Chen, Gong

Jukofsky, Lori

Martinelli, Giovanni

Pierceall, William

Reis, Bernhard

Seiter, Karen

English

The Touro College and University System

SUPPLEMENTARY APPENDIXAcute myeloid leukemia patients’ clinical response to idasanutlin (RG7388) is associated with pre-treatment MDM2protein expression in leukemic blastsBernhard Reis,1 Lori Jukofsky,2 Gong Chen,2 Giovanni Martinelli,3 Hua Zhong,2 W. Venus So,2 Michael J. Dickinson,4 Mark Drummond,5Sarit Assouline,6 Maneja Hashemyan, 2 Michel Theron,1 Steven Blotner,2 Je-Hwan Lee,7 Margaret Kasner,8 Sung-Soo Yoon,9Ruediger Rueger,10 Karen Seiter,11 Steven A. Middleton,2 Kevin R. Kelly,12 Norbert Vey,13 Karen Yee,14 Gwen Nichols,2 Lin-Chi Chen,2 andWilliam E. Pierceall21Roche Innovation Center Basel, Roche Pharma Research and Early Development, Switzerland; 2Roche Innovation Center New York, Roche Pharma Researchand Early Development, NY, USA; 3Seragnoli Institute of Hematology, Bologna University School of Medicine, Italy; 4Department of Haematology, Peter Mac-Callum Cancer Center, Melbourne, VIC, Australia; 5Beatson West of Scotland Cancer Center, Gartnavel General Hospital, Glasgow, United Kingdom; 6Divi-sion of Hematology, Jewish General Hospital, McGill University, Montreal, QC, Canada; 7Department of Hematology, Asian Medical Center, University ofUlsan College of Medicine, Seoul, South Korea; 8Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA; 9Department of Internal Medi-cine, Seoul National University School of Medicine, Seoul, South Korea; 10Roche Innovation Center Penzberg, Roche Pharma Research and Early Development,Germany; 11New York Medical College, Valhalla, NY, USA; 12University of Southern California, Norris Comprehensive Cancer Center, Los Angeles, CA,USA; 13Hematology, Institut Paoli Calmettes, Marseilles, France; and 14Princess Margaret Hospital, Toronto, ON, CanadaCorrespondence: william.pierceall@roche.comdoi:10.3324/haematol.2015.139717Supplemental Methods Flow cytometry Cells were stained according to the following procedure: 100 µL of donor whole blood was added to appropriately labeled tubes. Surface markers were subsequently stained by adding antibody fluorochrome conjugates; to facilitate population identification, respective isotype controls were added to control tubes. Tubes were then vortexed and incubated for 20 minutes in the dark at room temperature. After washing with phosphate-buffered saline (PBS) bovine serum albumin (BSA) buffer, tubes were centrifuged and decanted. Two milliliters of prewarmed 1× BD lyse/fix solution was then added and tubes were vortexed and incubated for 15 minutes in a 37°C water bath. After incubation, tubes were centrifuged and the supernatant was decanted and washed with PBS BSA buffer. Then 500 µL of perm buffer III was added and incubated according to the manufacturer’s specifications. After washing twice, the appropriate antibodies for intracellular staining or isotype controls were added with PBS/ BSA buffer and vortexed and incubated for 30 minutes in the dark at room temperature. These isotype controls were used to establish background staining and determine MDM2 percent cell positivity.  Tubes were washed twice with PBS/ BSA buffer and finally resuspended in 500 µL for acquisition in a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). A total of 250,000 events were acquired.  Electronic gating Flow cytometry analysis softwares Winlist and Flowjo were used. Initially, a gate is drawn around all cells with normal size (forwards scatter) and granular characteristics (side scatter): the total nucleated cell gate (TNC; Figure S1). Within this TNC subset, the AML blast population is then identified by drawing a region around cells with dim CD45 expression and low side scatter properties (CD45dim blasts in Figure S1). The blast population can be further refined by gating on CD34-positive or CD34-negative blasts (Figure S1; CD45dim/CD34+ or CD45dim/CD34− blasts, respectively). The threshold for CD34 positivity can be identified using an isotype control staining in that respective channel (not shown).    Figure S1. Identification of CD45dim/side scatter (SSC) low blasts and CD34+ blasts by electronic gating. The CD45dim general blast population and the more refined blasts that additionally express CD34 are displayed in dot plots as shown in Figure S2. The isotype control used instead of MDM2 staining can be used to set the threshold for MDM2 positivity (upper 2 panels in Figure S2.) The staining conditions, including the MDM2 recognizing antibody, are displayed at the bottom of Figure S2. The cell population falling in the regions defined as MDM2 positive is determined by calculating the percentage of MDM2-expressing cells within the respective blast parent population. TNC, total nucleated cell.  Figure S2. Identification of MDM2-positive cells in the CD45dim blast population and in the CD45dim/CD34+ blast population. Isotype controls (upper 2 dot plots) are used to draw the borders for MDM2 threshold positivity. SSC, side scatter.    Table S1. Patient information Characteristics CR Non-CR All P value No. of patients 26 66 92 NA Age, mean (SD), years 62 (11) 60 (13) 60 (12) .61 Sex, male (female), % 62 (38) 61 (39) 61 (39) 1 Race, white (nonwhite), % 73 (27) 89 (11) 85 (15) .98 ECOG PS 2 (1, 0), % 0 (46, 54) 9 (67, 24) 7 (61, 33) .017 TP53, mutant (wild type), % 4 (96) 26 (74) 2 (80) .018 Treatment, monotherapy (combination), % 20 (80) 42 (58) 36 (64) .084  All statistical analyses were conducted with R 3.0.2 software. The Wilcoxon rank-sum test was used to examine marginal association between the clinical responses and continuous variables such as MDM2 flow measurements and age. Fisher exact test was used to examine marginal association between the clinical responses and the categorical variables. Area under the concentration curve of receiver-operating characteristic was calculated to indicate complete remission (CR)/non-CR classification performance, with 95% CI calculated with bootstrapping. Logistic regression was adopted to adjust baseline demographic variables and treatment effects (monotherapy or combination therapy). Vuong test was used to compare models with different MDM2 flow measurements. All reported P values are 2 sided (P significant < .05). ECOG PS, Eastern Cooperative Oncology Group performance status; NA, not applicable.    

Acute Myeloid Leukemia Patients\u27 Clinical Response to Idasanutlin (RG7388) Is Associated with Pre-Treatment MDM2 Protein Expression in Leukemic Blasts

https://touroscholar.touro.edu/cgi/viewcontent.cgi?filename=0&article=1096&context=nymc_fac_pubs&type=additional

Acute Myeloid Leukemia Patients\u27 Clinical Response to Idasanutlin (RG7388) Is Associated with Pre-Treatment MDM2 Protein Expression in Leukemic Blasts

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