Immunoglobulin Heavy Chain Gene Rearrangements in Patients with Gaucher Disease

Summary Several studies support the evidence of increased incidence of hematological complications in Gaucher disease including monoclonal and polyclonal gammopathies and blood malignancies, especially multiple myeloma. Serum concentrations of immunoglobulins and PCR analysis of the IGH gene rearrangements were performed. The clonal PCR products were directly sequenced and analyzed with the appropriate database and tools. Serum monoclonal proteins were detected and identified by electrophoresis. Among 27 Gaucher patients, clonal IGH rearrangement was discovered in eight, with 5/8 having also serum monoclonal protein. Elevated immunoglobulins were detected in 9/27 patients. Follow-up data for 17 patients showed that the clonal rearrangement remained the same in four of them, however, in one patient it disappeared after the follow-up period. The remaining 12/17 patients were without previous IGH clonal rearrangement and remained so after the follow-up. Although clonal expansion may occur relatively early in the disease course, at least judging by the IGH gene rearrangements in Gaucher patients, the detected clones may be transient. A careful clinical follow-up in these patients is mandatory, including monitoring for lymphoid neoplasms, especially multiple myeloma.

Generalized Recalcitrant Pruritus as the Presenting Manifestation of Hypereosinophilic Syndrome

Background and Objective: Pruritus is a frequent occurrence in dermatology, and investigation is often unrevealing. The authors report the case of a 65-year-old man presenting with generalized recalcitrant pruritus as the presenting manifestation of hypereosinophilic syndrome. Methods and Results: A 65-year-old man developed intractable pruritus. Results of polymerase chain reaction clonal rearrangement were positive and led to the diagnosis and treatment. The patient also developed massive pulmonary embolism, which can be caused by chronic eosinophilia. Conclusions: This case highlights the importance of investigating patients with pruritus and unexplained persistent eosinophilia. Hypereosinophilic syndrome must be included in the differential diagnosis, which in this case presented initially as intractable pruritus. Polymerase chain reaction clonal rearrangement was key in reinforcing the diagnosis.

Minimal Residual Disease (MRD) Testing in Newly Diagnosed Multiple myeloma (MM) Patients: A Prospective Head-to-Head Assessment of Cell-Based, Molecular, and Molecular-Imaging Modalities

*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.

A case of Paraneoplastic Pemphigus associated with Castleman's disease

We present a case of PNP associated with Castleman's Disease. We have also reviewed the literature and described the characteristics of the two associated diseases. Gene clonal rearrangement was done to help diagnosis. We used, in addition, stereotactic radiosurgery which, as far as we know, has never before been employed to treat PNP associated with Castleman's Disease. This produced a good response, suggesting that it might be a good alternative treatment for PNP associated with Castleman's Disease when it is too difficult to operate.

Detection of Multiple Myeloma Cells in Peripheral Blood Using High-Throughput Sequencing Assay

Abstract 321 Background: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow (BM), and related organ or tissue impairment. MM patients are monitored during therapy and posttherapy using immunoglobulin, M-protein and free light chain assays. Assessing pathological myeloma cells using flow cytometry and RT-PCR has been shown to have superior prognostic value. However, the sensitivity of these techniques has generally limited their use to assessment of BM. In order to determine whether myeloma minimal residual disease could be detected in peripheral blood (PB), we assessed a cohort of MM patients using a sequencing based platform, LymphoSIGHT, with a sensitivity of 1 cancer cell per million leukocytes. Methods: We obtained from 4 sources (UCSF, NYU, Washington Univ, commercial source) pairs of BM and PB samples from 60 MM patients at different points of disease. BM samples were used to identify the clonal MM sequence and detection of that sequence in the PB was assessed. For some patients BM/PB sample pairs were obtained from >1 time point resulting in a total of 78 pairs. In addition blood and bone marrow plasma samples were available for 44 and 6 patients, respectively, to assess presence of the myeloma clonotype in cell free DNA. Altogether there were 206 samples. BM samples were either available as BM mononuclear cells (BMMC) or bead enriched CD138+ cells. Using universal primer sets, we amplified IgH@ variable (V), diversity (D), and joining (J) gene segments from genomic DNA and/or RNA samples, the incomplete IgH rearrangement (DJ), and IgK from genomic DNA. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype determination. Myeloma-specific IgH, IgK, and DJ clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in all PBMC (DNA and RNA), BM plasma (DNA), and PB plasma (DNA) samples using the same IgH and in some samples using the IgK sequencing assays. A quantitative and standardized measure of clone level among all leukocytes in each PB or BM sample was determined using internal reference DNA. Here we describe data on 46/60 patients; data from all 60 patients will be presented. Results: In BM samples, we detected the myeloma clonal rearrangement of at least one receptor (“calibrating receptor”) in 34/46 (74%) of MM patients (Table 1). The calibration rate varied by receptor, with 30/46 (65%) patients calibrating with IgH, 14/43 (33%) with IgH DJ, and 22/43 (51%) with IgK (Table 1). Identification of myeloma-specific clonal rearrangement is based on presence at high frequency and may not occur in samples from patients with low disease load (e.g., post-treatment). Of the 12 non-calibrating patients, only 3 had high disease load. The myeloma clonotype that was identified in the BM was also detected in PBMC in 22/30 (73%) and 28/30 (93%) patients with the DNA and RNA IgH analysis, respectively (Table 2). IgK DNA analysis showed the presence of the myeloma clonotype in 9/10 PBMC samples, all of which were concordant with IgH results. The myeloma clonotype that was identified in the BM was also detected in the cell-free BM and PB samples in 5/5 and 7/11 patients, respectively, using the IgH DNA assay. The evaluation of blood plasma and PBMC were at times complementary in detecting the myeloma. Conclusions: Results from the application of a high-throughput sequencing method for detection of myeloma-specific clonotypes in 46 MM patients are shown. A clonal rearrangement was detected in 74% of MM BM samples. Importantly, 93% of peripheral blood samples from 30 patients showed evidence of circulating myeloma in PBMC. Analysis of BM and PB samples from 14 additional MM patients as well as association of the level of myeloma in PBMC and BM with clinical measures is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Klinger:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

Abstract 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). 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). 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. 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.