In patient 21, a centromere 7 probe (CEP7, Abbott Laboratories) (cutoff was defined as 3%) was added to the fusion probe

In patient 21, a centromere 7 probe (CEP7, Abbott Laboratories) (cutoff was defined as 3%) was added to the fusion probe. Translocation probe; Abbott Arzoxifene HCl Laboratories, Abbott Park, IL) (cutoff was set to 3%). The 4 trials were GMALL Elderly 1/2003 (“type”:”clinical-trial”,”attrs”:”text”:”NCT00198978″,”term_id”:”NCT00198978″NCT00198978; German Multicenter Trial for Treatment of Elderly Patients With Newly Diagnosed Acute Lymphoblastic Leukemia), 07/2003 (“type”:”clinical-trial”,”attrs”:”text”:”NCT00198991″,”term_id”:”NCT00198991″NCT00198991; German Multicenter Trial for Treatment of Newly Diagnosed Acute Lymphoblastic Leukemia in Adults), MT103-206 (“type”:”clinical-trial”,”attrs”:”text”:”NCT01209286″,”term_id”:”NCT01209286″NCT01209286; Study of the bispecific T-cell engager [BiTE] Blinatumomab [MT103] in Adult Patients With Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia [ALL]), and Alcantara (“type”:”clinical-trial”,”attrs”:”text”:”NCT02000427″,”term_id”:”NCT02000427″NCT02000427; A Phase 2 Single Arm, Multicenter Trial to Evaluate the Efficacy of the BiTE Antibody Blinatumomab in Adult Subjects With Relapsed/Refractory Philadelphia Positive B-precursor Acute Lymphoblastic Leukemia). Patient characteristics are provided in supplemental Table 1, available on the Web site. In patient 21, a centromere 7 probe (CEP7, Abbott Laboratories) (cutoff was defined as 3%) was added to the fusion probe. The following populations were investigated on the basis of the approach described by Castor et al12: CD34+38C19C3C (HSCs and multipotential progenitor [MPP] cells), CD34+38+19C3C (myeloid and lymphoid progenitors), CD34+19+20C3C (leukemia cells without CD20 coexpression; leukemia associated immunophenotype [LAIP] 20C), CD34+19+20+3C (leukemia cells with CD20 coexpression; LAIP 20+), CD34C19+20+ (mature B cells), CD34C19C20C3+ (mature T cells), CD34+19C13/33+10C16C (early myeloid compartment), CD34C19C13/33+10C16C (late myeloid compartment), and CD34C19C13/33+10C16+ (mature myeloid compartment) (supplemental Cdh5 Figure 1; supplemental Tables 2-4; supplemental Methods). Cell sorting, FISH, array comparative genomic hybridization, molecular analysis of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, and real-time quantitative polymerase chain reaction were performed as described in supplemental Methods. The clinical study was approved by the local ethics committee (D448/14). Informed consent was obtained in accordance with the Declaration of Helsinki. Results and discussion Patient 21 presented with a relapse of fusion/monosomy 7Cpositive cells were found in the CD19+ leukemia compartments (CD34+19+20CCD3C and CD34+19+20+CD3C), but they were also found within the HSC and progenitor compartments (CD34+38C19C3C and CD34+38+19C3C) and within the myeloid compartment (CD34+19C13/33+10C16C, CD34C19C13/33+10C16C, and CD34C19C13/33+10C16+). fusionCpositive cells without monosomy 7 were also present at initial diagnosis within the myeloid but not within the CD19+ leukemia compartment. Within the HSC and progenitor cell compartments they remained at 4% and 3%, respectively, which was within the FISH and sorting purity cutoff range. The same analysis was performed in a second patient (patient 29) with positivity at initial diagnosis. Open in a separate window Figure 1. Leukemic involvement and evolution of and immunoglobulin heavy chain (IGH) and T-cell receptor (TRB) gene rearrangement patterns (figure not to scale). Patients with leukemia were screened at initial diagnosis for clonal IG and TR gene rearrangements. Two clonal IGH gene rearrangements (VH3-23-DH2-2-JH6 and VH6-1-DH3-22-JH4) and 1 clonal cross-lineage TRB gene rearrangement (DB2-JB2.7) were detected, and clone-specific real-time quantitative polymerase chain reaction (RT-qPCR) assays were established on the basis of sequence information. RT-qPCR and FISH showed dominance of the IGH R/R, TRB R/G, and translocation RT-qPCR revealed a subclonal IGH gene rearrangement (0.3%). A clonal evolution of the leukemic bulk with occurrence of a new dominant IGH/TRB gene rearrangement was excluded by IGH/TRB multiplex Arzoxifene HCl PCR, which has a sensitivity of about 1% to 5%. (C) Subclonal architecture of fusion and monosomy 7 in immunophenotypic compartments of patient 21 at initial diagnosis analyzed by FISH after FACS. Left: FISH results of each compartment. Orange circle, aberrant signal constellation; green circle, normal signal constellation; asterisk (*) indicates being within the range of the FISH and sorting purity cutoff. Right: Representative interphase nuclei showing the 2 2 different aberrant signal constellations in a false color display using MetaSystems software. FISH images were acquired using a 63/1.40 numeric aperture oil objective in a Zeiss Axioskop 2 fluorescence microscope (Axioskop 2). The meaning of signals is as follows: isolated red, fusion; blue, centromere 7. APC, allophycocyanin; B, mature B cells; CEP7, centromere 7 signal; F, fusion signal; FITC, fluorescein isothiocyanate; leukemia-associated immunophenotype 20? (LAIP 20?), leukemic bulk without CD20 coexpression; LAIP 20+, leukemic bulk with CD20 coexpression; M1, early myeloid compartment; M2, late myeloid compartment; M3, mature myeloid compartment; MPP, multipotent Arzoxifene HCl progenitor cells; n.d., not determined; neg., negative (not detected); PE, phycoerythrin; pos, positive; PRO, myeloid and lymphoid progenitors; SCT, stem cell transplantation; T, mature T cells. To investigate the clonal architecture of ALL systematically, pretherapeutic samples from an additional 25 patients with positivity (HSC/progenitor, myeloid, and leukemia involvement) as patients 21 and 29, which we now termed MPP pattern. In 13 (52%) of 25 patients, a second predominant pattern, referred to as B-lineage-pattern, was identified in which positivity was restricted to the B-lineage-determined CD34+19+ cells. In 23 of 25 evaluable patients, independent of the pattern of positivity, the mature B-cell compartment was negative, which indicated a lymphatic maturation stop of transcript mainly showed the MPP pattern (6 of 8) and much less frequently showed the B-lineage-pattern (1.