Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10−4 after induction predicts subsequent relapse. Likewise, MRD ≥ 10−4 in bone marrow prior to the initiation of conditioning for allogeneic hematopoietic cell transplantation (allo-HCT) predicts transplant failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus primed immunoglobulin (Ig) and T-cell receptor (TCR) amplification and high-throughput sequencing (HTS) permits use of a standardized algorithm for all patients and can detect leukemia at 10−6 or lower. We applied the Sequenta LymphoSIGHT™ HTS platform to quantification of MRD in 237 samples from 29 adult B-ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10−4 before HCT conditioning predicted post-HCT relapse (HR 7.7, 95% CI 2.0–30, p=0.003). In post-HCT blood samples, MRD ≥ 10−6 had 100% positive predictive value for relapse with median lead-time of 89 days (HR 14; 95% CI 4.7–44, p<0.0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention prior to overt relapse.
Mucormycosis has been reported in otherwise healthy individuals; however, it is primarily seen in immunocompromised patients, such as those with diabetes mellitus, malignancy, or chronic graft-versus-host disease, and has a high mortality rate. Because most cases of mucormycosis are associated with contiguous rhinocerebral infection, only 5 cases of isolated musculoskeletal Rhizopus infection have been reported in the literature. One patient underwent hematopoietic cell transplant, which resulted in a fatal outcome.This article describes the successful treatment of isolated Rhizopus osteomyelitis in a patient who underwent hematopoietic cell transplant using a combined surgical and medical approach. A 33-year-old woman with pre-B cell acute lymphoblastic leukemia underwent hematopoietic cell transplant with few complications but developed chronic graft-versus-host disease 8 months posttransplant. She was treated with high-dose steroids for 6 weeks before she was admitted for severe right tibial pain in the absence of trauma. Early detection, aggressive therapies, and a multidisciplinary surgical and medical team allowed for the microbiologically confirmed resolution of the infection. Treatment included multiagent antimicrobial therapy with amphotericin B, daptomycin, and ertapenem. Several surgical irrigation and debridement procedures were also performed, with the eventual placement of amphotericin-impregnated polymethylmethacrylate cement beads and small fragment titanium screws. The patient continued taking postoperative antifungal treatment for 7 months after discharge. Six months following the discontinuation of antifungal therapy, the team's multidisciplinary approach achieved a continued resolution of the patient's infection and a return to a fully ambulatory and radiographically proven recovery without limb loss.
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<0.0001) prior to clinical relapse. Amongst 50 contemporaneous PB and BM samples, 25 (50%) were concordantly MRD negative, 7 (14%) were MRD positive in BM but negative in PB, and 15 (30%) exhibited significantly higher MRD in BM versus PB (median 41-fold higher, range 1.1 – 411-fold). 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.
4104 Background: Although acute lymphoblastic leukemia (ALL) can be eradicated in some patients using allogeneic hematopoietic cell transplantation (allo-HCT), post-transplant relapse is common and associated with a dire prognosis. The ability to predict ALL relapse while disease burden is minimal might offer the opportunity for timely intervention. Minimal residual disease (MRD) may be quantified in ALL using flow cytometry or detection of molecular markers, including clonal gene aberrations such as BCR-ABL translocations. Here, we evaluated a novel approach for quantifying leukemic clones using ultrasensitive high-throughput sequencing (HTS) of the immunoglobulin heavy chain (IGH) gene. Methods: We amplified VDJ-rearranged IGH loci from genomic DNA extracted from peripheral blood mononuclear cells (PBMC) or bone marrow aspirates (BM) using V and J segment consensus primers. Amplified IGH molecules were sequenced with one million or more dedicated reads from a median 287,000 input genomes (range 11,000 – 2,900,000) 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. Thirty-six 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. In total, 160 samples (32 for disease clone ascertainment and 128 for MRD quantification) were subjected to IGH-HTS analysis. Results: The IGH locus was successfully amplified from PBMC and sequenced from 18/36 ALL patients. The remaining 18 patients did not exhibit an apparent disease clone using consensus IGH primers, suggesting that either the PBMC disease burden was below threshold for clone identification, or the IGH locus had not completed VDJ rearrangement in the cancer clone. Among 12 patients who achieved MRD negativity following allo-HCT, 8 ultimately relapsed with a median time to molecular disease progression of 189 days (range 77–689 days) and a median time to clinical relapse of 278 days (range 89–889 days). Four patients achieved persistent MRD negativity following HCT and 3 remain alive at a median 714 days (range 325–1008 days). One of the 4 long-term MRD negative patients died leukemia-free from cardiovascular disease at 1047 days following HCT. All patients with MRD detected more than 100 days following HCT relapsed and died (median survival 431 days; range 286–624 days). Across all 8 patients who relapsed after achieving molecular remission, the lead time between molecular disease detection by IGH-HTS and clinical relapse was a median 89 days (range 0–275 days) with significant likelihood of MRD detection in a PBMC sample at least one month (p<0.0001), three months (p<0.0001), and six months (p=0.01) prior to clinical relapse. Amongst 30 contemporaneous PBMC and BM samples, 16 were concordantly MRD negative, 5 were MRD positive in BM but negative in PBMC, and 9 exhibited significantly higher MRD in BM versus PBMC (median 14.2-fold higher). IGH-HTS exhibited higher sensitivity than histopathologic BM interpretation, with 38% (8/21) of BM biopsies deemed negative by histopathology being positive for MRD by IGH-HTS, and all such patients relapsed. Detection of MRD in BM aspirates 50–100 days following allo-HCT was predictive of relapse (p=0.03), irrespective of PBMC disease burden. Conclusions: We demonstrate the applicability of IGH-HTS using consensus primers for ultra-sensitive detection of MRD in ALL and a significant correlation between detection of molecular MRD and clinical relapse. IGH-HTS MRD quantification appears to provide a valuable lead-time prior to clinical relapse. This 3–6 month window could provide an opportunity to apply additional chemotherapy and/or immunotherapy while disease burden is small. Our finding that MRD sensitivity in BM is more than 14 times higher than in PBMC suggests marrow assessment may further improve the lead-time for predicting disease recurrence using IGH-HTS. The dismal outcome of patients with ALL, even in the setting of allo-HCT, demands further clinical study of both post-transplant disease monitoring and novel methods for treating ALL relapse in evolution. 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. Klinger:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.
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