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Transforming growth factor-β (TGF-β) plays an essential role in regulating immune responses through its immunossupressive effect on adaptive and innate immune cells. In addition, we and others have shown that alterations in cell-intrinsic metabolism profiles can significantly impact the function of immune cells. Previously, our group demonstrated that TGFβ-imprinting (TGFβi) decreases NK cell sensitivity to TGFβ through SMAD3 suppression and enhances a pro-inflammatory phenotype with hypersecretion of IFN-γ, TNF-α, and GM-CSF, which appeared to be mediated by an epigenetic mechanism. To evaluate this process, we conducted RNA-seq and ATAC-seq to evaluate the impact of chromosomal remodeling on gene expression. In addition to confirming low SMAD3 transcription, this data showed that TGFβi NK cells have decreased CD38 expression, which we confirmed at the protein level. Since CD38 is an ectoenzyme that regulates nicotinamide adenine dinucleotide (NAD+), a critical component of OXPHOS in both T and NK cells, we examined the effect of TGFβi on NK cell metabolism. TGFβi primary NK cells were generated from peripheral blood of healthy donors as previously described, and using a mitochondrial stress test assay, we observed higher oxygen consumption rates (OCR). However, in the glycolysis stress test assay we observed comparable extracellular acidification rates (ECAR) between Standard expanded and TGFbi NK cells, resulting in higher OCR/ECAR ratios in TGFbi NK cells. Since TGFβ has been shown to induce fragmented mitochondria, and inhibition of mitochondrial fragmentation improved mitochondrial metabolism, we evaluated subcellular structure of TGFβi NK cellsby transmission electron microscopy (TEM). We found no difference in mitochondrial fragmentation between STD and TGFβi NK cells. Together, these results show that TGFβi induces epigenetic reprograming of NK cells that results in increased OXPHOS through CD38 suppression. Moreover, the decreases CD38 expression in TGFβi NK cells could be a promising non-genetic alternative to generating CD38-negative NK cells to avoid fratricide in combination with DARA Figure Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The individual prognosis of patients with chronic lymphocytic leukemia (CLL) is extremely variable. Although clinical stages remain the basis for assessing prognosis in CLL, a number of biological markers, particularly serum markers, cytogenetic abnormalities, IgVH mutations, CD38 and ZAP-70 expression in leukemic cells offer important, independent prognostic information. Before being incorporated into daily practice, however, these markers require standardization and validation in large, prospective trials. Meanwhile, treatment of patients with CLL not included in clinical studies should be decided on the basis of classical NCI/CLL Working Group criteria. An important area of research in CLL prognostication is the identification of markers useful for predicting response to therapy and its duration. Among them, del(17p), reflecting P53 abnormalities, is particularly important. Also relevant is del(11q), which points to ATM defects. There is also some correlation between IgVH mutational status, ZAP-70 and CD38 expression and response to therapy and its duration, although these relationships need further investigation. Finally, there is increasing evidence that response to therapy, particularly in those cases in which minimal residual disease is eradicated, is associated with longer survival.

Background: Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western countries but very rare in the East Asia including Japan. The previous reports from Japan showed that CLL patients in Japan have different characteristics including atypical morphology and immunophenotypes compared to those of Western countries. We started the cooperative study of CLL between Japan and Austria from 2012. Aim: The aim of this retrospective study is to compare the characteristics of typical CLL patients in Japan and Austria. Material and Methods: Diagnostic procedures were first harmonized between sites and laboratories. Samples were exchanged between Japan and Austria, and morphology, flow cytometry (FCM), FISH and IGVH gene sequence were independently assessed. After technical agreement, we selected 86 Austrian CLL patients from the database of the Medical University of Vienna, and 80 Japanese patients from the CLLRSG-01 prospective observational study. All CLL cases had typical immunophenotypes (Matutes score 4 or 5). The analyses of morphology, FCM and FISH including the deletion in 11q22, 13q14, and 17p13, and trisomy 12 were performed. Immunoglobulin heavy chain variable region (IGHV) gene mutation was also analysed. Results: Median age of CLL patients was 62.5 years old (range: 35-93) in Austria and 66.4 years (range: 34-93) in Japan. Japanese patients were significantly older at diagnosis than Austrian patients (P = 0.027). No differences were found for sex and the Binet stage. Also, there were no differences in laboratory findings (lymphocyte and platelet counts and serum LDH and beta-2-microglobulin levels) except for hemoglobin (Hb) level (median: 13.8g/dL in Austria vs. 13.1g/dL in Japan, P = 0.005) between two countries. Immunophenotypic study by FCM showed higher proportion of CD38 expression in Austrian CLL patients (34% in Austria vs. 10% in Japan, P < 0.001). No differences were found for chromosomal aberrations by FISH analyses between Austria and Japan. del(13q14) was found in 62% and 56% (P = 0.456), trisomy 12 in 7% and 13% (P = 0.315), del(11q22) in 13% and 10% (P = 0.513) and del(17p13) in 7% and 3% (P = 0.179), in Austria and Japan, respectively. The IGHV mutation rate was higher in Japan (P = 0.002). Mutated IGHV was found in 61% of Austrian and 84% of Japanese patients. IGHV family usage profile was significantly different. In Austrian patients, VH1, 2, 3, 4, 5, 6, and 7 were 24%, 1%, 54%, 19%, 1%, 1% and 0%, respectively. However, in Japanese patients, they were 2%, 0%, 75%, 20%, 2%, 0% and 2%, respectively. The proportion of VH1 was surprisingly lower in Japan (P = 0.006). Notably, none of the Japanese patients used VH1-69, in contrast to the use of 10% in Austrian patients. Conclusion : Our analysis indicates that there are clear differences between Japanese and European CLL patients, in age, Hb level, CD38 expression and IGHV usage and mutations. The reason for particular IGHV usage should further be investigated. Disclosures Takizawa: Teijin: Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria; Chugai: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Janssen: Honoraria; Sumitomo Dainippon: Honoraria, Research Funding; MSD: Honoraria, Research Funding. Suzuki:Bristol-Myers Squibb: Honoraria; Kyowa Hakko kirin: Honoraria; Chugai: Honoraria. Hoermann:Gilead: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Ariad: Honoraria. Aoki:SymBio Pharmaceuticals: Consultancy. Suzumiya:Kyowa Hakko kirin: Research Funding; Eisai: Honoraria, Research Funding; Astellas: Research Funding; Takeda: Honoraria; Toyama Chemical: Research Funding; Chugai: Honoraria, Research Funding. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

To the editor: Immunoglobulin V genes and CD38 expression in CLL The finding that the mutational status of immunoglobulin (Ig) V genes correlates with identifiable disease subsets of chronic lymphocytic leukemia (CLL) and is of prognostic importance has been documented and mutually corroborated by

In chronic lymphocytic leukemia (CLL), chromosomes usually evade detailed cytogenetic analyses because cells poorly respond to the traditionally used set of mitogens. We applied novel technologies, such as stimulation of CLL cells either with CD40 ligand or with a combination of CpG-oligodeoxynucleotides and IL-2, to increase the freequncy of metaphase spreads for detailed chromosome analysis in 96 patients with CLL. This approach revealed that translocations occurred in 33 of 96 (34%) of our patients with CLL. The presence of translocations defined a new prognostic subgroup because these patients have significantly shorter median treatment-free survival (24 months vs 106 months; P < .001) and significantly inferior overall survival (OS; median, 94 months) than patients without translocations (346 months; P < .001). In multivariate analysis—including Binet stage, complex karyotype, CD38 expression, and 17p deletions—translocation proved to be the prognostic marker with the highest impact for an unfavorable clinical outcome (P < .001). In summary, we identified a new subgroup of patients with CLL defined by chromosomal trans-locations and poor prognosis. Our data may facilitate the identification of molecular events crucial for transforming activity in this disease and should have implications for risk-adapted clinical management of patients with CLL. (Blood. 2006;107:742-751)

Recent immunogenetic studies of chronic lymphocytic leukemia (CLL) suggest a dichotomy: those developing from naive, pregerminal B lymphocytes exhibiting germline configuration of Ig VHstatus (poor outcome) and those stemming from more mature, postgerminal center memory B cells with mutated Ig

The prognostic relevance of CD38 expression by clonal B cells from chronic lymphocytic leukemia (B-CLL) cases has been extensively reported. Expression of CD38 by a B cell indicates both its activation status and differentiation stage and remains fairly constant over time in most B-CLL cases although it has been reported to change with time in some cases and this change is associated with change in clinical behavior of the patient. Due to these reasons it has achieved a prominent place among other prognostic indicators such as Ig V gene mutation and ZAP-70 expression in assessment of B-CLL patients. To assess which aspects of the immunobiology of B-CLL cells are impacted by their expression of CD38 by clonal members “within” a B-CLL case we flow-sorted B-CLL cells from peripheral blood of 16 patients (% CD38 expression pre-sort, ranging from 1–90%), into CD38− and CD38+ subsets and quantified telomere lengths as a measure of their replicative history. As a measure of cellular activation we studied telomerase activity in these flow-sorted cells. In addition, expression of CD69 and CD62L (activation-associated), ZAP-70 (signaling-associated) and Ki-67 (cell cycle-associated) were studied on CD38− and CD38+ cell subsets by immunofluorecence and multi-color flow cytometry in 35 cases (including those used for cell sorting experiments). Telomerase activity was markedly higher in the CD38+ subset compared to the CD38− subset in each case (p< 0.01), although telomere lengths of the subsets did not differ significantly within individual cases. Simultaneously, the percentages of cells expressing CD69 and ZAP-70 were significantly higher (p

Comment on Deaglio et al, page [3042][1] Deaglio and colleagues in this issue of Blood provide an important series of investigations that establish that CD38 expression is not simply a prognostic factor unrelated to the biology of CLL that correlates rapid disease progression, poor response to

Since the first description by Damle et al,[1][1]several investigators demonstrated the independent prognostic significance of CD38 expression in predicting survival of patients suffering from B-cell chronic lymphocytic leukemia (B-CLL).[2-9][2] But despite confirming its prognostic value, Hamblin

We have read with great interest the papers by Ibrahim et al[1][1] and Hamblin et al[2][2] regarding conversion of CD38 expression and prognosis of B-CLL. The former described how one of their B-CLL patients showed a change of CD38 expression level from low (