Immunoglobulin and T Cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-

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Immunoglobulin and T Cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post- Transplantation Relapse and Survival Aaron C. Logan, Nikita Vashi, Malek Faham, Victoria Carlton, Katherine Kong, Ismael Buño, Jianbiao Zheng, Martin Moorhead, Mark Klinger, Bing Zhang, Amna Waqar, James L. Zehnder, David B. Miklos Biology of Blood and Marrow Transplantation Volume 20, Issue 9, Pages 1307-1313 (September 2014) DOI: 10.1016/j.bbmt.2014.04.018 Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Frequency of disease clonotype in diagnostic samples. At least 1 immunoreceptor clonotype suitable for use in MRD quantification was identified in 93% of patients with ALL. The number of leukemia-associated immunoreceptor locus rearrangements per patient are shown in (A). For each of the immunoreceptors tested, the frequency of dominant clonotypes in diagnostic specimens is shown in (B). Clonotype frequency greater than 5% (dotted line) was required to identify disease-associated immunoreceptor rearrangements. Biology of Blood and Marrow Transplantation 2014 20, 1307-1313DOI: (10.1016/j.bbmt.2014.04.018) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Comparison of molecular MRD sensitivity in bone marrow aspirates and peripheral blood. In 37 paired bone marrow (BM) aspirate and peripheral blood (PB) samples, MRD quantification was concordantly negative (0) in 18 (49%). MRD quantification was discordantly positive in BM while negative in PB in 4 (11%), and the median disease burden in these BM samples was 5.5 × 10−6 (range, 4.5 × 10−6 to 1.7 × 10−5). When MRD was detected in both sample sources, disease burden was higher (median, 8.4-fold) in BM than PB, as shown in (A). MRD quantification in all samples in this study is demonstrated in (B). Biology of Blood and Marrow Transplantation 2014 20, 1307-1313DOI: (10.1016/j.bbmt.2014.04.018) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Disease-free survival impaired by pre- or post-transplantation MRD. ALL disease burden ≥ 10−4 during the 30 days before transplantation strongly predicts likelihood of disease-free survival after transplantation (HR, 7.7; 95% CI, 2.0 to 30; P = .003) is shown in (A). (B) Shows MRD positivity (≥10−6) at any time through day +100 after transplantation predicts subsequent relapse (HR, 14; 95% CI, 4.7 to 44; P < .0001). Biology of Blood and Marrow Transplantation 2014 20, 1307-1313DOI: (10.1016/j.bbmt.2014.04.018) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Overall survival and time to event analysis. (A) Patients with persistent MRD negativity experienced significantly higher overall survival after hematopoietic cell transplantation (HCT) than patients with transient MRD negativity and failure to achieve MRD negativity (P < .0001). (B) shows the Kaplan-Meier estimate of time from molecular progression to clinical relapse and death for patients who relapsed. The median time from molecular progression to relapse was 89 days. Biology of Blood and Marrow Transplantation 2014 20, 1307-1313DOI: (10.1016/j.bbmt.2014.04.018) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions