ALBERT

Description: Background High throughput sequencing (HTS) of rearranged TCRB and IGH chains has been demonstrated as a means to detect malignant T and B cells at a frequency as low as 10E-6. However, onerous input requirements (typically 20-30ug gDNA input over multiple library preparations) have impeded widespread adoption of 10E-6 as a threshold for minimal residual disease (MRD) translational research studies. Here we demonstrate an optimized highly multiplex PCR approach for amplifying IGH chains from fresh or FFPE-preserved RNA or DNA input material. Coupled with automated clonotyping and clonal lineage detection, we demonstrate detection of malignant B cell clones at a frequency of 10E-6 from a single library preparation. Methods Rearranged IGH chains were amplified using multiplex framework 3 and joining gene primers targeting all human IGH variable and joining gene alleles in the IMGT database (Oncomine IGH-SR assay). Libraries were generated from 25 or 100ng total RNA or 2ug gDNA derived from (1) peripheral blood leukocyte (PBL) or bone marrow (BM) spiked with Ramos B-cell cell line and (2) PBL or BM spiked with synthesized chronic lymphocytic leukemia (CLL) rearrangements from literature. Sequencing analysis was performed using the Gene Studio S5 and Ion Reporter to identify clonotypes, track clones across samples, and identify B cell clonal lineages. Clonal lineages were defined such that lineage members have a shared variable and joining gene identity, identical CDR3 lengths, and CDR3NT sequences within 85% similarity of other lineage members. Automated rarefaction analysis in Ion Reporter was used to determine optimal sequencing depth. Results Ramos and synthesized spike in controls were detected at a frequency of 10E-5 using 25ng of PBL total RNA and sequencing to 3M reads depth, and 10E-6 using 100ng input and 10M reads depth. gDNA-based libraries required 2ug and 3M reads depth to detect spike-in rearrangements at a frequency of 10E-5, while 10E-6 was achieved by combining the results from four 2ug gDNA libraries, each sequenced to 3M reads depth. Input and sequencing depth requirements were consistent across PBL or bone marrow derived libraries. Rarefaction analysis confirmed that the sequencing depth was appropriate for the targeted limit of detection. Conclusions These results demonstrate routine detection of B cell malignancy IGH chains at a frequency of 10E-6 using a limited amount of RNA or DNA material, comparing favorably to existing HTS-based approaches. We anticipate this approach to become a routine component of rare clone tracking applications including those involving B-ALL and CLL, particularly where sample material is limited or a low limit of detection is of paramount importance. Disclosures Looney: Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.

Description: Background Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic mature B cells and a heterogenous disease course ranging from aggressive clonal expansion requiring early intervention to indolent disease that does not require treatment. Accumulating evidence suggests that the somatic hypermutation (SHM) status of the IGHV gene of the malignant clone may serve as a prognostic marker of clinical outcome, where a SHM frequency of 〉2% indicates a favorable outcome (SHM-M) while 〈 2% SHM (SHM-U) indicates comparatively poor prognosis. Current next-generation sequencing (NGS) approaches for analyzing SHM commonly rely on multiplex primers targeting the framework 1 (FR1) or leader region of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA template. Limitations include the potential for joining gene mutations to interfere with primer binding and an inability to evaluate isotype, which could potentially serve as an additional prognostic marker given the mechanistic link between somatic hypermutation and class-switch recombination. Here we present a novel method for translational research investigations of IGH chain SHM employing multiplex FR1 and isotype (constant gene) specific primers to amplify IGH chains from RNA template. We demonstrate performance of the assay via sequencing of synthesized CLL IGH chains derived from literature. Methods Multiplex PCR primers were designed to target all IGH variable genes and constant genes and alleles in the IMGT database (Oncomine IGH-LR assay). Primers were used to amplify IGH chains in samples consisting of 25ng healthy donor peripheral blood leukocyte (PBL) total RNA spiked with one of 7 synthesized monoclonal CLL rearrangements including three germline rearrangements (0% SHM), and four mutated rearrangements (SHM ranging from 3-9%), each expressed with a different isotype. Resultant libraries were sequenced on the Gene Studio S5 and analyzed via Ion Reporter to identify clonotypes, quantify SHM, and identify B cell clonal lineages. Automated downsampling analysis was used to confirm that libraries had been sequenced to an appropriate depth. Results 7 of 7 synthesized rearrangements were correctly classified as SHM-M or SHM-U, and in each instance the isotype was correctly identified by the software. Clonal lineage analysis indicated that each spike in rearrangement was monoclonal, consistent with expectation. In silico analysis revealed a high correlation between SHM estimates obtained using the entire v-gene sequence and those obtained from FR1-targeting primers (Pearson's correlation 〉.99). Conclusions These results support the robustness and reliability of multiplex FR1 and constant gene based IGH chain amplification, combined with clonotyping and lineage analysis, for the translational research characterization of somatic hypermutation in CLL and other B cell neoplasms, including efforts to understand the potential prognostic value of isotype. Disclosures Looney: Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.

Description: Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a 〉90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.