Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Hyun-Young Kim, M.D., In Young Yoo, M.D., Dae Jin Lim, M.T., Hee-Jin Kim, M.D., Sun-Hee Kim, M.D., Sang Eun Yoon, M.D., Seok Jin Kim, M.D., Duck Cho, M.D., and Kihyun Kim, M.D.. Ann Lab Med 2022;42:558-65. https://doi.org/10.3343/alm.2022.42.5.558

Mikyoung Park, M.D., Ph.D., Jihyang Lim, M.D., Ph.D., Ari Ahn, M.D., Ph.D., Eun-Jee Oh, M.D., Ph.D., Jaewoo Song, M.D., Ph.D., Kyeong-Hee Kim, M.D., Ph.D., Jin-Yeong Han, M.D., Ph.D., Hyun-Woo Choi, M.D., Ph.D., Joo-Heon Park, M.D., Kyung-Hwa Shin, M.D., Ph.D., Hyerim Kim, M.D., Ph.D., Miyoung Kim, M.D., Ph.D., Sang-Hyun Hwang, M.D., Ph.D., Hyun-Young Kim, M.D., Ph.D., Duck Cho, M.D., Ph.D., and Eun-Suk Kang, M.D., Ph.D.. Ann Lab Med 2024;44:222-34. https://doi.org/10.3343/alm.2023.0298

Objectives: Despite its utility, interpreting multiparameter flow cytometry (MFC) data for hematologic malignancy remains time-intensive and complex. This study evaluated the applicability of two dimensionality reduction (DR) algorithms, t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP), to MFC data of hematologic malignancy. Methods: A total of 237 samples were re-analyzed by t-SNE- and UMAP-based gating: 80 with acute leukemia orientation tube panel, 42 with B-cell lymphoma (BCL) panel, 45 with multiple myeloma (MM) panel, 40 and 30 with measurable residual disease (MRD) panels for B-cell acute lymphoblastic leukemia (B-MRD) and MM (MM-MRD), respectively. Each result was compared to the manual gating, and sensitivity and precision were assessed using BCL and B-MRD panels. Results: Compared to manual gating, DR-based gating demonstrated agreements over 95.0 % for all MFC panels, and quantitative correlations ( ρ ) exceeded 0.94. Both t-SNE- and UMAP-based gating showed a sensitivity and negative predictive value of 100 %. Also, in one sample each from the BCL and MM-MRD panels, DR-based gating identified populations that were missed by manual gating. Sensitivity evaluation showed that both t-SNE- and UMAP-based gating successfully identified MRD populations down to the lowest MRD level of 10 ^−5.30 when applying primary-gating strategy for CD19-positive population. Precision evaluation showed coefficient of variation below 10 % across all levels. Conclusions: This study shows that DR-based gating streamlines data interpretation and minimizes overlooked populations, demonstrating significant potential as a valuable tool in MFC analysis for hematologic malignancies.

Somatic CALR mutations occur in approximately 70% of patients with JAK2 V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). We evaluated the effects of the CALR mutant type and burden on the phenotype of CALR-mutated myeloproliferative neoplasms (MPN). Of the 510 patients with suspected or diagnosed MPN, all 49 patients detected with CALR mutations were diagnosed with ET (n = 32) or PMF (n = 17). The CALR mutant burden was significantly higher in PMF than in ET (45% vs. 34%), and type 1-like and type 2-like mutations were detected in 49% and 51% patients, respectively. Patients with MPN and type 2-like mutation showed a significantly higher median platelet count than those with type 1-like mutation. Particularly, patients with ET and type 2-like mutation had no thrombotic events, despite higher platelet counts. The effect of CALR mutant burden differed depending on the mutant type. A higher mutant burden tended to be associated with a cytopenic phenotype (i.e., lower hemoglobin levels and platelet counts) in patients with the type 1-like mutation and a proliferative hematological phenotype (i.e., higher platelet and neutrophil counts) in patients with the type 2-like mutation. This study suggests that the disease phenotype of MPN may be altered through CALR mutant burden and mutant type.

Parvovirus B19 (PV) infection is usually symptomless and can cause benign, short-lived conditions. Anemia associated with PRCA is the most representative hematologic manifestation, but neutropenia and thrombocytopenia have been rarely reported. Three patients were admitted to the hospital with neutropenia and thrombocytopenia. The accompanying symptoms were fever, myalgia, rash, or arthralgia, and all patients were previously healthy. Patients were positive for PV PCR and diagnosed with PV infection. Before the diagnosis of PV infection, 2 patients underwent BM study and almost absence of erythroid progenitor cells in BM aspiration were a clue for the PV infection. Other BM findings were hypocellular marrow and a few hemophagocytic histiocytes. Patients received supportive care with follow-up of CBC. All 3 patients spontaneously recovered from neutropenia and thrombocytopenia within 3 weeks without severe complications. The evaluation of PV infection should be considered in situations where there is neutropenia and thrombocytopenia in healthy individuals even without anemia as a differential diagnosis.