Acute Undifferentiated Leukemia Research Articles

Immunophenotypic profile of acute leukemia: Critical analysis and insights gained at a tertiary care center in India

To analyze the spectrum of various types and subtypes of acute leukemia. Two thousand five hundred and eleven consecutive new referral cases of acute leukemia (AL) were evaluated based on WHO classification. It included 1,471 cases (58%) of acute lymphoblastic leukemia (ALL), 964 cases (38%) of acute myeloid leukemia (AML), 45 cases (1.8%) of chronic myelogenous leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL, and 2 cases of acute undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%), which comprised of intermediate stage/CALLA positive (73%), early precursor/proBALL (3%). T-cell ALL constituted 24% (351 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), and AML t(8;21) (9%). CMLBC was commonly of myeloid blast crisis subtype (40 cases). B-cell ALL was the commonest subtype in children and AML in adults. Overall incidence of AML in adults was low (53% only). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. A minimal primary panel of nine antibodies consisting of three myeloid markers (CD13, CD33, and CD117), B-cell lymphoid marker (CD19), T-cell marker (CD7), with CD45, CD10, CD34, and HLADR could assign lineage to 92% of AL. Cytogenetics findings lead to a change in the diagnostic subtype of myeloid malignancy in 38 (1.5%) cases.

Significance of terminal deoxynucleotidyl transferase in patients with acute leukemia and malignant lymphoma: results obtained by immunofluorescence assay (author's transl)

Deoxynucleotidyl transferase (TdT) is of diagnostic and therapeutic importance in acute leukaemia and malignant lymphoma. Previous methods for determining TdT activity by biochemical assay are complex and require the use of radioactive substances. There now is available a specific antibody for immunofluorescent demonstration of TdT. The two methods were compared on samples from 43 patients with leukemia or malignant lymphoma. There was very good agreement. High TdT activity was found in 12 of 14 patients with acute lymphatic leukemia and in one of two with acute undifferentiated leukaemia. TdT activity was absent with acute and chronic myeloid leukaemia, chronic lymphatic leukaemia and non-Hodgkin's lymphoma of low malignancy. Immunofluorescence assay, although its results are of similar significance to that obtained with the biochemical tests, is simpler to do. Furthermore, it can be done on normal bone-marrow smear or lymph-node preparations and can be correlated with the morphological findings.

Aleukemic granulocytic sarcoma with associated T-cell lymphoblastic lymphoma in the same lymph node: Morphologic features and molecular signatures

The concept of bilineal acute leukemia is defined in the WHO classification, where, along with the undifferentiated acute leukemia and the biphenotypic acute leukemia, it is included in the group o...

Activated glucocorticoid receptor interacts with the INHAT component Set/TAF-Iβ and releases it from a glucocorticoid-responsive gene promoter, relieving repression: Implications for the pathogenesis of glucocorticoid resistance in acute undifferentiated leukemia with Set-Can translocation

Set/template-activating factor (TAF)-Ibeta, part of the Set-Can oncogene product found in acute undifferentiated leukemia, is a component of the inhibitor of acetyltransferases (INHAT) complex. Set/TAF-Ibeta interacted with the DNA-binding domain of the glucocorticoid receptor (GR) in yeast two-hybrid screening, and repressed GR-induced transcriptional activity of a chromatin-integrated glucocorticoid-responsive and a natural promoter. Set/TAF-Ibeta was co-precipitated with glucocorticoid response elements (GREs) of these promoters in the absence of dexamethasone, while addition of the hormone caused dissociation of Set/TAF-Ibeta from and attraction of the p160-type coactivator GRIP1 to the promoter GREs. Set-Can fusion protein, on the other hand, did not interact with GR, was constitutively co-precipitated with GREs and suppressed GRIP1-induced enhancement of GR transcriptional activity and histone acetylation. Thus, Set/TAF-Ibeta acts as a ligand-activated GR-responsive transcriptional repressor, while Set-Can does not retain physiologic responsiveness to ligand-bound GR, possibly contributing to the poor responsiveness of Set-Can-harboring leukemic cells to glucocorticoids.

Stem Cell Transplantation (SCT) Improves Survival in Acute Biphenotypic (ABL) and Acute Undifferentiated Leukemia (AUL): A Single Centre Experience.

Background: ABL and AUL are associated with an unfavourable outcome for patients (pts) treated with standard-dose therapy alone. Although high-dose therapy has been used successfully in this population, the optimal management of pts with ABL/AUL remains unclear. Methods: A retrospective review was performed involving 24 adult pts with ABL or AUL who were treated in Vancouver between 1984 and 2006. Kaplan-Meier estimates were utilized for event-free survival (EFS) and overall survival (OAS) and a multivariate analysis was performed to determine factors predictive of outcome. Characteristics: Utilizing the WHO criteria, 18 pts had ABL and 6 pts had AUL. There were 17 males and 7 females with a median age of 37 (range 22–75) years. Median white cell count (WCC) at presentation was 10.1 × 109(range: 0.9–196 × 109)/L. Seven pts had poor-risk karyotypes (3 pts complex, 3 pts with t(9,22), 2 pts with11q23 rearrangement, and 1 pt with monosomy 7),12 pts had standard-risk karyotypes, and 5 pts had an unknown karyotype. Induction chemotherapy consisted of Cytosine arabinoside (3–6/m2/day), Daunorubicin, Vincristine and Prednisone with one pt with t(9,22) also having received Imatinib Mesylate. Thirteen pts went on to receive high-dose therapy and SCT. Stem cell source was autologous in 3 pts (all with AUL) or a related (6 pts) or an unrelated donor (4 pts). Eight of 10 pts were in complete remission at the time of SCT, one was in relapse and one had primary refractory ABL. Conditioning was TBI-based in 10 pts and Busulfan-based in 3 pts. Results: EFS and OAS estimates for all 24 patients at 3 years were 25% (95% CI 13%–50%) and 32% (95% CI 17%–58%), respectively. The non-relapse mortality (NRM) for the whole group at 3 years was 43% (95%CI 15%–61%). On multivariate analysis, when compared to pts receiving only standard-dose chemotherapy, pts who underwent high-dose therapy had significantly improved EFS [39% (95% CI 19%–77%) vs. 9% (95% CI 1%–59%), p=.03] and OAS [46% (95% CI 26%–83%) vs.14% (95% CI 3–74%), p=0.01], respectively. Age, WCC at presentation and cytogenetic risk group were not found to significantly influence outcome. Conclusion: Although patient numbers are limited, this experience would suggest that patients with ABL/AUL who achieve complete remission with standard chemotherapy should be considered for high-dose therapy and stem cell transplantation. This approach provides them with the greatest probability of long-term event- free and overall survival.

SET-CAN, the Product of the t(9;9) in Acute Undifferentiated Leukemia, Causes Expansion of Early Hematopoietic Progenitors and Hyperproliferation of Stomach Mucosa in Transgenic Mice

Leukemia-specific chromosome translocations involving the nucleoporin CAN/NUP214 lead to expression of different fusion genes including DEK-CAN, CAN-ABL, and SET-CAN. DEK-CAN and CAN-ABL1 are associated with acute myeloid leukemia and T-cell acute lymphoblastic leukemia, respectively, whereas SET-CAN was identified in a patient with acute undifferentiated leukemia. In addition, SET is overexpressed in solid tumors of the breast, uterus, stomach, and rectum. Ectopic expression of SET-CAN inhibits vitamin-D(3)-induced differentiation of the human promonocytic U937cells, whereas ectopic SET expression induces differentiation. Here, we assessed the leukemogenic potential of SET-CAN in the hematopoietic system of transgenic mice. Although SET-CAN mice showed expansion of an early progenitor cell pool and partial depletion of lymphocytes, the animals were not leukemia-prone and did not show shortening of disease latency after retroviral tagging. This suggests that SET-CAN expression in acute undifferentiated leukemia might determine the primitive phenotype of the disease, whereas secondary genetic lesions are necessary for disease development. Surprisingly, SET-CAN mice developed spontaneous hyperplasia of the stomach mucosa, which coincided with overexpression of beta-catenin and vastly increased numbers of proliferating gastric mucosa cells, suggesting a role of SET-CAN in proliferation of certain epithelial cells.

Impairment of erythroid and megakaryocytic differentiation by a Leukemia‐Associated and t(9;9)‐derived fusion gene product, SET/TAF‐Iβ‐CAN/Nup214

SET-CAN associated with the t(9;9) in acute undifferentiated leukemia encodes almost the entire sequence of SET and the C-terminal two-third portion of CAN, including the FG repeat region. To clarify a role(s) of SET-CAN in leukemogenesis, we developed transgenic mice expressing SET-CAN under the control of the Gata1 gene hematopoietic regulatory domain that is active in distinct sets of hematopoietic cells. SET-CAN transgenic mice showed anemia, thrombocytopenia, and splenomegaly. A significant number of transgenic mice started dying after 6 months post-birth, being in good agreement with the fact that red blood cells and platelets decreased. We found that a significant number of c-kit+ myeloid cells appeared in peripheral blood in transgenic mice. Characterization of the bone marrow cells of transgenic mice indicated impairment in hematopoietic differentiation of erythroid, megakaryocytic, and B cell lineages by SET-CAN. Transgenic mice, in particular, exhibited a high population of the c-kit+Sca-1+Lin- fraction in bone marrow cells compared with that of the control littermates. Our results demonstrate that SET-CAN blocks the hematopoietic differentiation program--one of the characteristics of acute myeloid leukemia.

Liposomal Cytarabine in the Central Nervous System (CNS) Prophylaxis of Elderly Patients with Aggressive B-Cell Non-Hodgkin's Lymphoma (NHL) and Undifferentiated Acute Leukemia (UAL): Preliminary Results of a Single-Center Experience.

Background: CNS involvement in acute lymphoblastic leukemia/AUL is a well-recognized event and CNS prophylaxis is considered mandatory. In NHL, meningeal relapse occurs more rarely, ranging from 4–14% depending on histology, anatomical location and biological parameters. Flow cytometric analysis of cerebrospinal fluid (CSF), however, detected occult lymphomatous meningitis in 22% of NHL cases at risk at diagnosis (Hegde et al. Blood 2005; 105:496). CNS prophylaxis is currently recommended only in high-risk disease (stage IV/high IPI score) and in patients with extranodal NHL. Sustained-release liposomal cytarabine (DepoCyte®), which is licensed for meningeal relapse in NHL, has proved effective in treating lymphomatous and leukemic meningitis (Glantz et al. J Clin Oncol 1999; 17:3110; Sancho et al. Haematologica 2006; 91:ECR02). Intrathecal (IT) liposomal cytarabine is distributed throughout the CSF and has an extended half-life, allowing administration once every 2–4 weeks (Chamberlain et al. Arch Neurol 1995; 52:912). We therefore tested the efficacy of liposomal cytarabine in CNS prophylaxis for elderly patients with aggressive NHL or AUL, with the aim of testing the safety of IT treatment in elderly patients and the efficacy of liposomal cytarabine in preventing lymphoma/leukemia CNS relapse.Methods: From June to November 2005, 4 patients > 70 years of age entered the study. Diagnoses were: 2 stage IV, IPI 3, diffuse large B-cell lymphoma (DLBCL); 1 mantle cell lymphoma (MCL), and 1 AUL; 2 patients had extranodal bulky disease (1 psoas muscle, 1 retro-orbital plus paranasal sinus involvement). As first-line treatment, the 3 NHL cases were given R-CHOP every 21 days for 6 cycles. The patient with AUL received conventional 3-drug induction (vincristine/idarubicine/prednisone) every week for 3 weeks, followed by 3 courses of L-VAMP (vincristine/cytarabine/intermediate-dose methotrexate/leucovorin rescue) and then conventional maintenance (6-mercaptopurine/methotrexate and monthly re-induction with vincristine/prednisone). All patients received CNS prophylaxis with IT liposomal cytarabine 50 mg followed by systemic steroid injection. In NHL cases, IT therapy was given the day before systemic chemotherapy for a total of 4 administrations; in AUL, prophylaxis was given every 4 weeks during induction and maintenance for a total of 6 doses.Results: Three (2 NHL and 1 AUL) patients achieved a complete response (CR) and 1 (NHL) achieved a partial response, with response durations of 4, 5, 6+ and 8+ months, respectively. As of July 2006, after a median follow-up of 10 months (range 9–12), all patients were alive; 2 (1 DLBCL, 1 AUL) were in continuous CR, and 2 (1 DLBCL, 1 MCL) had progressive disease and were receiving second-line treatment. Isolated relapse of leukemia/lymphoma in the CNS was not seen. Liposomal cytarabine was well tolerated; no drug-related side effects or hematological toxicities were recorded.Conclusions: As occult CNS involvement has been shown to occur in >20% of newly diagnosed patients with high-risk NHL, flow cytometry and cytospin analysis of CSF at diagnosis should be implemented in order to adequately target CNS prophylaxis. Liposomal cytarabine should be the drug of choice for CNS prophylaxis, particularly in elderly patients.

FLJ/P1 Is a New Mitochondrial Dehydrogenase Important in Respiration and Apoptosis.

Leukemia is a neoplastic proliferation of cells of hematopoietic origin that arises following somatic mutation in a single hematopoietic stem cell, the progeny of which forms a clone of leukemia cells. Genetic alterations leading to leukemia transformation of a cell are often associated with major alterations of chromosomes that can be detected by studying cells of the leukemic clone in mitosis. One of these alterations is a chromosomal translocation that is often used to identify genes potentially involved in other type of rearrangements such as deletions. Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (ALL), in differentiated and undifferentiated acute myeloid leukemia (AML) and in chronic myeloid leukemia at progression to blast crisis (CML-BC). Recently, we have shown the presence of a myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222-229,1999). In addition, a new cell line has been established from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia 15:1471-1474, 2001). The molecular characterization of this translocation led to the identification of a new gene, FLJ13639, that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). Furthermore, three transcripts and proteins were found to be differentially expressed for this gene, where P1 is potentially the active form of dehydrogenase, while P2 and P3 are lacking the co-activator site. We previously reported that one of the consequences of the loss of FLJ13639 is the over-expression of CD24 that appears to provide leukemia cells with a proliferation and invasiveness advantage, as well as a certain degree of chemoresistance. In addition, data on patient samples indicated a correlation between survival and CD24High/FLJ/P1Low expression profile (Blood 106 (11), Nov 2005). We show that FLJ/P1 is a new mitochondrial protein that is important for the respiration and apoptosis processes. Restoration of the FLJ/P1 function induced CD24 down-regulation, decreased invasive potential as assessed by Matrigel assay and decreased chemoresistance. FLJ/P1 function restoration might represent a new potential therapy, that could, when combined with the assessment of FLJ/P1-CD234 expression profiles, allow the future development of personalized treatment.

Fusion of NUP98 and the SET binding protein 1 (SETBP1) gene in a paediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12)

Three NUP98 chimaeras have previously been reported in T cell acute lymphoblastic leukaemia (T-ALL): NUP98/ADD3, NUP98/CCDC28A, and NUP98/RAP1GDS1. We report a T-ALL with t(11;18)(p15;q12) resulting in a novel NUP98 fusion. Fluorescent in situ hybridisation showed NUP98 and SET binding protein 1(SETBP1) fusion signals; other analyses showed that exon 12 of NUP98 was fused in-frame with exon 5 of SETBP1. Nested polymerase chain reaction did not amplify the reciprocal SETBP1/NUP98, suggesting that NUP98/SETBP1 transcript is pathogenetically important. SETBP1 has previously not been implicated in leukaemias; however, it encodes a protein that specifically interacts with SET, fused to NUP214 in a case of acute undifferentiated leukaemia.

A tribute to Sidney Farber – the father of modern chemotherapy

Sidney Farber, world-renowned paediatric pathologist, made major contributions to his field but is acknowledged as the father of the modern era of chemotherapy. He recognised that folic acid stimulated leukaemic cell growth and enhanced disease progression. He hypothesised that folic acid antagonists would inhibit or arrest the proliferation of cancer cells. His landmark study, published in 1948, demonstrated that a number of folic acid antagonists, including 4-aminopteroyl-glutamic acid (aminopterin) produced temporary remissions in children with acute undifferentiated leukaemia. These observations lead to the development and use of other chemotherapeutic agents, either singly or, more effectively, in combination for treating childhood and adult malignancies. He introduced actinomycin D for the treatment of metastatic and localised Wilms tumour. Under his guidance and leadership, both the 'Jimmy Fund', one of the first comprehensive paediatric oncology treatment centres, and the Children's Cancer Research Foundation, which later became the Dana-Farber Cancer Institute, were founded.

CD117 Expression as an aid to identify immature myeloid cells and foci of ALIP in bone marrow trephines

American Journal of HematologyVolume 81, Issue 1 p. 79-79 Letters and CorrespondenceFree Access CD117 Expression as an aid to identify immature myeloid cells and foci of ALIP in bone marrow trephines Kikkeri N. Naresh, Kikkeri N. Naresh Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this authorIrvin A. Lampert, Irvin A. Lampert Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this author Kikkeri N. Naresh, Kikkeri N. Naresh Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this authorIrvin A. Lampert, Irvin A. Lampert Department of Histopathology, Hammersmith Hospital and Imperial College, London, UKSearch for more papers by this author First published: 20 December 2005 https://doi.org/10.1002/ajh.20452Citations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Valverde LR, Matutes E, Farahat N, Heffernan A, Owusu-Ankomah K, Morilla R, Catovsky D. C-kit receptor (CD117) expression in acute leukemia. Ann Hematol 1996; 72: 11– 15. 2Wells SJ, Bray RA, Stempora LL, Farhi DC. CD117/CD34 expression in leukemic blasts. Am J Clin Pathol 1996; 106: 192– 195. 3Siitonen T, Savolainen ER, Koistinen P. Expression of the c-kit proto-oncogene in myeloproliferative disorders and myelodysplastic syndromes. Leukemia 1994; 8: 631– 637. 4Bene MC, Bernier M, Casasnovas RO, Castoldi G, Knapp W, Lanza F, Ludwig WD, Matutes E, Orfao A, Sperling C, van't Veer MB. The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL). Blood 1998; 92: 596– 599. Citing Literature Volume81, Issue1January 2006Pages 79-79 ReferencesRelatedInformation

CD24High / FLJ/P1Low Expression Correlates with Bad Prognosis and Shorter Survival in Adult ALL.

Leukemia is a neoplastic proliferation of cells of hematopoietic origin that arises following somatic mutation in a single hematopoietic stem cell, the progeny of which forms a clone of leukemia cells. Genetic alterations leading to leukemia transformation of a cell are often associated with major alterations of chromosomes that can be detected by studying cells of the leukemic clone in mitosis. One of these alterations is a chromosomal translocation that is often used to identify genes potentially involved in other type of rearrangements such as deletions. Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (ALL), in differentiated and undifferentiated acute myeloid leukemia (AML) and in chronic myeloid leukemia at progression to blast crisis (CML-BC). Recently, we have shown the presence of a myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222–229,1999). In addition, a new cell line has been established from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia 15:1471–1474, 2001). The molecular characterization of this translocation led to the identification of a new gene, FLJ13639 , that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). Furthermore, three transcripts and proteins were found to be differentially expressed for this gene, where P1 is potentially the active form of dehydrogenase, while P2 and P3 are lacking the co-activator site. We report here that one of the consequences of the loss of FLJ13639 is the over-expression of CD24 that appears to provide leukemia cells with a proliferation and invasiveness advantage, as well as a certain degree of chemoresistance. FLJ13639 is a new mitochondrial protein that seems to be important for the respiration and apoptosis processes. In addition, semi-quantitative RT-PCR for CD24 and FLJ13639 /P1 was performed in a series of cell lines as well as 29 adult ALL samples at diagnosis. Fifty percent of the samples showed a CD24High / FLJ/P1Low profile whereas the remaining samples showed either a balanced expression or a CD24Low / FLJ/P1High profile. These preliminary data on patient samples indicated a correlation between survival and CD24High / FLJ/P1Low expression profile (p=0.04). The current median time survival for the CD24High / FLJ/P1Low group is 9 months whereas the CD24Low / FLJ/P1High group is 28 months. Therefore, this appears to be a new potential prognostic marker for adult ALL. [Display omitted]

Trisomy 21 in neoplastic cells

Trisomy 21 as an acquired clonal chromosome change has been described in 642 of the 10,625 human neoplasms with chromosome aberrations known from the cytogenetic literature. A total of 590 of the 642 cases (92%) are hematologic disorders and malignant lymphomas. The incidence of trisomy 21 is similar (4.1%-6.7%) in acute myeloid leukemia (AML), chronic myeloid leukemia, myeloproliferative disorders, myelodysplastic syndromes, chronic lymphoproliferative disorders, and malignant lymphomas; it is substantially higher (14.8%) in acute lymphocytic leukemia (ALL). In most cases, the extra chromosome 21 is present together with other numerical and/or structural changes. Acquired trisomy 21 is the only karyotypic abnormality in only 0.4%. Trisomy 21 has never been reported as the sole anomaly in a solid tumor. The cytogenetic literature contains information on 62 patients with constitutional trisomy 21 and a malignant disorder in which the tumor cells have been analyzed by banding techniques. Thirty-four of the 62 patients had AML, 16 had ALL, and 2 had acute undifferentiated leukemia. The 52 leukemic Down syndrome (DS) cases account for 1.4% of the total acute leukemias, an overrepresentation that parallels the generally increased risk of leukemia development in DS. Sixty-three percent of the ALL patients and 79% of those with AML had additional changes superimposed on constitutional trisomy 21. These included several of the characteristic primary leukemia-associated aberrations: 5q-, 7q-, +8, and t(8;21) in AML, and t(1;19), t(4;11), 6q-, and 14q + in ALL. Thus, it seems that the pattern of acquired karyotypic changes is similar in patients with DS and in individuals with a normal constitutional karyotype.

The oncoprotein I-2PP2A/SET negatively regulates the MEK/ERK pathway and cell proliferation

I-2PP2A/SET, the translocation breakpoint-encoded protein expressed in acute undifferentiated leukemia, was identified as an inhibitor of protein phosphatase 2A (PP2A). Induction of exogenous I-2PP2A/SET at a ratio of 1:1 to the endogenous protein resulted in suppression of cell proliferation. In contrast, siRNA-mediated depletion of I-2PP2A/SET resulted in enhanced cell proliferation. Depletion of I-2PP2A/SET was accompanied with a decrease in the number of cells in G1 and an increase in cells in S phase. To examine the mode of action by which I-2PP2A/SET suppresses cell proliferation, we determined the effect of over-expressed I-2PP2A/SET on ERK activation. I-2PP2A/SET suppressed activation of ERK following EGF stimulation but did not affect activation levels of stress kinases, JNK and p38. By contrast, knocking down I-2PP2A/SET by siRNA resulted in enhancement of ERK and MEK activations, suggesting that I-2PP2A/SET negatively regulates MEK/ERK. These data suggest that I-2PP2A/SET negatively regulates cell growth by inhibiting the G1/S transition and inhibiting the MEK/ERK pathway stimulated by external stimuli. These data demonstrate that I-2PP2A/SET potentially functions as a tumor suppressor.

Profiling of nuclear extract proteins from human neuroblastoma cell lines: the search for fingerprints

Purpose Neuroblastoma (NB) commonly presents with advanced disease at diagnosis and is associated with poor survival. If identified early, however, survival is improved suggesting a benefit of early detection. The authors have used proteomics technology in an attempt to identify novel markers that permit early detection of NB and characterize its molecular makeup. Methods Three different human NB cell lines SK-N-AS, SK-N-DZ, and SK-N-FI were subjected to series of biochemical fractionation steps to extract nuclear proteins. These proteins were analyzed for differential expression by 2-dimensional polyacrylamide gel electrophoresis. Polypeptides of interest were subsequently identified by liquid chromatography–linked tandem mass spectrometry. Results Multiple proteins were identified in these human NB cell lines including SET (a ubiquitous nuclear protein), stathmin (a cytosolic signal transduction protein), and grp94 (a heat shock protein). SET is a putative oncogene associated with the chromosomal translocation (6;9) leading to acute undifferentiated leukemia. Stathmin is an oncogene found in greater abundance in leukemic cells compared to nonleukemic cells. A total of 94-kDa glucose-regulated protein has been shown to be protective in human breast cancer cells in vitro and related with the occurrence, differentiation, and progression of human lung cancer. The first protein has not been previously associated with NB. Conclusions The identification of these 3 previously unrecognized cancer-related potential biomarkers in human NB cell lines may prove useful in developing diagnostic tests. The proteomic methodology of 2-dimensional polyacrylamide gel electrophoresis/mass spectrometry also provides an improved opportunity to understand the natural history of NB and develop novel chemotherapeutic agents for this prevalent childhood malignancy with a dismal outcome.

The CALM/AF10 Fusion: Molecular Analysis of the Fusion Transcripts in 13 Cases of AML and ALL; Gene Expression Profiling Reveals HOX Gene Deregulation.

The t(10;11)(p13;q14) is a recurring translocation associated with the CALM/AF10 fusion gene which is found in undifferentiated leukemia, acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma with poor prognosis. The CALM/AF10 fusion protein was reported to be the most common fusion protein in T-ALL with TCR gamma delta rearrangement. We have analyzed samples from 9 patients with different types of leukemia: case 1 (AML M2), case 2 (AML M0), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), case 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), with a t(10;11) translocation suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM mRNA by RT-PCR and sequence analysis. All these patients were found positive for the CALM/AF10 fusion.In addition, we analyzed a series of twenty-nine patients with T-ALL with gamma delta rearrangement. Among these patients, four were positive for CALM/AF10 transcripts, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. We found three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 11). There was no correlation between disease phenotype and breakpoint location. The patients were 5 to 46 years old (median 25). Ten CALM/AF10 positive patients were further analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent of the pattern seen in leukemias with rearrangements of the MLL gene, and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. It is known that alhambra, the Drosophila homologue of AF10 can act on polycomb group responsive elements, which play a critical role in the regulation of the HOX gene clusters. It is thus conceivable that the CALM/AF10 fusion proteins acts in a dominant negative fashion on wild type AF10 function relieving the repression that is presumably normally exerted by AF10 on the expression of HOX genes.

NUP98-HOXD13 Transgenic Mice Develop Myelodysplastic Syndrome, Acute Myeloid Leukemia, and Pre-T Lymphoblastic Leukemia.

The NUP98 gene is located at chromosome 11p15 and encodes the 98 kd component of the nuclear pore complex; this protein normally functions as a docking protein involved in nucleocytoplasmic transport. NUP98 is fused to at least 15 different partner genes by chromosomal translocation in a wide spectrum of hematological malignancies including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and pre-T lymphoblastic leukemia (pre-T LBL). Over half of the known NUP98 gene fusions involve fusions to a HOX family member; these fusions invariably retain the amino terminal FG repeats of NUP98 and the homeodomain DNA-binding region of the HOX partner. The NUP98-HOXD13 fusion was initially identified in a patient with MDS that subsequently transformed to erythroleukemia, and has subsequently been identified in AML M1 and M2 patients as well. To model this disease in vivo, we generated transgenic mice which expressed the NUP98-HOXD13 (NHD13) fusion from vav regulatory elements.The NHD13 transgene is ubiquitously expressed in hematopoietic tissues such as thymus, spleen, and bone marrow, and is not expressed in other tissues. Serial CBCs from clinically healthy mice aged 4–7 months demonstrated a progressive neutropenia, lymphopenia, anemia, and macrocytosis. Peripheral blood smears showed signs of dysplasia including giant platelets and hypersegmented neutrophils; bone marrow exam showed an increase number of dysplastic binucleate erythroblasts and increased apoptosis, consistent with a diagnosis of MDS. 10/10 (100%) of the NHD13 mice died of hematologic disease by 14 months of age; in contrast, none of the non-transgenic control littermates developed evidence of hematologic disease. We classified the hematologic diseases according to the Bethesda proposals. Three mice died with MDS, two mice had pre-T LBL, two had acute undifferentiated leukemia, one had megakaryocytic leukemia, one had myeloid leukemia with maturation, and one had both pre-T LBL and erythroid leukemia.The malignant blasts from mice with pre-T LBL showed monoclonal T-cell receptor B gene rearrangements and were positive for CD3, 4, and 8. The mouse with megakaryocytic leukemia had serial CBCs documenting a platelet count of 3.2 million/uL, rising to >15million/uL at the time of death. This mouse had CD41+ megakaryocytes and megakaryoblasts invading the liver and spleen, and an osteosclerotic bone marrow reminiscent of chronic idiopathic myelofibrosis (CIMF). The mouse with concurrent pre-T LBL and erythroid leukemia had replacement of the thymus and infiltration of the lung with T-lymphoblasts which had a clonal TCRB gene rearrangement; interestingly, the spleen, liver, and bone marrow of this mouse were invaded with erythroblasts that were negative for CD3 and TCRB gene rearrangements.We conclude that the NHD13 transgene consistently induces an MDS, of variable severity, in these mice. Some mice die of severe anemia due to MDS, and MDS transforms into an acute non-lymphoid leukemia in other mice. Still other mice die of pre-T LBL which we believe evolves in the thymus separately from the MDS. These data demonstrate that the NHD13 fusion gene is transforming in both lymphoid and myeloid cells.

SET (TAF-IB) Gene Is Highly Expressed in Acute Leukemia Patients without Associated with Methylation Status.

SET gene, also known as TAF-I beta, was originally identified as a component of the SET-CAN fusion gene, which results from t(9;9) translocation, in a patient with acute undifferentiated leukemia (AUL). SET gene encodes a nuclear phosphoprotein that ubiquitously expressed. There is an accumulating data that suggest a role for SET in gene silencing either through prevention of histone acetylation as a subunit of inhibitor of acetyltransferases (INHAT) complex or through inhibition of DNA demethylation. SET also inhibits the activity of protein phosphatase 2A, which involves in regulation of cell proliferation and differentiation, and blocks DNase activity of the tumor metastasis suppressor NM23-H1. Taken together, available data suggest that SET might play a role in tumorogenesis via tumor suppressor gene silencing or inhibition of apoptosis.In this study, we investigated SET gene expression level in bone marrow samples of 77 patients with acute leukemia (57 acute lymphoblastic leukemia (ALL) and 26 acute myeloid leukemia (AML)) and 5 control bone marrow samples from healthy volunteers using quantitative real-time RT-PCR. The ALL patient ages ranged 10months – 17 years, with a median of 6 years and the AML patient ages ranged 1–72 years, with a median of 18 years. For determination of the prognostic significance of SET gene expression in ALL patients, the association between patient's clinical characteristics and the SET gene expression level was assessed usind the Pearson's chi-square test or Fisher's Exact test. Overall survival (OS) in 48 patients and relapse-free survival (RFS) in 37 patients with ALL at 6 years (median follow-up 35 months, range 1–79 months) were analyzed according to Kaplan-Meier method. We also studied methylation of various genes (p15, p16, p73, SOCS1, RAR beta, E-Cadherin, GSTP1, DAP-Kinaz, ER and 5-HIC) using methylation spesific PCR and COBRA analysis in AML samples to examine whether high SET gene expression play a role in tumorogenesis via gene silencing.Here we demonstrate that 54.4% of ALL patients and 53.8% of AML patients show two fold and higher SET expression level compare to control samples (p=0.005 and p=0.016 respectively). There were no significant association between SET gene expression level and age, peripheral WBC count, sex, FAB group and immunophenotype (P=0.823, P=0.182, P=1.00, P=0.132, P=0.751) in ALL. The OS was not significantly different between high (83.10% ± 6.92%) and low SET expressed patients (51.14% ± 18.73%) (log-rank=3.36, P=0.067) and the probability of RFS was not significantly different between high (81.20% ± 7.60%) and low SET expressed patients (80.00% ± 17.89%) with ALL (log-rank=0.01, P= 0.92). There was no statisticall association between methylation index and SET gene expression level (P=0.618).Our data suggest that high level of SET expression may play an important role in leukemogenesis. Further analyses are required to determine prognostic significance of SET gene expression different types of leukemia.

Aberrant intracellular localization of SET-CAN fusion protein, associated with a leukemia, disorganizes nuclear export.

The SET-CAN fusion gene is the product of a chromosomal rearrangement found on 9q34 associated with an acute undifferentiated leukemia. SET-CAN encodes an almost complete SET protein fused to the C-terminal two-thirds of CAN. SET is also known as TAF-Ibeta, a histone chaperone and intracellular inhibitor of protein phosphatase 2A, whereas CAN is identical to Nup214, a nucleoporin protein. To obtain insight into the leukemogenic function of SET/TAF-Ibeta-CAN/Nup214, we have examined its subcellular localization. Immunofluorescence analyses showed that SET/TAF-Ibeta and CAN/Nup214 are found in the nucleus and the nuclear envelope, respectively, whereas the majority of SET/TAF-Ibeta-CAN/Nup214 is localized in the nucleus. SET/TAF-Ibeta-CAN/Nup214 interacted with hCRM1, one of the nuclear export factors, and caused aberrant intracellular localization of hCRM1. In cells expressing SET/TAF-Ibeta-CAN/Nup214, a protein containing a nuclear export signal accumulated in the nucleus. The export of this protein was partially restored by overexpression of hCRM1. These results suggest that aberrantly localized molecules associated with SET/TAF-Ibeta-CAN/Nup214 may be involved in oncogenesis.