ALBERT

Description: Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G 〉 T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Description: Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012).  Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy and kappa chain (IGH and IGK), as well as T-cell receptor beta, delta and gamma.  The assay was performed on genomic DNA isolated from cells from the bone marrow, cerebrospinal fluid, or testes.  The test was first done at the time of relapse to identify the malignant clonotype, which was monitored at subsequent time points. The patients were ineligible for clinical trials and concurrently underwent MRD testing using flow cytometry. The sequencing assays were performed to show feasibility of the approach. Results  Patient one was a 14 y/o ALL relapse patient who was not in morphologic remission after standard re-induction therapy. The malignant clonotype was identified on a bone marrow aspirate from relapse; follow-up MRD tests were done using both flow cytometry and deep sequencing five times throughout salvage therapy with 5-aza-2'-deoxycytidine, suberoylanilide hydroxamic acid and high dose cytarabine over 75 days; the last two MRD data points showed 0.6% and 6% by ClonoSIGHT MRD and 0.4% and 1.3% by flow cytometry MRD. Morphologic remission with count recovery was used as the criteria to direct this patient to SCT. Patient two was a 9 y/o with ALL, for whom MRD was used to test for relapsed disease in multiple tissues.  This patient experienced three isolated testicular relapses (M1 marrow and no CNS involvement) at the time of each relapse. The ClonoSIGHT assay was used on tissue from a testicular biopsy to identify the malignant clone(s).  Testing of the bone marrow and cerebrospinal fluid did not detect the malignant clones in those sites. This patient underwent therapeutic orchiectomy and 4-week systemic re-induction resulting in a fourth complete remission and now is under evaluation for consolidation therapy with a SCT. A third patient was an 8 y/o with a combined bone marrow and testicular ALL relapse, who was in morphologic remission in the marrow after re-induction therapy and testicular radiotherapy. Prior to undergoing SCT the patient had negative MRD by flow cytometry but had 0.008% MRD using the ClonoSIGHT MRD assay.  The fourth patient was a 15-yo with ALL relapse at 9 years from first remission, treated with a four-drug re-induction and Berlin-Frankfurt-Münster based consolidation and maintenance therapy.  This patient was MRD negative by both flow cytometry and ClonoSIGHT MRD at end of re-induction as well as end of consolidation and remains in remission. Conclusions We have shown the feasibility of using sequencing-based tests for monitoring MRD in children with relapsed ALL in medullary (bone marrow) and extramedullary compartments (testes and CSF).  Further studies are needed to establish the prognostic value of MRD detected by the ClonoSIGHT assay in both medullary and extramedullary sites that are below the limit of detection of PCR and flow cytometry. These sequencing-based tests may provide a useful tool to develop risk stratification schemas for drug development in relapsed childhood ALL. Disclosures: Weng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Description: 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.

Description: The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

Description: Key Points HTS identifies MRD at the conventional clinical cutoff in more patients than FC, and these patients have worse outcomes. A subset of B-ALL patients essentially cured using current chemotherapy is identified at end of induction by HTS.

Description: Background Early response to induction chemotherapy is a significant prognostic factor in the outcome of children with acute lymphoblastic leukemia (ALL). High throughput sequencing (HTS) of rearranged immune receptor (TCR and Ig) genes offers the possibility of a more accurate, sensitive, and standardized approach to determination of early response to therapy.In this study, we investigate the ability of an HTS assay to risk stratify children with B-ALL at the end of induction therapy in comparison with flow cytometry (FC), assess the impact of increased MRD sensitivity on risk group assignment, evaluate the significance of MRD discordance between HTS and FC, and identify a novel subset of patients having an inferior outcome. Methods 619 paired Pretreatment and End of Induction (Day 29) samples from patients with B-ALL enrolled on Children's Oncology Group (COG) clinical trials AALL0331 (standard-risk, SR) and AALL0232 (high-risk, HR) having minimal residual disease (MRD) at Day 29 of less than 0.1% by flow cytometry were assayed by high throughput sequencing of CDR3 regions of IGH and TCRG. Dominant clonal CDR3 sequences in the pretreatment samples were quantitated in the paired Day 29 samples as residual disease of total nucleated cells without knowledge of the FC results. The relationship of residual disease determined by HTS and FC to 5-year event-free and overall survival (EFS and OS) was evaluated using Kaplan-Meier statistics. Results HTS detected a dominant clonal sequence in 93.2% of Pretreatment B-ALL samples, providing an informative cohort of standard-risk (N=282) and high-risk (N=297) patients. Using a threshold of 0.01% on the combined cohort, HTS and FC show identical EFS and OS for MRD positive (77.7% ± 0.04, 91.6% ± 0.03) and negative (92.5% ± 0.02, 96.3% ± 0.01) subsets, see Figure 1. Interestingly, reducing the HTS threshold from 0.01% to 0.0001% results in an improvement in EFS for the HTS MRD positive subset in both standard (80.1% -〉 88.2%) and high-risk (75.3% -〉 84.8%) patients, likely due to major reductions in the number of patients otherwise scored as MRD negative using the higher threshold of 0.01%(70.9% -〉 27.0% SR and 78.5% -〉 36.7% HR). This reflects the much more favorable outcome of the large cohort of patients with MRD between 0.0001% and 0.01% compared to those 〉0.01%. Little improvement in EFS is seen for HTS MRD negative patients with a reduction in MRD threshold. Maximal difference in EFS is achieved at an HTS threshold of 0.01%. Importantly, the subset of SR patients with no detectable residual clonal sequence at any level (19.9% of total) show an excellent EFS (98.1% ± 0.02) and OS (100% ± 0), different from the similar subset of HR patients (30.0% of total) showing less favorable EFS (92.7% ± 0.04) and OS (95.1% ± 0.03). Patients discordant for MRD at a threshold of 0.01%, either HTS+/FC- (N=55) or HTS-/FC+ (N=17), show intermediate EFS compared with concordantly positive or negative patients. Of interest, patients lacking a detectable clonal IgH sequence (N=42) show a significantly inferior EFS (78.5% ± 0.08 vs. 89.3% ± 0.02, p=0.01) but not OS. Conclusions HTS is equivalent to FC in its ability to risk stratify patients with childhood B-ALL at End of Induction therapy using a MRD threshold of 0.01%. Reducing the HTS MRD threshold below 0.01% does not improve risk stratification, but does allow identification of a small subset of MRD negative standard-risk patients virtually certain to be cured with current therapy. Patients discordant for MRD between HTS and FC have an outcome intermediate between that seen for concordant patients. Patients lacking a detectable clonal IgH sequence, presumably representing a more primitive form of leukemia, show a significantly inferior outcome. Figure 1. Equivalence of outcomes by high throughput sequencing and flow cytometry for B-ALL patients at a residual disease threshold of 0.01%. Figure 1. Equivalence of outcomes by high throughput sequencing and flow cytometry for B-ALL patients at a residual disease threshold of 0.01%. Disclosures Wood: Pfizer: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement. Kirsch:Adaptive Biotechnology: Employment. Crossley:Adaptive: Employment, Equity Ownership. Williamson:Adaptive Biotechnology: Employment. Borowitz:HTG Molecular: Consultancy; BD Biosciences: Research Funding; Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Robins:Adaptive Biotechnology: Employment.

Description: 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.