Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for transplant-eligible patients with multiple myeloma (MM). Among factors that influence outcome after autoHSCT, it has been suggested that the number of natural killer (NK) cells plays an important role. However, the impact that different NK cell subsets and their phenotype could have in disease progression after autoHSCT are less clear. For this reason, we have phenotypically and functionally characterized NK cells during immune system reconstitution after autoHSCT in 54 MM patients. Shortly after leukocyte recovery, an extensive redistribution of NK cell subsets occurs in these patients. In addition, NK cells undergo a profound phenotypic change characterized, among others, by their increased proliferative capacity and immature phenotype. Importantly, MM patients who showed lower frequencies of the mature highly differentiated NKG2A-CD57+ NK cell subset at +30 and +100 days after autoHSCT experienced superior progression-free survival and had a longer time to the next treatment than those with higher frequencies. Our results provide significant insights into NK cell reconstitution after autoHSCT and suggest that the degree of NK cell maturation after autoHSCT affects the clinical outcome of MM patients treated with this therapeutic strategy.

Introduction We evaluated the value of hematopoietic progenitor cells (HPCs) counted in Sysmex XN analyzers to predict the mobilization and collection of CD34+ cells in apheresis for stem cell transplantation. Methods Eighty patients who underwent stem cell transplantation were enrolled (50 autologous and 30 allogeneic). In the autologous group, patients were considered poor mobilizers when the CD34+ count was <10 × 106/L or <20 × 106/L in patients with multiple myeloma who were going to undergo two transplants. ROC curves were generated, and HPC cutoffs were calculated. Results The correlation between the HPC and CD34+ cell counts was good. Two algorithms were proposed. In the first algorithm, samples collected the day before apheresis, negative and positive HPC cutoffs were selected to detect poor and good mobilization and, therefore, the need or not to administer plerixafor. In the second algorithm, samples collected pre‐apheresis, the negative HPC cutoff was an indication to delay apheresis; an HPC higher than the optimal cutoff was an indication to start apheresis. When the HPC values were between these cutoffs, there was an indication to count CD34+ cells for a better decision‐making. Finally, in samples collected pre‐apheresis, HPC counts could be used to predict patients who would have poor CD34+ cell collections. In the allogeneic group, all the donors mobilized well, and very few needed two apheresis procedures. Conclusions The HPC count is useful for decision‐making in the management of patients subjected to apheresis procedures to collect peripheral blood stem cells.

Malignant pleural effusion and peritoneal carcinomatosis are frequent causes of effusion. Cytological evaluation (PAP-stained slides followed by immunocytochemistry, IHC, if applicable) is currently the gold standard for the diagnosis of malignant effusions, but its sensitivity varies between 40 and 80%, being a time-consuming technique. Although flow cytometry (FC) is not routinely used in the diagnosis or follow-up of nonhematopoietic neoplasms, it has the advantage of being rapidly applicable to fresh samples, potentially decreasing the time for the diagnosis. The main objective of this study was to assess the utility of FC as a confirmatory tool in the diagnosis of neoplastic effusions, based on the expression of EpCAM antibody in tumor cells versus the cytological evaluation. In this work 1,535 serous fluids were collected, of which 101 (68 pleural, 33 ascites) were selected through a screening algorithm and sent to the FC and cytological evaluation. Seventy-three of these samples (46 pleural, 27 ascites) were considered malignant as determined by clinical, cytological and radiological criteria. According to our data, 75% (55/73) of these samples were positive by Cytology/IHC and 74% (54/73) by FC. We noticed that, although the sensitivity, specificity, and area under the curve were similar, the turnaround time was shorter when using FC. Moreover, these results clearly improved by combining both techniques. We conclude that FC provides information about malignant effusions faster than immunohistochemical staining, and we believe that performing both techniques in parallel would improve diagnostic performance.