Therapeutic effects of erlotinib ex vivo and in vivo. (A,B) Representative FACS diagrams of CD34⁺ bone marrow cells from a healthy volunteer and an AML patient with a normal karyotype. Cells were cultured with medium plus DMSO or erlotinib for 3 to 6 days, followed by the assessment of cell death with 2 distinct methods, the DiOC₆(3)/PI and annexin V/PI staining of dying (DiOC₆(3)^low PI⁻ or AnnV⁺PI⁻) and dead cells (PI⁺). Numbers in each quadrant are the percentages of total cells gated within the quadrant. (C-F) NPM-1 and p14^ARF translocation induced by erlotinib in AML cells (D,F), but not in normal CD34⁺ bone marrow cells (C,E). Cells were treated and assessed as described in Figure 5A,D. Error bars represent SD. (G,H) Induction of myeloid differentiation by erlotinib in malignant myeloblasts. CD34⁺ bone marrow cells from a high-risk MDS (G,H) and an AML patient (H) were cultured for 6 days in the presence of 0.02% DMSO or 10 μM erlotinib. CD11b surface exposure was measured by FACS analysis while excluding apoptotic cells. (G) The morphology and percentage of cells exhibiting signs of erlotinib-induced differentiation were determined by Wright-Giemsa staining as described in Figure 3. (I) Quantitative comparison of apoptosis induction in a cohort of normal controls and different subgroups of MDS/AML patients. Results are depicted as a box plot, giving the increase in apoptosis induced by erlotinib (10 μM, 3 days) in vitro/ex vivo as compared with DMSO-only–treated controls in the indicated subgroups. K indicates karyotype; NS, not significant; *P < 0.01. The horizontal bar is the mean, the box the 25^th percentile, and the whiskers the extremes. (J,K) Comparison of the ex vivo and in vivo effects of erlotinib on a patient with high-risk MDS. A patient diagnosed with metastatic EGFR-positive NSCLC and MDS RAEB-2 was treated with a monotherapy of erlotinib for the NSCLC. (J) Apoptosis-inducing effect of erlotinib on the patient's CD34⁺ bone marrow cells ex vivo (3 days, 10 μM) as assessed by staining with DiOC₆(3)/PI. (K) Routine blood examinations before and during monotherapy with erlotinib demonstrated the in vivo effect of erlotinib on platelet and neutrophil counts resulting in a hematologic improvement. D-32 indicates day 32 before erlotinib treatment; D0, start of erlotinib therapy; D+34, day 34 under erlotinib treatment.