Debra M. Lillington

Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34− fraction

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Therapy-Related Myelodysplasia and Secondary Acute Myelogenous Leukemia After High-Dose Therapy With Autologous Hematopoietic Progenitor-Cell Support for Lymphoid Malignancies

PURPOSEnTo evaluate the incidence of and risk factors for therapy-related myelodysplasia (tMDS) and secondary acute myelogenous leukemia (sAML), after high-dose therapy (HDT) with autologous bone marrow or peripheral-blood progenitor-cell support, in patients with non-Hodgkins lymphoma (NHL).nnnPATIENTS AND METHODSnBetween January 1985 and November 1996, 230 patients underwent HDT comprising cyclophosphamide therapy and total-body irradiation, with autologous hematopoietic progenitor-cell support, as consolidation of remission. With a median follow-up of 6 years, 27 (12%) developed tMDS or sAML.nnnRESULTSnMedian time to development of tMDS or sAML was 4.4 years (range, 11 months to 8.8 years) after HDT. Karyotyping (performed in 24 cases) at diagnosis of tMDS or sAML revealed complex karyotypes in 18 patients. Seventeen patients had monosomy 5/5q-, 15 had -7/7q-, seven had -18/18q-, seven had -13/13q-, and four had -20/20q-. Twenty-one patients died from complications of tMDS or sAML or treatment for tMDS or sAML, at a median of 10 months (range, 0 to 26 months). Sixteen died without evidence of recurrent lymphoma. Six patients were alive at a median follow-up of 6 months (range, 2 to 22 months) after diagnosis of tMDS or sAML. On multivariate analysis, prior fludarabine therapy (P =.009) and older age (P =.02) were associated with the development of tMDS or sAML. Increased interval from diagnosis to HDT and bone marrow involvement at diagnosis were of borderline significance (P =.05 and.07, respectively).nnnCONCLUSIONntMDS and sAML are serious complications of HDT for NHL and are associated with very poor prognosis. Alternative strategies for reducing their incidence and for treatment are needed.

Segmental uniparental disomy is a commonly acquired genetic event in relapsed acute myeloid leukemia

Despite advances in the curative treatment of acute myeloid leukemia (AML), recurrence will occur in the majority of cases. At diagnosis, acquisition of segmental uniparental disomy (UPD) by mitotic recombination has been reported in 15% to 20% of AML cases, associated with homozygous mutations in the region of loss of heterozygosity. This study aimed to discover if clonal evolution from heterozygous to homozygous mutations by mitotic recombination provides a mechanism for relapse. DNA from 27 paired diagnostic and relapsed AML samples were analyzed using genotyping arrays. Newly acquired segmental UPDs were observed at relapse in 11 AML samples (40%). Six were segmental UPDs of chromosome 13q, which were shown to lead to a change from heterozygosity to homozygosity for internal tandem duplication mutation of FLT3 (FLT3 ITD). Three further AML samples had evidence of acquired segmental UPD of 13q in a subclone of the relapsed leukemia. One patient acquired segmental UPD of 19q that led to homozygosity for a CEBPA mutation 207C>T. Finally, a single patient with AML acquired segmental UPD of chromosome 4q, for which the candidate gene is unknown. We conclude that acquisition of segmental UPD and the resulting homozygous mutation is a common event associated with relapse of AML.

Proposals for standardized Protocols for cytogenetic analyses of acute leukemias, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, and myelodysplastic syndromes

The impact of cytogenetic characterization based on chromosome banding analyses and fluorescence in situ hybridization on clinical decision making has increased dramatically during recent years. Therefore, laboratory techniques have to be optimized to provide reliable results for optimal patient care. In addition, quick and correct results save time and money by preventing unnecessary additional diagnostics and suboptimal treatment approaches. It was our aim to present proposals for standardized protocols to improve the diagnosis, and hence the treatment outcome, of hematologic malignancies.

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34+CD38− stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34+CD38+ progenitors were reduced. Residual normal CD34+ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.

Upregulation of lipocortin 1 inhibits tumour necrosis factor‐induced apoptosis in human leukaemic cells: a possible mechanism of resistance to immune surveillance

The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A2 (cPLA2). The steroids dexamethasone (Dex) and 1,25(OH)2u2003D3 both render U937 leukaemic cells resistant to TNF‐induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF‐induced activation of cPLA2. Dex had no direct effect on cellular cPLA2 levels, but facilitated cPLA2 degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA2 inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex‐induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF‐sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF‐induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative‐feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune‐mediated killing.

Novel chromosome findings in bladder cancer cell lines detected with multiplex fluorescence in situ hybridization

Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82, 253J, and SCaBER. Alterations of chromosome 9 are the most frequent cytogenetic and molecular findings in transitional cell carcinomas of all grades and stages, while changes of chromosomes 3, 4, 8, 9, 11, 14, and 17 are also frequently observed. In the present study, alterations previously described, including del(8)(p10), del(9)(p10), del(17)(p10), and overrepresentation of chromosome 20, as well as several novel findings, were observed. These novel findings were a del(15)(q15) and isochromosome 14q, both occurring in three of nine cell lines examined. These abnormalities may reflect changes in bladder tumor biology. M-FISH represents an effective preliminary screening tool for the characterization of complex tumor karyotypes.

Characteristics of human primary mantle cell lymphoma engraftment in NSG mice.

Mantle cell lymphoma (MCL) is a clinically heterogeneous, but often aggressive lymphoma characterized by the IGH:CCND1 translocation and cyclin D1 (CCND1) over‐expression. Chromosomal instability, due to disrupted DNA damage response, in conjunction with abnormal activation of cell survival mechanisms underlies the aggressive clinical course in MCL (Jares etxa0al, 2012). n nIn recent years, improved understanding of lymphoma biology has led to the development of a number of small molecule inhibitors. However, the relative rarity of MCL (incidence 0·55 per 100xa0000) (Smedby & Hjalgrim, 2011) poses a challenge in effectively evaluating these drugs in patients. n nIn vitro studies have been limited by the difficulty of culturing primary MCL cells. Murine models of MCL cell lines are relatively easy to establish in SCID or NOD/SCID/IL2Rγ null (NSG) mice (Wang etxa0al, 2007, 2008a; Weston etxa0al, 2010) but have their limitations. Until a couple of years ago, the only primary mouse model of human MCL described in the literature was established by injection of primary MCL cells into subcutaneous human bone grafts implanted in SCID mice (SCID‐Hu model) (Wang etxa0al, 2008b). Recently, however, disseminated models of human primary MCL have been established in NSG mice (Iyengar etxa0al, 2012; Klanova etxa0al, 2014). We report our experience here in further detail, focussing on the characteristics of MCL engraftment in this model. n nWe used 8‐ to 12‐week‐old NSG mice that were sub‐lethally irradiated (3·75xa0Gy) 24xa0h prior to transplantation. Before undertaking xenograft studies with primary cells, we used the MCL cell line JEKO‐1 to assess kinetics, disease burden and distribution of MCL cells in NSG mice. JEKO‐1 cells were transduced with firefly luciferase and injected intravenously into irradiated mice at two doses – 0·5xa0×xa0106 and 2xa0×xa0106 cells. Bioluminescent imaging was performed at weekly intervals following injection of D‐luciferin. All mice became ill with marked weight loss and had to be sacrificed by day 29. Bioluminescence was observed in the bone marrow and spleen in all mice. Mice injected with the higher cell dose had more rapid disease progression, developed hind leg weakness and had bioluminescence in the central nervous system (CNS) on imaging, indicating involvement (Figxa01). n n n nFigure 1 n nLongitudinal quantitative analysis of bioluminescent imaging (BLI): NSG mice were injected intravenously with the JEKO‐1 cell line transduced with luciferase (Luc) reporter constructs. (A) Representative dorsal view (upper panels) and ventral ... n n n nFollowing this, seven cryopreserved primary MCL samples were identified from the Barts Cancer Institute tissue bank. An additional fresh primary sample derived from a splenectomy was included in the cohort. Ethical approval was obtained from East London and the City Local Research Ethics Committee. Written informed consent was obtained from patients according to the Declaration of Helsinki. All samples had a classical MCL phenotype with CD5/CD20 positivity and were confirmed to have the IGH:CCND1 translocation by fluorescence inxa0situ hybridization (FISH). Irradiated NSG mice were injected intravenously with a dose of 107 unselected MCL cells each. Flow cytometry was performed for mouse CD45 and human CD45, CD3, CD5 and CD20 on peripheral blood samples taken from mice at 3, 6 and 12xa0weeks. Mice were sacrificed at 20xa0weeks, or earlier if they met Home Office guidelines, and tissue was harvested for immunohistochemistry (IHC). Cells were flushed from mouse femur for flow cytometry. n nAt 20xa0weeks, MCL cells were found in the bone marrow and spleen of mice injected with 2 out of the 7 cryopreserved primary samples. Both samples that engrafted had blastoid morphology and one was obtained from a patient with relapsed disease. FISH for IGH:CCND1 on cell suspensions prepared from spleen of NSG mice further confirmed engraftment. None of the mice that engrafted appeared to have bowel involvement as assessed by IHC. Lymphadenopathy was not found at sacrifice. Scattered human CD20‐positive cells were seen in the liver but this was not a consistent feature. Mice remained relatively well until sacrifice (Figxa02A–F). As a next step, secondary transplantation of MCL cells isolated from NSG spleen (107 cells per mouse) was undertaken. Once again, engraftment was seen in mouse spleen and bone marrow on sacrifice at 20xa0weeks (Figxa02G). n n n nFigure 2 n nEngraftment of human primary mantle cell lymphoma (MCL) in NSG mice: T‐cell depleted human primary mononuclear cells were injected intravenously at a dose of 107 cells per mouse. Peripheral blood was sampled from mice at 3, 6 and 12xa0weeks, ... n n n nIn addition to the two cryopreserved samples, evidence of engraftment was also seen in the spleen of NSG mice injected with the fresh primary sample (non‐blastoid). Interestingly, there appeared to be co‐existence of MCL cells and T‐cells in the spleen of mice injected with fresh MCL cells, with tumour cells concentrated around blood vessels. However, these mice had to be sacrificed at 7xa0weeks due to illness and T‐cell infiltration was found in the liver and bone marrow, without evidence of MCL. We found a similar proliferation of T cells but without evidence of MCL in one of the seven cryopreserved samples that had high T‐cell content (>10%), indicating T‐cell depletion may be important in this scenario. n nTherefore, similar to the recent report by Klanova etxa0al (2014), we demonstrate human primary MCL engraftment in NSG mice. In contrast to their study where mice were injected with a variable cell dose (1–8xa0×xa0107 cells), we injected all mice with a fixed dose of 107 cells. This may explain the lower rate of engraftment in our study. Both cryopreserved samples that engrafted in our study had blastoid morphology, suggesting that a higher cell dose may be required for engraftment of non‐blastoid MCL in this model. n nIn our experiments, mice with primary MCL engraftment were not visibly ill at 20xa0weeks and disease burden was heaviest in the spleen. In contrast, disease progression was rapid in the JEKO‐1 xenograft, with CNS involvement and hind leg weakness developing by 4xa0weeks. These findings mirror those of Klanova etxa0al (2014), and are important considerations when designing pre‐clinical experiments involving these models. The longer overall survival of NSG mouse models of primary human MCL could be an advantage for pre‐clinical testing of newer agents, which often require longer periods of administration for efficacy. n nFinally, our study demonstrates, similar to the findings of Klanova etxa0al (2014), that secondary transplantation can be successfully carried out in this model, highlighting the self‐renewal and tumour‐initiating capacity of primary MCL cells. In summary, this NSG model of human primary MCL is a promising inxa0vivo model for both pre‐clinical drug testing and further understanding MCL biology. Our research provides further insight into the advantages and limitations of this model, which will be crucial for its effective use in pre‐clinical research.