T. S. K. Wan

Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSCloALDHbr cell population was identified (ALDH+AML) (median: 14.89%, range: 5.65–48.01%), with the majority of the SSCloALDHbr cells coexpressing CD34+. In another 29 cases, there was undetectable (n=23) or rare (⩽5%) (n=6) SSCloALDHbr population (ALDH−AML). Among other clinicopathologic variables, ALDH+AML was significantly associated with adverse cytogenetic abnormalities. CD34+ BM cells from ALDH+AML engrafted significantly better in NOD/SCID mice (ALDH+AML: injected bone 21.11±9.07%; uninjected bone 1.52±0.75% vs ALDH−AML: injected bone 1.77±1.66% (P=0.05); uninjected bone 0.23±0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSCloALDHbr population (median: 2.92%, range: 0.92–5.79%), but none of the ALL cases showed this fraction. In conclusion, SSCloALDHbr cells in ALDH+AML might denote primitive LSC and confer an inferior prognosis in patients.

Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion : a molecular cytogenetics study

TEL/AML1 gene fusion that results from a cryptic t(12;21) is the most common genetic aberration in childhood B-lineage acute lymphoblastic leukemia (ALL). While the translocation may initiate the leukemic process, critical secondary genetic events are currently believed to be pivotal for leukemogenesis. We investigated 12 cases of childhood ALL with TEL/AML1 gene fusion by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) and documented additional or secondary genetic changes in seven patients (58%). Three patients showed extra copies of chromosome 21 including a case in which the trisomy 21 (+21) clone was distinct from the one harboring TEL/AML1 gene fusion. Interestingly, one patient without +21 showed amplification of the AML1 gene on chromosome 21q, supporting the contention that AML1 amplification may be an important additional genetic event. Gene expression study by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) in two of these four patients showed an increase in AML1 transcripts that paralleled the increase in gene copy number. Deletion of the normal TEL allele was detected in two patients, with one of them showing loss of chromosome 12 together with duplication of the der(12)t(12;21). Finally, one patient showed duplication of the fusion signal. Our findings confirm that additional or secondary genetic changes including AML1 amplification are commonly encountered in childhood ALL with TEL/AML1 gene fusion, which are envisaged to play significant roles in disease progression.

Cytogenetics and molecular genetics of childhood leukemia

Childhood leukemia is the commonest form of childhood cancer and represents clonal proliferation of transformed hemopoietic cells as a result of genetic changes. Molecular characterization of these changes, in particular chromosomal translocations, has yielded a wealth of information on the mechanisms of leukemogenesis. These findings have also allowed the development of sensitive assays for the identification of underlying molecular defects, which is applicable to disease diagnosis and to monitor response to treatment. Genetic alterations in childhood leukemia are powerful prognostic indicators. TEL‐AML1 fusion and hyperdiploidy >50 chromosomes are associated with a good prognosis in childhood acute lymphoblastic leukemia, whereas BCR‐ABL fusion and MLL rearrangements are associated with a poor prognosis. Hence cytogenetic and molecular genetic classification of childhood leukemia will significantly improve the ability of clinicians to predict therapeutic response and prognosis, which paves the way for risk stratification based on clinical and genetic features. Finally, deciphering of genetic lesions in leukemia has allowed elucidation of the molecular basis of current treatment, as typified by the success of all‐trans retinoic treatment in acute promyelocytic leukemia, and has identified targets for novel therapeutic approaches. It is envisaged that efforts in characterization of molecular defects in childhood leukemia will ultimately be translated into better clinical outcome for patients. Copyright

Complex Variant 15;17 Translocations in Acute Promyelocytic Leukemia: A Case Report and Review of Three-Way Translocations

Complex variant 15;17 translocations are increasingly recognized in acute promyelocytic leukemia (APL). We report a novel three-way translocation in APL involving chromosomes 15, 17, and X in the form of t(X;17;15)(q13;q12;q21). Southern blot analysis showed retinoic acid receptor alpha (RARA) gene rearrangement at intron 2. Clinical and morphologic findings are typical of APL, and a complete remission was attained with a course of conventional chemotherapy. A review of three-way complex variants of 15;17 translocation in the literature reveals 21 published cases in addition to ours. PML/RARA fusion was observed in all 8 cases in which molecular genetic analysis had been performed. More cases need to be analyzed to determine if clustering to particular chromosomal bands occurs in variant translocations, and whether APL cases harboring complex 15;17 variants differ clinically from those with classical 15;17 translocation.

Trisomy 21 as the sole acquired karyotypic abnormality in acute myeloid leukemia and myelodysplastic syndrome

We report five cases of myeloid disorders in which trisomy 21 (+21) was found as the sole acquired karyotypic abnormality, comprising two cases of acute myeloid leukemia (AML) and three cases of myelodysplastic syndrome (MDS). In this series, MDS patients with +21 presented as high grade disease, which included two cases of refractory anemia with excess of blasts (RAEB) and one case of refractory anemia with excess of blasts in transformation (RAEBt), and showed rapid disease progression. Significant thrombocytopenia was observed in all three patients, and bone marrow examination showed a marked reduction in megakaryocytes. AML patients with +21 included one case each of AML-M2 and M4. Despite the poor prognosis reported in AML patients with +21 as the sole abnormality, the patient in our series who was able to complete intensive treatment was cured of disease. The role of +21 in leukemogenesis is reviewed.

Near-haploid common acute lymphoblastic leukaemia of childhood with a second hyperdiploid line: a DNA ploidy and fluorescence in-situ hybridization study

Near‐haploidy is a rare cytogenetic finding in childhood acute lymphoblastic leukaemia (ALL) and is associated with a poor prognosis. A second hyperdiploid line, occurring presumably by endoreduplication of the near‐haploid stemline, is often observed. We present a case of common ALL in relapse characterized morphologically by a dual population of small and large lymphoblasts. Cytogenetic analysis supplemented with fluorescence in‐situ hybridization (FISH) studies localized near‐haploidy and hyperdiploidy to the small and large blast population respectively. DNA ploidy determination confirmed two abnormal clones with near‐haploidy as the predominant one. A novel t(9;12)(q11;q13) was present in the near‐haploid clone and was duplicated in the hyperdiploid clone. This finding identified cells bearing near‐haploidy to be the clonogenic population following malignant transformation and confirmed endoreduplication as the mechanism for the presence of associated hyperdiploidy.

The role of trisomy 8 in the pathogenesis of chronic eosinophilic leukemia

A case of chronic eosinophilic leukemia (CEL) manifesting as spinal cord compression by an extradural eosinophilic chloroma in a 32-year-old Chinese man was presented, who subsequently developed extramedullary transformation at the skin and then peritoneal cavity. Cytogenetic study of bone marrow cells at diagnosis showed a clonal karyotypic abnormality of trisomy 8 (+8), which on fluorescence in situ hybridization (FISH) was shown to be present in a clone of abnormal eosinophils, hence showing the neoplastic nature of the eosinophilic proliferation. There was another population of abnormal eosinophils that did not show +8. At blastic transformation, all blast cells in ascitic fluid were shown by FISH to harbor +8. These findings suggest that +8 in this case may have arisen from clonal evolution and is not the primary genetic event in leukemogenesis, but +8 most probably imparts a further survival advantage to the clone responsible for subsequent blastic transformation.

Cytogenetic Characterization of Childhood Hepatoblastoma

We describe the cytogenetic abnormalities in two cases of childhood hepatoblastoma. The first case was of fetal histology with squamous metaplasia, and cytogenetic study showed an add(5)(q31). Although an association between hepatoblastoma and familial adenomatous polyposis is recognized, the breakpoint in this case is distal to 5q21 and most probably does not involve the APC gene at that location. The second case was of macrotrabecular histology, and cytogenetic study showed an unbalanced translocation in the form of der(4)t(1;4)(q12;q34) in a hyperdiploid clone. Including our case, der(4)t(1;4)(q12;q34) has been recognized in four cases of hepatoblastoma, and it may be the first recurrent translocation in this tumor. Understanding the molecular mechanism and clinical significance of this translocation awaits analysis of more cases.

Methylation of TET2, CBL and CEBPA in Ph-negative myeloproliferative neoplasms

A loss-of-function mutation of TET2, CBL and CEBPA has been implicated in the pathogenesis or leukaemic transformation of myeloproliferative neoplasm. As tumour suppressor genes may potentially be inactivated by promoter hypermethylation, the authors studied the methylation status of these genes in three cell lines and diagnostic marrow samples from 45 patients with myeloproliferative neoplasm (MPN) (essential thrombocythaemia, N=34; polycythaemia vera, N=7 and primary myelofibrosis, N=4) by methylation-specific PCR. TET2 was heterozygously methylated in MEG-01 and K562 but completely unmethylated in HEL. On the other hand, both CBL and CEBPA were completely unmethylated in all three cell lines. In the primary marrow samples, methylation of TET2 occurred in two (5.9%) patients with essential thrombocythaemia (4.4% of all patients), both without JAK2 V617 mutation, but not in polycythaemia vera or primary myelofibrosis. There was no association between TET2 methylation with the type of MPN (p=0.713). Hypermethylation of CBL or CEBPA was not detected in any patients. In summary, methylation of TET2, CBL and CEBPA is infrequent in MPN at diagnosis. The role of methylation of these genes at the time of leukaemic transformation warrants further study.

Acquired pericentric inversion of chromosome 9 in essential thrombocythemia.

(1999) and Verma (1999), regarding the classification systems of polymorphic pericentric inversions involving the secondary constriction (qh) region of chromosome 9. While human chromosome 9 with a pericentric inversion involving the qh region is relatively common as a constitutional genetic aberration without any apparent phenotypic consequences, it has not been recognized as an acquired abnormality in cancer. Here we describe a case of essential thrombocythemia that showed an acquired inv(9)(p11q13) on cytogenetic analysis of bone marrow cells. The acquired nature of the inversion was confirmed by a normal constitutional karyotype on phytohemagglutinin (PHA)-stimulated culture of peripheral blood lymphocytes. A 48-year-old Chinese man was incidentally found to have a high platelet count during a routine blood test. Physical examination showed a slightly enlarged spleen just palpable below the left costal margin. Subsequently, complete blood counts revealed: 12.8 g hemoglobin/dl, 8.7×109 leucocytes/l (differential: 69% neutrophils, 26% lymhocytes, 5% monocytes), and 1380×109 platelets/l. Numerous platelet clumps and giant platelets were noted. Bone marrow aspiration and trephine biopsy showed a hypercellular marrow with trilineage hyperplasia. Megakaryocytes were plentiful, showing clustering and localization toward paratrabecular areas. The overall picture was consistent with essential thrombocythemia; reactive causes of thrombocytosis were excluded. Polymerase chain reaction revealed an absence of BCR/ABL chimeric transcript. Cytogenetic study performed on short-term, overnight synchronized and nonsynchronized cultures of marrow cells supplemented by a direct harvest showed the following karyotype: 46,XY,inv(9)(p11q13)[3]/46,XY[11]. PHA-stimulated culture of peripheral blood lymphocytes in remission showed a normal 46,XY karyotype in 110 metaphases analyzed, thus confirming that the inv(9qh) was an acquired abnormality (Fig.1). The patient was given hydroxyurea, which provided adequate control of platelet production. Polymorphic inversion of the 9qh region is considered to be a normal variation of human chromosome 9. This notwithstanding, recent studies indicate that the pericentric region of chromosome 9 may be etiologically linked to schizophrenia (Kunugi et al. 1999; Miyaoka et al. 1999) and Walker-Warburg syndrome (Baltaci et al. 1999). Different forms of inv(9qh) with differential breakpoints may further be characterized by in situ hybridization, utilizing a series of probes for satellite DNA (Ramesh and Verma 1996; Samonte et al. 1996). The localization of breakpoints on chromosome 9 may lead to cloning of disease-susceptibility genes for these conditions. Secondly, whether constitutional pericentric inversion of chromosome 9 is a predisposing factor for cancer remains controversial. There is evidence to suggest that T. S. K. Wan · S. K. Ma · L. C. Chan Acquired pericentric inversion of chromosome 9 in essential thrombocythemia Hum Genet (2000) 106 :669–670 Digital Object Identifier (DOI) 10.1007/s004390000312