Kit Fai Wong

Identification of del(6)(q21q25) as a recurring chromosomal abnormality in putative NK cell lymphoma/leukaemia

The putative natural killer (NK) cell lymphomas/leukaemias are a group of recently characterized haematolymphoid malignancies sharing an immunophenotype of CD3/Leu4− CD3ɛ+ CD56+, and a genotype of germline T‐cell receptor genes. They frequently present in extranodal sites and exhibit a highly aggressive clinical course. Information on the cytogenetic or molecular events leading to the tumourigenesis in this group of tumours is very scarce. In this study we analysed the cytogenetic findings of seven patients with CD56‐positive putative NK cell lymphoma/leukaemia. Three cases, including one nasal, one extranasal and one leukaemic form, showed a common region of deletion at 6q21‐q25, suggesting that this may be a non‐random chromosomal aberration.

FLT-3 aberrations in acute promyelocytic leukaemia: clinicopathological associations and prognostic impact.

FLT‐3 aberrations that occur as an internal tandem duplication (ITD) or a mutation at the activation‐loop position 835, D835, are common in acute promyelocytic leukaemia (APL). We investigated the clinicopathological associations and prognostic impact of FLT‐3 aberrations in a cohort of APL patients. FLT‐3 exons 11 and 12 were amplified by polymerase chain reaction (PCR), and the ITD was recognized as an increase in the size of the PCR product. FLT‐3 exon 17 was amplified, and D835 mutation was identified by loss of an EcoRV site, followed by DNA sequencing. Of 82 patients studied, FLT‐3 aberrations were detected in 35 cases (43%) at diagnosis (ITD: 16; D835 mutation: 18; ITD + D835 mutation: 1). FLT‐3 ITD, but not D835 mutations, was significantly associated with higher presentation white blood cell count (WBC) and microgranular morphology. Early/induction deaths were related to male sex and high presentation WBC. There was a trend for FLT‐3 ITD to be associated with non‐remission (P = 0·06). For disease‐free survival, high WBC was the only significant adverse factor. Male sex, high WBC and FLT‐3 ITD were significant adverse factors for overall survival. These findings have important implications on the possible use of FLT‐3 inhibitors in the treatment of APL.

Aberrant promoter CpG methylation as a molecular marker for disease monitoring in natural killer cell lymphomas

Summary. Natural killer (NK) cell lymphomas lack suitable clonal markers for tumour cell detection, making the monitoring of minimal residual lymphoma difficult. Aberrant promoter CpG methylation occurs frequently in NK cell lymphomas. The objective of this study was to assess the potential of aberrant methylation as a surrogate tumour marker. Twenty‐five primary tumours and 105 serial biopsies taken at various time points after treatment were examined using a methylation‐specific polymerase chain reaction (MSP) for a panel of genes, comprising p73, p16, hMLH1, RARβ and p15, previously shown to be methylated in NK cell lymphomas. All samples underwent independent morphological examination, supplemented by immunostaining for CD56 and in‐situ hybridization for Epstein–Barr‐virus‐encoded RNA. Primary tumours showed the frequent methylation of the genes p73 (92%), p16 (71%), hMLH1 (61%), RARβ (56%) and p15 (48%). MSP results in serial post‐treatment biopsies were correlated with clinicopathological findings. Results were concordant in 89 follow‐up samples (18 samples, histology positive/MSP positive; 71 samples, histology negative/MSP negative) and discordant in 16. Fifteen samples were histology negative/MSP positive, and tumour involvement was subsequently confirmed (positive re‐biopsies or relapses at the same sites), indicating that MSP was more sensitive for minimal lymphoma detection. One sample was histology positive/MSP negative; a subsequent histological review and continuous clinical remission of the patient did not support tumour involvement. Our findings suggest that MSP for aberrantly methylated genes is a potentially valuable molecular marker for detecting either residual or relapsed disease in NK cell lymphoma patients.

Chromosomal abnormalities in T-cell large granular lymphocyte leukaemia: report of two cases and review of the literature

Summary. Cytogenetic information on T‐cell large granular lymphocyte leukaemia (T‐LGL; large granular lymphocytosis) is limited. We report two cases of T‐LGL with unusual karyotypic aberrations. The first case showed a novel inv(7)(p15q22) as the sole chromosomal abnormality, while the second case showed an inv(14)(q11q32) with evidence of clonal evolution. The breakpoints 7p14–p15 and 14q11 coincide with the T‐cell receptor (TCR)‐γ and TCR‐α/TCR‐δ gene loci respectively. This is the first report describing the possible involvement of T‐cell receptor genes in karyotypic aberrations in T‐LGL.

Refractory cytopenia with t(1;7), + 8 abnormality and dysplastic eosinophils showing intranuclear Charcot-Leyden crystals: A fluorescence in situ hybridization study

A case of refractory cytopenia and marrow eosinophilia showing t(l;7) translocation and concomitant trisomy 8 is reported. The eosinophils were dysplastic, and showed the unique feature of intranuclear Charcot‐Leyden crystal formation, giving rise to a‘lip‐like’appearance. We speculate that this unusual cytologic feature resulted from abnormal precipitation of Charcot‐Leyden crystal protein in the eosinophils. By fluorescence in situ hybridization using a chromosome 8 specific a‐satellite probe, the abnormal eosinophils were shown to have derived from the abnormal clone. We postulate that the dysplastic clone might have retained a differentiation potential and be responsive to normal haemopoietic stimuli.

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.

Human H7N9 avian influenza virus infection: a review and pandemic risk assessment

China is undergoing a recent outbreak of a novel H7N9 avian influenza virus (nH7N9) infection that has thus far involved 132 human patients, including 37 deaths. The nH7N9 virus is a reassortant virus originating from the H7N3, H7N9 and H9N2 avian influenza viruses. nH7N9 isolated from humans contains features related to adaptation to humans, including a Q226L mutation in the hemagglutinin cleavage site and E627K and D701N mutations in the PB2 protein. Live poultry markets provide an environment for the emergence, spread and maintenance of nH7N9 as well as for the selection of mutants that facilitate nH7N9 binding to and replication in the human upper respiratory tract. Innate immune suppression conferred by the internal genes of H9N2 may contribute to the virulence of nH7N9. The quail may serve as the intermediate host during the adaptation of avian influenza viruses from domestic waterfowl to gallinaceous poultry, such as chickens and related terrestrial-based species, due to the selection of viral mutants with a short neuraminidase stalk. Infections in chickens, common quails, red-legged partridges and turkeys may select for mutants with human receptor specificity. Infection in Ratitae species may lead to the selection of PB2-E627K and PB2-D701N mutants and the conversion of nH7N9 to a highly pathogenic avian influenza virus.

Acute promyelocytic leukaemia with cryptic PML-RARA fusion.

A 68-year-old female presented with gum bleeding and multiple bruises. Peripheral blood counts showed pancytopenia: haemoglobin 71 g/l, platelet count 1Æ3 · 10/l and leucocytes 1Æ3 · 10/l with 15% neutrophils, 54% lymphocytes and 31% abnormal promyelocytes. The abnormal promyelocytes were heavily granulated and some had bilobed nuclei. Many faggot cells were found. The bone marrow was hypercellular and was packed with abnormal promyelocytes (top left). Cytochemical study showed that the abnormal promyelocytes were strongly positive for myeloperoxidase and chloroacetate esterase. Coagulation screening tests showed: prothrombin time 14Æ8 s (normal 10–12Æ2 s), activated partial thromboplastin time 27Æ1 s (normal 26Æ5–36Æ5 s) and D-dimer >400 ng/ml FEU (normal <200 ng/ml FEU). A provisional diagnosis of acute promyelocytic leukaemia was made. However, cytogenetic study performed by overnight fluorodeoxyuridine-synchronized culture of marrow cells showed 46,XX[20]. Reverse transcription polymerase chain reaction on RNA extracted from the marrow cells detected the PML-RARA fusion transcript, and DNA sequence analysis showed that the breakpoint in the PML gene was at the bcr1 in exon 6. Fluorescence in situ hybridization (FISH) with dual colour dual fusion probes (SpectrumOrange labelled PML and SpectrumGreen-labelled RARA) showed 46,XX.ish ins(17;15)(q21Æ1;q22)(RARA+,PML+). Nuclear FISH showed two orange PML, one green RARA and one yellow PMLRARA fusion signals (top right). Metaphase FISH shows a single PML-RARA fusion signal in chromosome 17, indicating insertion of the PML gene on 15q into the RARA gene on 17q (bottom). The diagnosis of acute promyelocytic leukaemia with cryptic PML-RARA was confirmed. The patient was treated with all-trans-retinoic acid. The t(15;17)(q22;q12) is detectable in only about 90% of APL with molecular evidence of PML-RARA fusion. In most instances, absence of the t(15;17) is a reflection of failed cytogenetic study, but about 2–3% of cases are due to cryptic PMLRARA fusion as a result of insertion of the PML gene into the RARA gene or vice versa, which is only demonstrable by FISH.

Application of tri-colour, dual fusion fluorescence in situ hybridization (FISH) system for the characterization of BCR-ABL1 fusion in chronic myelogenous leukaemia (CML) and residual disease monitoring

BackgroundWe studied the application of the BCR-ABL1 + 9q34 tri-colour dual fusion fluorescence in situ hybridization (FISH) system in the characterization of fusion signal pattern and the monitoring of residual disease in chronic myelogenous leukaemia (CML). The signal constellation on metaphases with the tri-colour dual fusion system was defined. The knowledge of various signal patterns obtained from the different genetic rearrangements was further applied to the analysis of hybridization signals on interphase nuclei.MethodsBCR-ABL1 dual colour, dual fusion FISH (D-FISH) was performed on diagnostic samples of 22 CML patients. The tri-colour FISH system was performed on cases that showed aberrant signal patterns other than the classical 1 green (G) 1 orange (O) 2 fusions (F). Using the aqua band-pass filter, random signal overlap in interphase nuclei would be indicated by the presence of an aqua signal (ASS1), while genuine fusion was represented by the absence of the ASS1 signal.ResultsUsing the D-FISH system, the signal patterns could be categorized into 4 groups: group 1 (n = 17) showed the classical 1G1O2F; group 2 (n = 2) showed 2G1O1F indicating ABL1 deletion; group 3 (n = 1) showed 1G2O1F indicating BCR deletion; group 4 (n = 2) with 1G1O1F indicating reciprocal ABL1-BCR deletion. The tri-colour dual fusion system correlated with the D-FISH system for cases with der(9) deletion. The added aqua-labelled ASS1 probe was useful in differentiating random signal overlap from genuine BCR-ABL1 fusion in the interphase cells (group 4).ConclusionAlthough the D-FISH probe was valuable in establishing the different patterns of aberrant signals and monitoring patients with the classic 2-fusion signals in CML, the tri-colour dual fusion probe should be used for patients with der(9) deletion to monitor response to treatment.

Detection of FIP1L1‐PDGFRA fusion by FISH

A 20-year-old male student presented with left upper quadrant abdominal discomfort, general malaise, shortness of breath on exertion, gum bleeding and petechiae over both lower limbs. Physical examination showed splenomegaly of 4 cm below the left costal margin. Peripheral blood (PB) counts were: haemoglobin (Hb) 74 g/l, leucocytes 69 · 10/l with 50% eosinophils (top left), and platelets 55 · 10/l. Lactate dehydrogenase was raised and stool samples were repeatedly negative for parasites. Bone marrow could not be aspirated. Trephine biopsy showed increased cellularity with increased granulopoiesis predominated by cells of the eosinophilic lineage, megaloblastoid erythropoiesis, reduced but normal megakaryocytes and moderate reticulin fibrosis. Cytogenetic study performed on the marrow cells showed a normal karyotype and BCR-ABL gene fusion was negative by fluorescence in situ hybridisation (FISH). A diagnosis of hypereosinophilic syndrome was made. A computed tomography scan of the thorax and cardiac echocardiogram were unremarkable. The patient was managed conservatively. The eosinophilia persisted for 3 years: Hb 83 g/ l, leucocyte count 31 · 10/l (eosinophils 70%) and platelet count 47 · 10/l. Again there was a dry-tap. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and no excess blasts (bottom left). Nested reverse transcription polymerase chain reaction performed on the PB showed the presence of a FIP1L1-PDGFRA (F-P) fusion transcript. The diagnosis was then revised to chronic eosinophilic leukaemia. A commercially available 4q12 tricolour rearrangement FISH probe (Vysis, Downers Grove, IL, USA) was used to detect the F-P gene fusion on interphase nuclei from the cytogenetic preparation at diagnosis. Results showed 50% normal cells (top right) with two tri-colour (green, orange and aqua) fusion signals and 40% cells with one tri-colour signal and another green-aqua fusion signal lacking an orange signal (middle right) that represented cryptic interstitial deletion of 4q12 and F-P gene fusion. Another 10% of cells showed one tri-colour signal, one green-aqua fusion signal and one relocated orange signal (bottom right), suggesting insertion of 4q12 sequences to another chromosomal region followed by deletion. The patient had a complete response to imatinib therapy, with shrinkage of the spleen and normalisation of the blood counts.