Annie Pang

Preferential methylation of Wnt inhibitory factor-1 in acute promyelocytic leukemia: an independent poor prognostic factor

Preferential methylation of Wnt inhibitory factor-1 in acute promyelocytic leukemia: an independent poor prognostic factor

T-Cell Large Granular Lymphocyte Leukemia of Donor Origin After Allogeneic Bone Marrow Transplantation

A 39-year-old man with chronic myeloid leukemia in accelerated phase underwent allogeneic bone marrow transplantation (BMT). At 6 months after BMT, lymphocytosis (WBC count, 23,100/microL [23.1 x 10(9)/L]; 80% (0.80) large granular lymphocytes [LGLs]) occurred. The LGLs were CD3+CD4-CD8+, with clonally rearranged T-cell receptor gamma gene, and of donor origin, as shown by analysis of polymorphic microsatellite markers. Epstein-Barr virus was not present. The diagnosis, therefore, was consistent with T-cell large granular lymphocytic (T-LGL) leukemia. Corticosteroids controlled the LGL count, but progressive pancytopenia led to death 4 months later. Retrospective analysis showed that the T-LGL leukemia apparently had arisen as early as 3 months after BMT. The distinguishing features of this case included donor origin, neoplastic nature, and the aggressive fatal outcome.

Low frequency of rearrangements of the homeobox gene HOXA9/t(7;11) in adult acute myeloid leukemia.

Translocation (7;11)(p15;p15) is uncommon in acute myeloid leukemia (AML) and may have a predilection to occur in Asian patients. It results in a reciprocal fusion of the homeobox gene HOXA9 and the nucleoporin gene NUP98. To ascertain the molecular features and the clinicopathological and prognostic significance of t(7;11)(p15;p15), 208 adult Chinese patients with AML were screened by Southern blot analysis with an HOXA9 cDNA probe and reverse transcription–polymerase chain reaction for NUP98/HOXA9. Three cases were found to have rearrangement of the HOXA9 gene. Two cases were found to have an NUP98/HOXA9 fusion transcript, with a breakpoint at NUP98 different from that previously described. The remaining case had no rearrangement of NUP98, nor was NUP98/HOXA9 detected. All positive cases had refractory AML with poor treatment outcome. In conclusion, t(7;11)/HOXA9 rearrangement was a rare event (3/208, 1.5%) in AML, even in a population where it was considered to be more prevalent. Two breakpoints in NUP98 may occur, and this is of importance in the design of primers to amplify t(7;11). Genes Chromosomes Cancer 25:70–74, 1999.

Aberrant promoter methylation of the retinoic acid receptor alpha gene in acute promyelocytic leukemia

The retinoic acid receptor alpha (RARA) gene is disrupted by PML/RARA fusion in acute promyelocytic leukemia (APL). The P2 promoter of RARA, controlling the RARα2 isoform, contains an RA-responsive element and may be targeted in APL. To test whether aberrant methylation of P2 was involved, 47 APL at diagnosis, 16 APL at first relapse, 50 acute myeloid leukemia (AML) and 22 acute lymphoblastic leukemia (ALL) were tested by methylation-specific polymerase chain reaction. RARA P2 methylation was highly associated with APL (APL: 25/63 vs AML/ALL: 2/75, P<0.0001). P2 methylation occurred at similar frequencies in APL at diagnosis and relapse, suggesting it was an initiating leukemogenic event. In the APL line NB4, RARα2 was not expressed, with the untranslocated RARA shown to be P2 methylated. 5-Azacytadine treatment of NB4 led to progressive P2 demethylation and re-expression of RARα2, confirming that RARA methylation collaborated with PML/RARA in totally suppressing RARα. In APL, RARA P2 methylation was unrelated to gender, age, presenting leukocyte counts and additional cytogenetic aberrations. For APL patients receiving all-trans retinoic acid for induction, P2 methylation did not affect the complete remission rates and survivals. RARA is the first myeloid-specific transcription factor shown to be dysregulated by both translocation and aberrant methylation.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency in elderly Chinese women heterozygous for G6PD variants

deficiencies [Yoshida et al., 1971]. Although chronic hemolysis is absent, oxidative hemolysis caused by drugs and infections may occur [Beutler, 1994]. Clinically overt G6PD deficiency in females is rare and is mostly attributed to homozygosity or compound heterozygosity for G6PDvariants. Other rare causes included the 45,X genotype, clonal hematopoeisis [Au et al., 2002] or extreme X chromosome inactivation [Beutler, 1994]. We describe two elderly Chinese womenwith severe hemolysis TABLE I. Index Patients, G6PD Deficient Females, and Their Family Members

t(8;16)(p11;p13) predisposes to a transient but potentially recurring neonatal leukemia

A Chinese girl presented with generalized papular rash and monocytic leukemia 19 days after birth. Cytogenetic analysis showed t(8;16)(p11.2;p13.3) as the sole chromosomal abnormality. Spontaneous regression of the leukemia was observed after 2 months, although the t(8;16) translocation persisted cytogenetically. This was followed 7 months later by the development of acute myeloid leukemia with maturation and cytogenetic evolution with extra chromosomes 4 and 8. Molecular study showed that the reciprocal MYST3 and CREBBP gene fusion characteristic of t(8;16) translocation persisted throughout the clinical course, even during spontaneous regression of the neonatal leukemia, and after chemotherapy-induced remission of the subsequent acute myeloid leukemia. The genetic lesion only became undetectable at the molecular level at the age of 20 months. The possible role of MYST3 and CREBBP gene fusion in the pathogenesis of the leukemia is discussed.

G6PD deficiency from lyonization after hematopoietic stem cell transplantation from female heterozygous donors

To determine whether during hematopoietic stem cell transplantation (HSCT), X-chromosome inactivation (lyonization) of donor HSC might change after engraftment in recipients, the glucose-6-phosphate dehydrogenase (G6PD) gene of 180 female donors was genotyped by PCR/allele-specific primer extension, and MALDI-TOF mass spectrometry/Sequenom MassARRAY analysis. X-inactivation was determined by semiquantitative PCR for the HUMARA gene before/after HpaII digestion. X-inactivation was preserved in most cases post-HSCT, although altered skewing of lyonization might occur to either of the X-chromosomes. Among pre-HSCT clinicopathologic parameters analyzed, only recipient gender significantly affected skewing. Seven donors with normal G6PD biochemically but heterozygous for G6PD mutants were identified. Owing to lyonization changes, some donor–recipient pairs showed significantly different G6PD levels. In one donor–recipient pair, extreme lyonization affecting the wild-type G6PD allele occurred, causing biochemical G6PD deficiency in the recipient. In HSCT from asymptomatic female donors heterozygous for X-linked recessive disorders, altered lyonization might cause clinical diseases in the recipients.

Integrity of blood-brain barrier in primary CNS NK/T cell lymphoma: implications in the treatment of other primary CNS lymphomas.

Dear Editor, Extranodal NK/T cell lymphoma is rare. It occurs worldwide, but shows a predilection for Asian and South American populations [1]. Lymphoma cells are invariably infected with clonal episomal Epstein-Barr virus (EBV). Quantification of EBV DNA is an accurate surrogate biomarker of lymphoma load [2]. A 40-year-old woman presented with cognitive decline and was managed as limbic encephalitis for nearly a year. Gradual deterioration led to a brain biopsy, which showed NK/T cell lymphoma. Her neuroimaging findings were described before [3]. On referral, she was unresponsive to external stimuli. There was extensive disease involving bilateral basal ganglia, bilateral frontotemporal lobes and brainstem (Fig. 1a). Positron emission tomography-computed tomography did not show disease involvement outside the central nervous system (CNS). Bilateral bone marrow examination was normal. She was treated with the SMILE protocol (steroid, methotrexate, ifosfamide, L-asparaginase, etoposide) [4]. After two courses, magnetic resonance imaging showed encephalomalacic changes with parenchymal loss in previously affected sites. On completion of six courses of treatment, she had become responsive to external stimuli, but with minimal motor improvement. Quantitative polymerase chain reaction (Q-PCR) of EBV DNA, calibrated against the World Health Organization standard (lowest detection limit 2.5 IU), was performed in the plasma and cerebrospinal fluid (CSF) as described [2, 5, 6]. On referral, CSF was practically acellular (cell count 6×10/L) and negative morphologically for lymphoma cells, but was positive for EBV DNA at 2.9×10 IU/mL. The concomitant plasma was negative for EBV DNA. Plasma EBV DNA was quantified daily during the first week of SMILE, and then every 3–4 days thereafter. On day 24, after the first course of SMILE, plasma EBV DNA became transiently detectable at 1.5×10 IU/mL, but was negative subsequently. After two courses of SMILE, CSF became negative for EBV DNA (Fig. 1b). In EBV-associated malignancies, apoptosis of neoplastic cells leads to release of EBV DNA, comprising fragments of naked DNA of less 200 bp [7]. Each basepair in doublestranded DNA is about 650 Da in molecular weight, so that EBV DNA released from neoplastic cells is less than 130 kDa in size. We showed previously that in isolated CNS involvement by NK/Tcell lymphoma, EBVDNAwas confined to the CSF and not detectable in the circulation [6]. Therefore, we had a unique opportunity to investigate the integrity of the blood-brain barrier (BBB) in this case of primary CNS lymphoma, with EBV DNA as a molecular marker. On presentation, despite the extensive lymphoma involvement, EBV DNAwas still confined to the CSF. More importantly, during treatment of the lymphoma with high-dose chemotherapy penetrating efficiently the BBB, including methotrexate (2 g/m, CSF/plasma drug concentration 10%) and ifosfamide (1.5 g/m, CSF/plasma drug concentration 100 %) [8], the BBB had remained intact. About 3 weeks after initiation of the first course of SMILE, there was only a short and transient period when EBV DNA leaked through the BBB into the circulation. Our observation is of interest in the treatment of primary CNS lymphoma of B cell lineage. Rituximab does not penetrate the CNS under normal circumstances. In primary CNS B cell lymphomas, current regimens call for the use of weekly * Yok-Lam Kwong ylkwong@hkucc.hku.hk

Concomitant Epstein-Barr virus (EBV)-positive B-cell and EBV-negative T-cell posttransplant lymphoproliferative disorders after renal allografting: pathogenetic implications.

Posttransplant lymphoproliferative disorders (PTLD) develop in organ allograft recipients because of the immunodeficient state induced by immunosuppressive drugs used to prevent graft rejection. In B-cell PTLD, EpsteinBarr virus (EBV) plays an important etiologic role, as unchecked EBV replication in infected B cells ultimately leads to neoplastic transformation. In T-cell PTLD, however, EBV is often not involved, so that it remains unclear what the pathogenetic mechanism(s) may be. A 30-year-old man received a cadaveric renal allograft for chronic glomerulonephritis. Five years later, he presented with a generalized seizure. A computered tomography showed a 4-cm frontal lobe lesion (arrow, Fig. 1A). Biopsy showed monotonous sheets of malignant large lymphoid cells, expressing CD20 and EBV-encoded small RNA, consistent with monomorphic B-cell PTLD. Further staging investigations, including computed tomography of thorax and abdomen and bilateral marrow biopsies, were negative. Furthermore, circulating EBV DNA as a surrogate biomarker (1) was negative, showing that there was no extracranial involvement. Cyclosporine was stopped. Local radiotherapy (30 Gy) and intrathecal anti-CD20 antibody rituximab (2) (30 mg twice weekly eight doses) produced a complete remission. However, graft rejection ensued, requiring hemodialysis and erythropoietin (EPO: 4000 IU weekly) treatment. Six months later, a blood count showed hemoglobin (Hb): 4.0 g/dL, undetectable reticulocytes, white cell count: 3 10/L, 15% large granular lymphocytes (LGLs) (Fig. 1B), and platelet count : 108 10/L. A marrow biopsy showed pure red cell aplasia (PRCA) with virtually absent erythropoiesis (Fig. 1C). Serologic tests for parvovirus B19 and antierythropoietin antibody were negative. The abnormal LGLs were CD3 ve, CD4 ve, CD8 ve, and EBV-encoded small RNA-negative. To confirm the clonal nature of the T-LGLs, polymerase chain reaction (PCR) for T-cell receptor gamma (TCR ) gene was performed as