Conatumumab is a monoclonal antibody specific for death receptor 5 (DR5) that activates caspases leading to DNA fragmentation and tumor-cell death. Like other Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) receptor therapies, conatumumab is currently being evaluated in clinical trials across a variety of tumor types. However, molecular evidence of on-target drug activity in tumors is often an elusive goal for clinical investigation. Here we evaluated a translational approach using a relevant biopsy method, fine needle aspirates (FNAs), to study the on-target pharmacodynamics of conatumumab pre-clinically. As detected by laser scanning cytometry, druginduced caspase-3 activation in FNA biopsies of Colo205 xenografts correlated well with activated caspase-3 in conventional section-based samples. Furthermore, in tumor-bearing mice, surrogate assays of serum caspase-3/7 activity and serum drug exposure correlated with in situ caspase-3 activation. We found that one advantage of FNA sampling over other sampling techniques was the ability to measure caspase activity on a per cell basis using DNA content information. To adapt the utility of FNAs for measuring pharmacodynamic markers in humans, detection of activated caspase-3 was multiplexed with EpCAM to characterize mock and clinical FNAs from colorectal and nonsmall cell lung cancer patients. These data suggest that FNA sampling is a practical method to cytometrically evaluate tumors for pharmacological impact in a clinical setting. '

Objectives This study aims to retrospectively assess C‐lectin‐like molecule 1 (CLL‐1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL‐1 bimodal associations with leukemia and patient‐specific characteristics. Methods Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL‐1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL‐1‐negative subpopulation of CLL‐1 bimodal AML samples. Results The frequency of a bimodal pattern of CLL‐1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL‐1 expression was most prevalent in patients with MDS‐related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia‐associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL‐1− subpopulation may consist of pre‐B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)‐leukemic mutations were detected in both CLL‐1+ and CLL‐1− subfractions of bimodal samples (N = 3). Conclusions C‐lectin‐like molecule 1 bimodality occurs in about 25% of AML patients and the CLL‐1− cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL‐1‐targeted therapies and warrant further investigation.