Cell Cycle Deregulation Determines Acute Transformation In Chronic Type Adult T-Cell Leukemia/Lymphoma

Introduction Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1-induced neoplasm with four clinical subtypes; acute, lymphoma, chronic and smoldering. Although chronic and smoldering subtypes are regarded as indolent ATL, about half of these cases progress to acute type ATL and subsequent death. Therefore, cases of indolent ATL also have poor prognosis and acute transformation is a predictive indicator for patients with indolent ATL. However, the molecular pathogenesis of acute transformation remains unknown. In the present study, oligo-array comparative genomic hybridization (CGH) and comprehensive gene-expression profiling (GEP) were applied to 27 and 35 cases of chronic and acute type ATL, respectively, in an effort to delineate the molecular pathogeneses of ATL, and especially the molecular mechanism of acute transformation. Materials and Methods All DNA and RNA used in this study were extracted from purified CD4-positive cells. Oligo-array CGH analyses and comprehensive GEP analyses were performed on 27 and 35 cases of chronic and acute type ATL, respectively. Subsequently, we established Tet-OFF ATL cell lines for functional analyses. Results Oligo-array CGH revealed that genomic loss of 9p21.3 was significantly characteristic of acute type ATL, but not chronic type ATL (p-value= 0.039). Although the minimal common deleted region of 9p21.3 contained MTAP, CDKN2A and CDKN2B, the expression level of only CDKN2A was reduced with genomic loss of 9p21.3 (Figure 1). Moreover, analysis of serial samples of a chronic type ATL patient showing acute transformation also revealed that reduction of CDKN2A expression by 9p21.3 loss was associated with acute transformation in this case. CDKN2A contains two known variants, INK4a and ARF. Re-expression of INK4a and ARF suppressed proliferation of Tet-OFF ATL cell lines, while the suppression efficiency of INK4a was stronger than that of ARF (Figure 2). In cell-cycle assays, the induction of INK4a and ARF decreased the proportion of S-phase cells. Additionally, re-expression of INK4a also increased the amount of apoptotic cells in induced cell lines, while re-expression of ARF did not have this effect. Since CDKN2A is a well-known cell cycle regulator, deregulation of the cell-cycle might be involved in acute transformation of chronic type ATL. In fact, deregulation of the cell-cycle pathway has been reported as a predictive indicator for the outcome in diffuse large B-cell lymphoma patients (Cancer Cell, 22:359-372). Therefore, we examined whether chronic ATL patients had alterations in cell-cycle related genes and found that chronic ATL patients could be divided into two groups. The group possessing alterations in these genes (referred to as “Cell cycle Alteration”) showed poorer prognosis compared with the group lacking such alterations (referred to as “Clean”) (p-value= 0.037) (Figure 3). Additionally, patients with such alterations tended to have earlier progression to acute type ATL. Conclusion These findings indicated that cell cycle-related genes play an important role in acute transformation and should serve as good prognostic markers for chronic type ATL. Disclosures: No relevant conflicts of interest to declare.

Identification of FOXO3 and PRDM1 as tumor-suppressor gene candidates in NK-cell neoplasms by genomic and functional analyses

Oligo-array comparative genomic hybridization (CGH) and gene-expression profiling of natural killer (NK)–cell neoplasms were used in an effort to delineate the molecular pathogenesis involved. Oligo-array CGH identified two 6q21 regions that were most frequently deleted (14 of 39 or 36%). One of these regions included POPDC3, PREP, PRDM1, ATG5, and AIM1, whereas the other included LACE1 and FOXO3. All genes located in these regions, except for POPDC3 and AIM1, were down-regulated in neoplastic samples, as determined by gene-expression analysis, and were therefore considered to be candidate tumor-suppressor genes. A20 and HACE1, the well-known tumor-suppressor genes located on 6q21-23, were included as candidate genes because they also demonstrated frequent genomic deletions and down-regulated expression. The Tet-Off NK cell line NKL was subsequently established for functional analyses. Seven candidate genes were transduced into Tet-Off NKL and forced re-expression was induced. Re-expression of FOXO3 and PRDM1 suppressed NKL proliferation, but this was not the case after re-expression of the other genes. This effect was confirmed using another NK cell line, SNK10. Furthermore, genomic analyses detected nonsense mutations of PRDM1 that led to functional inactivation in one cell line and one clinical sample. PRDM1 and FOXO3 are considered to play an important role in the pathogenesis of NK-cell neoplasms.

Myelodisplastic Syndrome with Trysomy 21 IN A Patient with Constitutional Submicroscopic 21Q22 Deletion.

Abstract 4831 Introduction Previous studies showed that chromosomal and genomic aberrations leading to activation of oncogenes or haploisufficiency of tumor suppressor genes are well-known pathogenic mechanisms in cancer. Additional copies of chromosme 21 are frequently found in Myelodisplastic Syndrome (MDS) and in Acute Myeloid Leukemia (AML); the presence of these chromosomal abnormalities and the high incidence of acute leukemias in subjects with constitutional trisomy 21, suggest that genes on chromosome 21, including RUNX1/AML1, play a particular role in leukemogenesis and hematopoiesis. We describe a patient with syndromic trombocytopenia ( average platelet count= 70000/mm3), psychomothor delay, microcephaly and low stature, that developed a progressive anemia and became transfusion-dependent at seventeen years of age. Materials and methods Cytogenetic analysis was performed on bone marrow cells and on peripheral blood lymphocytes, with standard techniques and evaluated with Giemsa-trypsin-Giemsa banding according to International System for Human Cytogenetic Nomenclature (ISCN 2005). Fluorescent In Situ Hybridization (FISH) experiments was performed on bone marrow samples with LSI AML1/ETO Dual Color, Dual Fusion Translocation and, at the same time, the High-resolution oligo array-CGH (Agilent Human Genome CGH Microarray 44B) was performed on the DNA of the patient. Results The bone marrow cells showed marked dysplastic morphology and the following abnormal karyotype: 46,XX[14]/47,XX,+21[6]; the peripheral blood karyotype was normal. The High-resolution oligo array-CGH demonstrated a constitutional de novo microdeletion of one chromosome 21. The interstitial deletion was found to be approximately 4,4Mb (Megabases), extending from 32,29 Mb to 36,51 Mb on band 21q22.11-12, involving MRAP, IFNAR2, IFNGRR2, KCNE2, KCNE1 and RUNX1 genes. The FISH performed on bone marrow cells, revealed two orange signals representing normal copies of ETO and one green signal for AML1 in 60% interphase cells and two orange signals and two green signals in the remaining 40% cells. The first pattern of signals, for AML1, is related to cells with karyotype 46,XX, while the second pattern of signals is related to cells with karyotype 47,XX,+21. These results indicate that in the myelodisplastic clone the third chromosome 21 are not deleted on band 21q22. Conclusion Three cases were recently published of syndromic thrombocytopenia with 21q22 constitutional deletion, including RUNX1, and variable degree of dysmorphic features and mental delay. One of the three patients developed LMA at the age of six years. Our results further support the fundamental role, in the pathogenetic mechanism of syndromic trombocytopenia and MDS/AML, of the numerical abnormalities of chromosome 21 associated with submicroscopic rearrangement of RUNX1 and other dosage-sensitive unknown genes on chromosome 21. Disclosures No relevant conflicts of interest to declare.