Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation.

INTRODUCTION: The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. MATERIALS AND METHODS: In this study, we selected two AML samples that had been fixed in methanol for 12-18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. RESULTS: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. CONCLUSIONS: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

A transcriptomic signature predicting septic outcome in patients undergoing autologous stem cell transplantation.

Autologous hematopoietic stem cell transplantation is the standard treatment for multiple myeloma and relapsed or refractory lymphomas. After autologous hematopoietic stem cell transplantation, hematologic reconstitution and infectious complications are the two most critical issues. Although many patients develop infectious complications after therapeutic intensification, it remains impossible to predict infection for each individual. The goal of this work was to determine and identify a predictive transcriptomic signature of systemic inflammatory response syndrome and/or sepsis in patients receiving autologous hematopoietic stem cell transplantation. High-throughput transcriptomic and bioinformatics analysis were performed to analyze gene expression modulation in peripheral blood mononuclear cells in 21 patients undergoing autologous hematopoietic stem cell transplantation for hematological malignancies (lymphoma or multiple myeloma). Transcriptomic analysis of peripheral blood mononuclear cells samples collected just after conditioning regimen identified an 11-gene signature (CHAT, CNN3, ANKRD42, LOC100505725, EDAR, GPAT2, ENST00000390425, MTRM8, C6orf192, LOC10289230, and XLOC-005643) that was able to early predict (at least 2-7 days before its occurrence) the development of systemic inflammatory response syndrome or sepsis. The possibility of systemic inflammatory response syndrome or sepsis occurrence early prediction (2-7 days before occurrence) opens up new therapeutic strategies based on preemptive antibiotic and/or antifungal prophylaxis adapted to the specific risk profile of each patient.

Natural killer cells in patients with polycythemia vera.

Natural killer cells (NK) are pivotal cells of innate immunity. They are potent antileukemic cytotoxic effectors. A defect in their cytotoxicity has been described in some hematopoietic malignancies such as acute myeloid leukemia, multiple myeloma and myelodysplastic syndromes. This defect is at least partially linked to a decreased or absent expression of some activating NK cells molecules, more particularly the so-called natural cytotoxicity receptors. In the present study, we more particularly focused our attention on NK cells of polycythemia vera, a myeloproliferative disease characterized by the presence of mutated JAK2 tyrosine kinase. The polymerase chain reaction analysis of NK cells from patients showed that they expressed the mutated form of JAK2. In polycythemia vera the proportion of NK was increased compared to healthy donors. The proliferative and cytotoxic abilities of NK cells from patients were similar to healthy donors. Expression of activating or inhibitory receptors was comparable in patients and donors, with nonetheless an imbalance for the inhibitory form of the CD158a,h couple of receptors in patients. Finally, the transcriptomic profile analysis clearly identified a discriminant signature between NK cells from patients and donors that could putatively be the consequence of abnormal continuous activation of mutated JAK2.

Distribution of lymphocyte subpopulations in patients with polycythemia vera.

Polycythemia vera (PV) is a disease with slow development. Nonetheless, the immune response is unable to eliminate the uncontrolled proliferating hematopoietic cells, leading to treatment of patient by phlebotomy and/or cytotoxic drugs. In addition, a higher incidence of cancers is observed in PV patients, independently of treatment. These observations argue for an impaired immune response in PV. In order to investigate this hypothesis, we studied the distribution of lymphocyte subpopulations in PV, and showed a significant decrease in T, TCR γ/δ and B cells together with an increase in natural killer (NK) cells.

Expression of activating receptors on natural killer cells from AIDS-related lymphoma patients.

BACKGROUND: Abnormal NK phenotype and cytotoxic functions have been described in acute myeloid leukemia, chronic lymphocytic leukemia, myeloma and myelodysplastic syndromes. Defective NK cytotoxicity is due to decreased expression of the Natural Cytotoxicity Receptors (NCRs), 2B4/CD244/p38, or NKG2D. This prompted us to test the expression of these molecules on circulating NK cells from patients with AIDS-related lymphomas (RL) in comparison with HIV + patients without lymphoma, healthy subjects and HIV-negative patients with lymphoma. METHODS: Blood samples were analyzed by flow cytometry for NCRs, 2B4/CD244/p38 and NKG2D expression on NK cells defined as CD3-/CD56+ lymphocytes. We also analyzed by quantitative PCR specific RNA for NKp30/NCR3 and NKp46/NCR1. RESULTS: We could not detect any defect in NKp46/NCR1 expression between all groups. NKp44/NCR2, NKp30/NCR3 and NKG2D had lower expression in AIDS-RL in comparison with HIV + patients without lymphoma when compared to patients with similar (>0.3 G/L) CD4+ lymphocyte levels. Expression of 2B4/CD244/p38 was lower in AIDS-RL than in HIV-negative lymphoma. Comparison of specific NKp30/NCR3 and NKp46/NCR1 RNA showed increased steady state levels, despite decreased surface expression for NKp30/NCR3, suggesting abnormal post-transcriptional regulatory mechanisms. CONCLUSIONS: We show a more pronounced defect in NK activating molecule when HIV infection is associated with lymphoma than when only one condition (HIV positivity or lymphoma) is present. Defective NK phenotype, in addition to CD4+ depletion and dysfunction, may participate to the increased incidence of lymphoma in HIV patients.

Differential expression of natural killer cell activating receptors in blood versus bone marrow in patients with monoclonal gammopathy.

In monoclonal gammopathies (MG) and multiple myeloma (MM), normal natural cytotoxicity receptors (NCR) expression (NCR1/NKp46, NCR2/NKp44, NCR3/NKp30) is observed in natural killer (NK) cells. Nonetheless, except in plasma cell leukemia, few tumor plasmocytes are present in PB, while NK studies have been performed on peripheral blood (PB). For this reason we focused our attention on NK from bone marrow (BM). Our study demonstrates that the down-regulation of NCR3/NKp30 is only detectable in NK from BM but not in PB, and shows a drastic decrease of both NKG2D and CD244/2B4/p38 expression in NK from BM in comparison with PB. In conclusion, our data more precisely describe the mechanism of immune escape of MG/MM from innate immunity since we show a drastic down regulation of 3 major activating NK receptors (NCR3/NKp30, NKG2D and CD244/2B4/p38) at the site of tumor, i.e BM, that was undetectable in PB. Further studies regarding immune regulatory drugs in MG/MM will imperiously require the assessment of immune cell status not only in PB but also in BM to obtain more relevant data regarding anti-tumor efficacy.

Natural killer cells modulation in hematological malignancies.

Hematological malignancies (HM) treatment improved over the last years resulting in increased achievement of complete or partial remission, but unfortunately high relapse rates are still observed, due to remaining minimal residual disease. Therefore, sustainment of long-term remission is crucial, using either drug maintenance treatment or by boosting or prolonging an immune response. Immune system has a key role in tumor surveillance. Nonetheless, tumor-cells evade the specific T-lymphocyte mediated immune surveillance using many mechanisms but especially by the down-regulation of the expression of HLA class I antigens. In theory, these tumor-cells lacking normal expression of HLA class I molecules should be destroyed by natural killer (NK) cells, according to the missing-self hypothesis. NK cells, at the frontier of innate and adaptive immune system, have a central role in tumor-cells surveillance as demonstrated in the setting of allogenic stem cell transplantation. Nevertheless, tumors develop various mechanisms to escape from NK innate immune pressure. Abnormal NK cytolytic functions have been described in many HM. We present here various mechanisms involved in the escape of HM from NK-cell surveillance, i.e., NK-cells quantitative and qualitative abnormalities.

Hematological malignancies escape from NK cell innate immune surveillance: mechanisms and therapeutic implications.

Hematological malignancies treatment improved over the last years resulting in increased achievement of complete or partial remission, but unfortunately high relapse rates are still observed. Therefore, sustainment of long-term remission is crucial. Immune system has a key role in tumor surveillance. Natural killer (NK) cells, at the frontier of innate and adaptive immune system, have a central role in tumor cells surveillance as demonstrated in the setting of allogenic stem cell transplantation. Nevertheless, tumor cells develop various mechanisms to escape from NK cells innate immune pressure. Abnormal NK cytolytic functions have been described in nearly all hematological malignancies. We present here various mechanisms involved in the escape of hematological malignancies from NK cells surveillance: NK cells quantitative deficiency and NK cell qualitative deficiency by increased inhibition signaling or decreased activating stimuli. A challenge of immunotherapy is to restore an efficient antitumor response. A combination of classical therapy plus immune modulation strategies will soon become a standard of care for hematological malignancies.