 | 10,036 | 0.5 | 0.2 | Alive and well | | 6 | 863,757 | 474,938 | 27.5 | 28.1 | Relapse | | 7 | 31,536 | 73 | 0.1 | 0.03 | Relapse | Table 1. Comparison of MRD results using sequencing and ASO-PCR methods Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. [1]: #fn-2

Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between 1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

Abstract 1436 Background: The use of flow cytometry or real-time PCR-based methods to detect minimal residual disease (MRD) in children with Acute Lymphoblastic Leukemia (ALL) is a powerful tool for risk-adapted therapy stratification. However, current protocols for MRD detection incorrectly anticipate leukemia-free survival in 20–30% of low and intermediate risk patients. In this study, we present a new method for MRD detection using next-generation sequencing of the variable region of the immunoglobulin heavy chain (IgH) gene that can overcome two important limitations of current approaches as: (1) it detects lower levels of leukemia cells and (2) it identifies multiple evolved clones. Methods: To capture IgH sequences, we developed a set of multiplexed primers that allow the amplification of all known alleles of each V and J segment. We optimized the protocol to minimize amplification bias between primers. The products were then sequenced using the Illumina platform to obtain >1 million reads per sample. Using a streamlined algorithm, the data were used to calculate the frequency of clonotypes in each sample in a very sensitive and specific manner. Serial dilution experiments have also shown that this technology has a sensitivity of 0.0001%, or about 2 orders of magnitude better than flow cytometry. Results: To establish the ability to identify the leukemic clone, as well as the frequency of evolved clonotypes at diagnosis, we performed a pilot study with 24 diagnostic bone marrow samples (standard risk n=17, high risk n=6, very high risk n=1) from children with ALL. In these samples, single high-frequency clones were identified in 6 samples and multiple high-frequency clones were detected in 12 patients. Thus, we identified high-frequency clones in 75% (18/24) of samples. This is in agreement with previously published reports of PCR methods for VJ amplification. Next we compared the sequences of samples with multiple high-frequency clonotypes and found that, in all cases, the evolution was consistent with the previously described mechanism of V replacement. The evolved clones shared the same J segment allele, the number of bases deleted in the J segment, and at least part of the NDN sequence. Interestingly, in one patient there were 6 clonotypes with a frequency >1%. We then assessed whether the unique sequences present in these 6 clonal populations appeared in other lower frequency clonotypes from the same patient. We identified hundreds of distinct but related clones, consistent with active ongoing evolution of the leukemia. To further validate that the identified clonotypes in this patient were indeed leukemic, we sorted out the normal and malignant B cells, followed by repeat IgH sequencing. All the sequences suspected to have arisen by V replacement were enriched in the leukemic population and virtually absent from normal B cells. Finally, we sought to assess whether the evolved clones could have variable expression of surface markers used for MRD detection. In one patient, the different evolved clonotypes had variable CD38 expression based on IgH sequencing of blasts sorted by CD38. Further characterization of these clonotypes may reveal distinct underlying biology, as well as differing propensities for relapse. To further assess the associations between IgH sequence and immunophenotype, we are performing flow cytometry and sequencing of sorted subpopulations in additional diagnostic ALL samples (n=50). Conclusion: These findings suggest that this new technology may offer superior sensitivity and specificity for MRD detection, as well as more accurate stratification for risk-adapted therapies in children with ALL. Disclosures: Faham: Sequenta Inc: Employment, Equity Ownership. Willis:Sequenta Inc: Employment, Equity Ownership. Moorhead:Sequenta Inc.: Employment. Carlton:Sequenta Inc.: Employment. Zheng:Sequenta Inc.: Employment. Klinger:Sequenta Inc.: Employment.

Abstract 2542 Background: Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR. Methods: We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient's unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR. Results: CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days). Conclusions: Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.

Abstract 2540 Background: Minimal Residual Disease (MRD) assessment is increasingly used for treatment stratification since it is a strong predictor of outcome in Acute lymphoblastic leukemia (ALL). The most widely used MRD assays include flow cytometric detection of aberrant immunophenotypes and PCR amplification of patient-specific antigen-receptor sequences. The latter approach has proven to provide reliable clinical information but requires the development of patient-specific reagents which is laborious, time-consuming, and generates assays with variable sensitivities. In addition, this methodology may miss clonal changes that can occur during the course of the disease, such as the emergence of subclones as well as genetic evolution. To overcome these limitations, we developed a universal amplification assay with a sequencing readout that eliminates the need for patient-specific reagents, allows the assay to detect leukemic cells that have genetically evolved, and has a higher sensitivity than conventional tests. Methods and Results: To amplify all the IgH sequences, we developed a PCR assay to amplify all alleles of all the V and J segments with very low amplfication bias. Amplified molecules were then subjected to clonal sequencing to obtain >1 million reads to measure the frequency of the different IgH clonotypes in the sample. It should be noted that current next generation sequencing costs of this deep sequencing are similar to those of an MRD test conducted by flow cytometry. We tested the sensitivity of the method by in serial dilutions of genomic DNA from a leukemia sample known to carry two IgH clonotypes with into genomic DNA obtained from peripheral blood sample from a healthy donor. The material from the dilution series was then sequenced and analyzed to measure the level of these clones. The leukemic clonotypes could be readily detected even when diluted 1 million fold. To directly compare the our method to established MRD assays in ALL, we studied diagnostic and follow-up samples from 10 ALL patients whose MRD levels have been previously assessed by both real-time PCR amplification of IgH genes and flow cytometry. The results of these tests were not disclosed until completion of the deep sequencing analysis. The follow up samples were collected during (n = 3) or at the end of remission induction therapy (n = 4), or during continuation therapy (n = 3). Samples were processed similarly to identify the leukemia-specific sequence in the diagnostic samples and determine the level of these sequences in the follow up samples. The sequencing-based method identified all 5 samples that were MRD-positive according to flow cytometry and PCR ( Figure 2 ), with highly concordant estimates of MRD levels. Notably, among the remaining 5 samples, scored as MRD-negative by both flow cytometry and conventional PCR, the sequencing method detected residual leukemic sequences at a very low level (∼10−6) in one of the samples. The other 4 samples were MRD-negative by all three methods. Studies with a larger cohort of ALL samples are ongoing. Download : Download high-res image (28KB) Download : Download full-size image Figure 1 . Detection of diluted leukemic clonotypes. The X axis shows the –log10 of the leukemia sample dilution factor and the Y axis depicts the –log10 frequency of the 2 detected clonotypes. All the dilution through sequencing are done in duplicate and plotted. The leukemia clonotypes are reproducibly detected even when diluted 1 million fold. Download : Download high-res image (45KB) Download : Download full-size image Figure 2 . MRD detection by three different methods MRD detection by three different methodologies in 5 MRD positive patients is shown in log scale. The X axis depicts the MRD level using flow cytometry. The Y axis shows the MRD level as determined by real time PCR and sequencing. Contrary to conventional PCR-based MRD testing, the sequencing technology allows for the detection of leukemic clones that evolve by V replacement or other mechanisms. In this study, we identified clonotypes in several of the diagnostic samples that appeared to be the result of V replacement. These and other newly appearing related clones can be monitored in subsequent samples using the generic amplification and sequencing assay. Conclusions: We developed a highly sensitive and specific MRD detection method based on next-generation sequencing of IgH genes. This method has substantial advantages over conventional PCR MRD in that it eliminates the need for patient-specific reagents, can follow genetic evolution, and has potential for higher sensitivity. Disclosures: Faham: Sequenta Inc: Employment, Equity Ownership. Willis: Sequenta Inc: Employment, Equity Ownership. Moorhead: Sequenta Inc: Employment, Equity Ownership. Carlton: Sequenta Inc: Employment, Equity Ownership. Zheng: Sequenta Inc: Employment, Equity Ownership.

Abstract 3068 Mantle cell lymphoma (MCL) has a poor long-term prognosis. Though autologous transplant prolongs survival, novel and mechanistically distinct therapies are needed to target residual, myeloablation-resistant tumor cells that result in relapse. Trials of CpG-based vaccines for low-grade lymphoma have shown induction of anti-tumor T cells and clinical responses [Brody J. et al, J Clin Oncol. 2010 Oct 1;28(28) :4324–32]. In a pre-clinical model, we developed the immunotransplant maneuver combining: 1) CpG-based vaccination, 2) harvest of vaccine-primed T cells, 3) myeloablation with stem cell rescue, and 4) T cell re-infusion. Immunotransplant amplifies the proportion of anti-tumor T cells by an order of magnitude and cures even bulky, systemic lymphoma burden [Brody J. et al, Blood. 2009 Jan 1;113(1) :85–94]. METHODS: We initiated a phase I/II study of immunotransplant for newly diagnosed MCL patients to test the hypothesis that immunotransplant will amplify anti-tumor T cells as in the pre-clinical model. Anti-tumor T cells are assessed by co-culturing autologous tumor with peripheral blood T cells and measuring their production of: IFNg, TNF, IL2, CD137, perforin and granzyme by multiplex surface and intracellular flow cytometry. A secondary endpoint is measurement of molecular residual disease (MRD) using both standard allele-specific oligonucleotide (ASO) qPCR as well as high-throughput sequencing (HTS) of the entire IgH repertoire. The study is powered to detect a 50% improvement in sustained molecular remission rate compared to recent trials of standard transplant [Pott C. et al, Blood 2010 Apr 22;115(16) :3215–23, Geisler C. et al, Blood 2008 Oct 1;112(7) :2687–93]. Using the same HTS technology, we have also initiated studies of the entire TCR β repertoire as an alternate approach of tracking the amplification of vaccine-induced T-cells. RESULTS: Accrual has been rapid with 25 patients enrolled in 22 months and 13 patients completing the complete protocol so far. Flow-cytometric immune response testing has demonstrated that immunotransplant amplifies the proportion of tumor-reactive T cells in 83% of patients thus far. Notably, we have observed some patients with primarily CD8 T cell responses, some with CD4 T cell responses, and some with a combination of the two. In some cases, tumor-reactive T cells have been tested for reactivity to autologous, non-malignant B cells and have demonstrated a significant proportion that are tumor-specific. TCR β repertoire sequencing has also demonstrated instances of significant clonal amplification after immunotransplantation, some exceeding three orders of magnitude. In extreme cases, these have yielded dominant clones comprising as much as 50% of a patient's entire peripheral blood T cell repertoire post-transplant. HTS of the IgH repertoire has been an effective measurement of MRD bypassing the assay individualization of ASO qPCR and has been shown to be more sensitive than conventional flow cytometry. CONCLUSIONS: Pre-clinically, amplification of anti-tumor T cells correlates with cure of even myeloablation-resistant disease. The reiteration of anti-tumor T cell amplification in our preliminary patient data raises the possibility that immunotransplant may improve clinical outcomes. Ongoing MRD testing should suggest whether certain patterns of T cell response –measured functionally per flow cytometry or clonally per HTS- correlate with clinical benefit and whether the cohort has a better-than-expected molecular remission rate. Disclosures: Moorhead: Sequenta, Inc.: Employment. Zheng:Sequenta, Inc.: Employment. Klinger:Sequenta, Inc.: Employment. Faham:Sequenta Inc: Employment, Equity Ownership. Advani:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding.