Biphenotypic acute leukemia: a case report

We describe an uncommon case of acute leukemia in which leukemic blasts expressed myeloid antigens and cyCD79alpha molecule. In this 49-year old male patient, two distinct blast populations were detected in peripheral blood and bone marrow samples: one of small size resembling lymphoblasts and another with pink cytoplasmic granules resembling myeloblasts. Cytochemical reaction for myeloperoxidase was negative in both cell types. Conventional cytogenetic analysis showed a normal karyotype (46 XY) in all metaphases studied, while gene rearrangement analysis by seminested PCR of the immunoglobulin heavy chain (Ig-H) and T-cell-gamma chain (TCR-gamma) receptor, showed a germline configuration of the TCR and clonal rearrangement of Ig-H chain genes. Multicolour cytofluorimetric analysis showed that bone marrow and peripheral blood blasts expressed CD19, CD79alpha bright, CD22 and terminal deoxynucleotidyl transferase (TdT) as lymphoid markers, CD13, CD117, CD15 as myeloid markers, CD34, HLA-DR as stem cell markers. CD33 myeloid antigen was expressed by 50% of the blastic population. No differences in the immunophenotypic profile were detected in the two blast populations which were identified by morphology. According to EGIL (European Group of Immunological Classification of Leukemias) and WHO (World Health Organization) criteria, a diagnosis of biphenotypic acute leukemia (BAL) was made. The patient was treated with AML induction therapy followed by autologous stem cell transplantation, but relapse free survival was 6 months. The patient died a few weeks later due to unresponsiveness to salvage chemotherapy regimens. We conclude that patients with BAL should have a risk stratification with treatment tailored to their immunophenotype and gene rearrangement profiles.     

Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia.

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.     

急性髓系白血病免疫表型特征及遗传学特征分析

目的:研究急性髓系白血病免疫表型特征以及遗传学特征。方法:选取2011年1月到2014年5月我院收治的急性髓系白血病患者169例,采用流式细胞术和相关的单克隆抗体来分析所有患者的骨髓免疫表型,采用染G染色体显带技术分析患者的核型,根据淋系抗原(lym Ag)的表达将患者分为lym Ag+组和lym Ag-组。结果:抗原CD13、CD33、CD117以及MPO等髓系抗原最常在急性髓系白血病患者中表达,其中CD117在M3型病例中表达为85.7%(24/28),而CD34、HLA-DR双阴性、较强的自发荧光、CD13、CD33和MPO对M3型的诊断也具有一定的价值;其中47.9%(81/169)的患者伴随着淋系抗原表达,以CD7和CD56为主;60.4%(102/169)的患者伴随着核型异常;而伴随着t(8:21)的M2患者中的CD15、CD19和CD56的表达显著增强,而t(15:17)均发生于M3型患者中;而lym Ag+组患者CD34的阳性患者为77.8%(63/81)显著高于lym Ag-组的47.7%(42/88),两组比较差异具有统计学意义(P0.05)。结论:免疫表型对急性髓系白血病的诊断具有重要的意义,且免疫表型和异常核型存在密切的联系。     

Two methods for the quantitative analysis of surface antigen expression in acute myeloid leukemia (AML)

The expression of lineage molecules (CD13 and CD33), c-Kit receptor (CD117), CD34, HLA-DR and adhesion molecule CD49d was assessed in acute myeloid leukemia (AML) blast cells from 32 cases, using direct and indirect quantitative cytometric analysis. High correlation (r=0.8) was found between antigen expression intensity values calculated by direct analysis method (ABC) and by indirect analysis method (RFI). Moreover, the differences in expression intensity of CD13, CD117 and CD34 antigens were found between leukemic and normal myeloblasts. This may be helpful in identification of leukemic cells in the diagnostics of minimal residual disease after treatment in AML patients.     

A Novel Application of Furazolidone: Anti-Leukemic Activity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.     

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33

BACKGROUND: AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS: In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS: The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION: These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.     

Pediatric acute leukemia presenting as bilateral renal enlargement. Report of a case with fine needle aspiration cytologic features suggestive of megakaryocytic differentiation

BACKGROUND: Fine needle aspiration (FNA) cytologic diagnosis of acute myeloid leukemia involving the kidney has rarely been reported, but acute leukemia with cytologic features suggestive of megakaryocytic differentiation has not been described before. CASE: An 8-month-old male presented with an abdominal swelling, bilateral cervical and inguinal lymphadenopathy and enlarged left epididymis. Ultrasonography of the abdomen revealed a space-occupying lesion in the liver and bilateral enlargement of the kidneys. FNA smears from the right kidney and right submandibular lymph node showed numerous blast cells. Since rare blast cells were positive for myeloperoxidase, a cytodiagnosis of involvement by acute myeloid leukemia (AML) was made. However, following the hematologic diagnosis of acute megakaryoblastic leukemia (M7) from peripheral blood and bone marrow smear examination, FNA smears were reviewed. There were cytoplasmic blebs or protrusions in the blast cells and cytologic features suggestive of their differentiation toward micro-megakaryocytes and megakaryocytes. There was also evidence of shedding of platelets, including numerous giant platelets. The reviewed FNA cytodiagnosis was suggestive of AML (M7). CONCLUSION: Extramedullary involvement by acute megakaryoblastic leukemia (M7) can be suspected based on cytomorphologic features in FNA smears.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Targeted immunotherapy in acute myeloblastic leukemia: from animals to humans

Immunity against acute myeloid leukemia (AML) is demonstrated in humans by the graft-versus-leukemia effect in allogeneic hematopoietic stem cell transplantation. Specific leukemic antigens have progressively been discovered and circulating specific T lymphocytes against Wilms tumor antigen, proteinase peptide or fusion-proteins produced from aberrant oncogenic chromosomal translocations have been detected in leukemic patients. However, due to the fact that leukemic blasts develop various escape mechanisms, antileukemic specific immunity is not able to control leukemic cell proliferation. The aim of immunotherapy is to overcome tolerance and boost immunity to elicit an efficient immune response against leukemia. We review different immunotherapy strategies tested in preclinical animal models of AML and the human trials that spurred from encouraging results obtained in animal models, demonstrate the feasibility of immunotherapy in AML patients.     

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.     

The peculiarities of cytogenetic changes in different types of myelodysplastic syndrome

A cytogenetic study of bone marrow aspirate from 32 patients with different types of myelodysplastic syndrome (MDS) has been carried out. The patients were from eight regions of Ukraine. Chromosome deletions prevailed in the spectrum of karyotype changes. The largest number of chromosome abnormalities was revealed in patients with a refractory anemia with an excess of blasts (66.6% of cases). Chromosomal changes that involved three or more chromosomes occurred among 27% of all karyotype changes examined by us. Transformation of myelodysplastic syndrome to acute myeloid leukemia (AML) was found in 5 patients (45.4% of the cases) among 11 patients with abnormal karyotypes. We propose that cytogenetic confirmation of increased apoptosis in the bone marrow from the myelodysplastic syndrome patients is a phenomenon of chromosome fragmentation. The risk of transformation of myelodysplastic syndrome to acute myeloid leukemia was measured with the use of a new international score system, IPSS.     

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML.MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells.ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells.ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.     

Chromosomal rearrangements with a common breakpoint at 6p23 in five cases of myeloid leukemia

Rearrangement of the short arm of chromosome 6 with a breakpoint at 6p23 was found in five patients with myeloid leukemia. Three of them had different morphological variants of AML (M2, M3, M4) and two blastic crisis of Ph1 negative and Ph1 positive CML. Identical translocation, t(6;9)(p23;q34), was revealed in two patients. One of them had AML (M2), the other blastic crisis of Ph1 negative CML. The blast cells of the last patient were morphologically similar to those in the M2 variant of AML. Translocation (6;9)(p23;q34) was also detected in two AML patients of Rowley and Potter (1976). The role of the breakpoint at 6p23 in myeloid malignancies needs further investigation.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

Acquired pericentric inversion of chromosome 9 in acute myeloid leukemia

Pericentric inversion of chromosome 9 involving the qh region is relatively common as a constitutional genetic aberration without any apparent phenotypic consequences. However, it has not been established as an acquired abnormality in cancer. Among the three patients reported so far in the literature with acquired inv(9), only one had acute myeloid leukemia (AML). Here we describe an unique case where both chromosomes 9 presented with an acquired pericentric inversion with breakpoints at 9p13 and 9q12 respectively, in a AML patient with aberrant CD7 and CD9 positivity. Additionally, one der(9) also showed short arm deletion at 9p21 to the centromeric region and including the p16 gene. The constitutional karyotype was normal. This is probably the first report describing an acquired inv(9) involving both chromosomes 9 in AML. The possible significance of this inversion is discussed.     

Murine models based on acute myeloid leukemia-initiating stem cells xenografting

Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.     

Combinatorial antigen targeting strategies for acute leukemia: application in myeloid malignancy

Background aimsEfforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse.MethodsFor co-transduction with the authors’ previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens.ResultsThe in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors.ConclusionsOverall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.