Incidence of myeloid malignancies by subtype in Hong Kong and comparisons with Asian and white men and women in the United States.

Data on incidence rates of myeloid malignancies for subtypes based on the World Health Organization (WHO) classification are lacking in Asian populations. We compared age-adjusted incidence rates for 27 myeloid malignancy WHO-defined subtypes in Hong Kong (HK) (2014-2016) with those for Asian and white individuals living in the United States (U.S.) (2010-2016). Except for overall acute myeloid leukemia (AML) (2.23 cases per 100,000) and myeloproliferative neoplasms (MPNs) (2.10 cases per 100,000), rates of all subtypes were <1 case per 100,000 person-years in HK. Overall rates of AML, myelodysplastic syndrome (MDS), and MDS/MPN were lower in HK compared to white and Asian individuals in the U.S., but the patterns by specific subtype varied. For these three broad groupings of myeloid malignancies, rates in U.S. Asians were intermediate to those in HK and white individuals in the U.S. These results suggest the possibility of a multifactorial etiology for specific myeloid malignancy subtypes that should be evaluated in future epidemiological studies.

Extensive amyloid deposits in bone marrow aspirate smears.

Amyloid light-chain (AL) amyloidosis is the most common form of systemic amyloidosis. It can cause progressive organ dysfunction and eventually death, mainly due to cardiac involvement. Amyloidosis may rarely present as extensive amorphous, purplish-blue deposits in marrow aspirate smears. Demonstration of congophilic property and apple-green birefringence under polarized light in aspirate smears can allow a rapid diagnosis of amyloidosis.

Clinicopathological features and outcome of chronic lymphocytic leukaemia in Chinese patients.

Chronic lymphocytic leukaemia (CLL) is uncommon in Chinese population and its biology, genetics and treatment outcome in Chinese patients have not been comprehensively investigated. In this study, we studied the clinicopathological features and outcome of 212 Chinese patients with newly diagnosed CLL in Hong Kong and Singapore. The median age at diagnosis was 64 years. The majority of patients presented with early-stage disease (Binet stage A, 56.1%). Del(13)(q14) was the most frequent abnormality (41.7%) detected by fluorescence in situ hybridization (FISH) analysis. Del(17p) and TP53 gene mutations were detected in 7.8% and 8.2% of patients, respectively. MYD88 mutations were found at a higher frequency (11.5%) than expected. CLL with unmutated variable region of the immunoglobulin heavy chain genes (IGHV) occurred in only 31.2% of cases, and was associated with advanced-stage disease (p <0.01) and adverse FISH abnormalities (p<0.01). With a median follow-up of 39 months, the median overall survival (OS) was 108 months. The presence of del(17p) or TP53 mutations was associated with a significantly shorter time to first treatment and an inferior OS (p <0.01). Unmutated IGHV was also associated with a significantly shorter time to treatment (p <0.01). Among patients who required treatment, the median OS and progression-free survival (PFS) were 107 and 23 months, respectively. The presence of del(17p) was associated with a significantly inferior OS and PFS (p <0.01). In summary, Chinese CLL patients had similar genetic aberrations at diagnosis compared with those of Western populations. FISH abnormalities are major factors affecting outcome.

Immunohistochemical detection of cytoplasmic nucleophosmin in formalin-fixed paraffin-embedded marrow trephine biopsies in acute myeloid leukaemia.

AIMS: Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalisation of nucleophosmin (NPMc+) are the most common genetic abnormality in acute myeloid leukaemia (AML). In this study, we tested whether immunohistochemical (IHC) detection of cytoplasmic NPM1 (cNPM1) in formalin-fixed bone marrow trephine biopsies correlated with NPM1 mutations and the prognostic impact of NPM1 and fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutations was also assessed. METHODS: A total of 71 Chinese adult de novo AML cases were evaluated for cNPM1 by IHC where the bone marrow trephines were fixed in 10% buffered formalin and decalcified by 5% EDTA. NPM1 and FLT3-ITD gene mutations were also investigated using PCR, fragment analysis and direct DNA sequencing. RESULTS: IHC analysis of cNPM1 had a very good sensitivity (86.7%) and excellent specificity (96.4%) for NPM1 mutation. The positive predictive value was 86.7% and the negative predictive value was 96.4%. NPM1 mutations and FLT3-ITD were closely associated (p = 0.003). Patients with mutated NPM1 and without FLT3-ITD mutation have a longer overall survival (p = 0.042) than patients with both NPM1 and FLT3-ITD mutations. CONCLUSIONS: Our results showed that IHC detection of cNPM1 in formalin-fixed trephine biopsies correlated well but not entirely with NPM1 mutation. Furthermore, NPM1 mutations were significantly more frequent in FLT3-ITD than FLT3-wild-type cases.

Genetic association between germline JAK2 polymorphisms and myeloproliferative neoplasms in Hong Kong Chinese population: a case-control study.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies that can be characterised by a somatic mutation (JAK2V617F). This mutation causes the bone marrow to produce excessive blood cells and is found in polycythaemia vera (~95%), essential thrombocythaemia and primary myelofibrosis (both ~50%). It is considered as a major genetic factor contributing to the development of these MPNs. No genetic association study of MPN in the Hong Kong population has so far been reported. Here, we investigated the relationship between germline JAK2 polymorphisms and MPNs in Hong Kong Chinese to find causal variants that contribute to MPN development. We analysed 19 tag single nucleotide polymorphisms (SNPs) within the JAK2 locus in 172 MPN patients and 470 healthy controls. Three of these 19 SNPs defined the reported JAK2 46/1 haplotype: rs10974944, rs12343867 and rs12340895. Allele and haplotype frequencies were compared between patients and controls by logistic regression adjusted for sex and age. Permutation test was used to correct for multiple comparisons. With significant findings from the 19 SNPs, we then examined 76 additional SNPs across the 148.7-kb region of JAK2 via imputation with the SNP data from the 1000 Genomes Project. RESULTS: In single-marker analysis, 15 SNPs showed association with JAK2V617F-positive MPNs (n = 128), and 8 of these were novel MPN-associated SNPs not previously reported. Exhaustive variable-sized sliding-window haplotype analysis identified 184 haplotypes showing significant differences (P < 0.05) in frequencies between patients and controls even after multiple-testing correction. However, single-marker alleles exhibited the strongest association with V617F-positive MPNs. In local Hong Kong Chinese, rs12342421 showed the strongest association signal: asymptotic P = 3.76 × 10-15, empirical P = 2.00 × 10-5 for 50,000 permutations, OR = 3.55 for the minor allele C, and 95% CI, 2.59-4.87. Conditional logistic regression also signified an independent effect of rs12342421 in significant haplotype windows, and this independent effect remained unchanged even with the imputation of additional 76 SNPs. No significant association was found between V617F-negative MPNs and JAK2 SNPs. CONCLUSION: With a large sample size, we reported the association between JAK2V617F-positive MPNs and 15 tag JAK2 SNPs and the association of rs12342421 being independent of the JAK2 46/1 haplotype in Hong Kong Chinese population.

A comparison of high resolution melting, allele-specific priming and Sanger sequencing for the detection of BRAFV600E mutation in hairy cell leukaemia from different haematological specimens.

The BRAFV600E mutation is a highly sensitive and specific marker for the diagnosis of hairy cell leukaemia (HCL) and a potential therapeutic target. We assessed the performance of high resolution melting (HRM), allele-specific priming (ASP) and Sanger sequencing (SS) for BRAFV600E detection in 17 unenriched samples from 15 HCL patients: blood (n = 7), marrow aspirate (n = 3), ethylenediaminetetraacetic acid (EDTA)-decalcified trephine biopsy (n = 2), formic acid (FA)-decalcified trephine biopsy (n = 5). Our results showed that for blood and marrow aspirate samples, both HRM and ASP had a very high analytical sensitivity (1%) in clinical specimens and excellent diagnostic sensitivity (100%) and specificity (100%) in analysable samples. Sanger sequencing had a lower analytical sensitivity (4%), resulting in false-negative analysis in samples with a low tumour cell percentage. High resolution melting was technically the simplest and had the shortest turn-around time (2 hours). Analysis of decalcified trephine biopsies was more difficult because of suboptimal DNA preservation. Although Sanger sequencing was least demanding on sample DNA quality for a successful analysis, none of the three techniques showed satisfactory diagnostic performance on trephine biopsies. Therefore, careful selection of a suitable sample type and testing platform is important to optimise the detection of this important mutation in HCL.

Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia.

BACKGROUND: TP53 mutation/deletion is uncommon in chronic lymphocytic leukemia (CLL). We postulated that components of TP53-centered tumor suppressor network, miR-34b/c, in addition to DAPK1 and miR-34a might be inactivated by DNA hypermethylation. Moreover, we tested if miR-34b/c methylation might correlate with miR-203 or miR-124-1 methylation in CLL. METHODS: miR-34b/c, miR-34a and DAPK1 methylation was studied in 11 normal controls, 7 CLL cell lines, and 78 diagnostic CLL samples by methylation-specific polymerase chain reaction. MEC-1 cells were treated with 5-Aza-2'-deoxycytidine for reversal of methylation-associated miRNA silencing. Tumor suppressor properties of miR-34b were demonstrated by over-expression of precursor miR-34b in MEC-1 cells. RESULTS: miR-34b/c promoter was unmethylated in normal controls, but completely methylated in 4 CLL cell lines. miR-34b/c expression was inversely correlated with miR-34b/c methylation. Different MSP statuses of miR-34b/c, including complete methylation and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. 5-Aza-2'-deoxycytidine treatment resulted in promoter demethylation and miR-34b re-expression in MEC1 cells. Moreover, over-expression of miR-34b resulted in inhibition of cellular proliferation and increased cell death. In primary CLL samples, miR-34a, miR-34b/c and DAPK1 methylation was detected in 2.6%, 17.9% and 34.6% of patients at diagnosis respectively. Furthermore, 39.7%, 3.8% and 2.6% patients had methylation of one, two or all three genes respectively. Overall, 46.2% patients had methylation of at least one of these three genes. Besides, miR-34b/c methylation was associated with methylation of miR-34a (P = 0.03) and miR-203 (P = 0.012) in CLL. CONCLUSIONS: Taken together, miR-34b/c is a tumor suppressor miRNA frequently methylated, and hence silenced in CLL. Together with DAPK1 methylation, miR-34b/c methylation is implicated in the disruption of the TP53-centered tumor suppressor network. Moreover, the association of miRNA methylation warrants further study.

Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia--implications on constitutive activation of NFκB pathway.

BACKGROUND: The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL). METHODS: Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction. RESULTS: The promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2'-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04). CONCLUSIONS: Of the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.

Pediatric acute myeloid leukemia with t(8;16)(p11;p13), a distinct clinical and biological entity: a collaborative study by the International-Berlin-Frankfurt-Munster AML-study group.

In pediatric acute myeloid leukemia (AML), cytogenetic abnormalities are strong indicators of prognosis. Some recurrent cytogenetic abnormalities, such as t(8;16)(p11;p13), are so rare that collaborative studies are required to define their prognostic impact. We collected the clinical characteristics, morphology, and immunophenotypes of 62 pediatric AML patients with t(8;16)(p11;p13) from 18 countries participating in the International Berlin-Frankfurt-Münster (I-BFM) AML study group. We used the AML-BFM cohort diagnosed from 1995-2005 (n = 543) as a reference cohort. Median age of the pediatric t(8;16)(p11;p13) AML patients was significantly lower (1.2 years). The majority (97%) had M4-M5 French-American-British type, significantly different from the reference cohort. Erythrophagocytosis (70%), leukemia cutis (58%), and disseminated intravascular coagulation (39%) occurred frequently. Strikingly, spontaneous remissions occurred in 7 neonates with t(8;16)(p11;p13), of whom 3 remain in continuous remission. The 5-year overall survival of patients diagnosed after 1993 was 59%, similar to the reference cohort (P = .14). Gene expression profiles of t(8;16)(p11;p13) pediatric AML cases clustered close to, but distinct from, MLL-rearranged AML. Highly expressed genes included HOXA11, HOXA10, RET, PERP, and GGA2. In conclusion, pediatric t(8;16)(p11;p13) AML is a rare entity defined by a unique gene expression signature and distinct clinical features in whom spontaneous remissions occur in a subset of neonatal cases.

IL10 and TNF variants and risk of non-Hodgkin lymphoma among three Asian populations.

Genetic variation in immune-related genes, such as IL10 and TNF, have been associated with the development of non-Hodgkin lymphoma (NHL) in Caucasian populations. To test the hypothesis that IL10 and TNF polymorphisms may be associated with NHL risk in Asian populations, we genotyped 20 single nucleotide polymorphisms (SNPs) within the IL10 and TNF/LTA loci in three independent case-control studies (2635 cases and 4234 controls). IL10 rs1800871, rs1800872, and rs1800896 were genotyped in all three studies, while 5 of the remaining SNPs were genotyped in two studies, and 12 in a single study. IL10 rs1800896 was associated with B cell lymphoma [per-allele odds ratio (OR) = 1.25, 95 % confidence interval (CI) 1.08-1.45; p trend = 0.003], specifically diffuse large B cell lymphoma (DLBCL) (per-allele OR = 1.29, 95 % CI 1.08-1.53; p trend = 0.004), as well as T cell lymphoma (per-allele OR = 1.44, 95 % CI 1.13-1.82; p trend = 0.003). TNF rs1800629, which was genotyped in only two of our studies, was also associated with B cell lymphoma (per-allele OR = 0.77, 95 % CI 0.64-0.91; p trend = 0.003), specifically DLBCL (per-allele OR = 0.69, 95 % CI 0.55-0.86; p trend = 0.001). Our findings suggest that genetic variation in IL10 and TNF may also play a role in lymphomagenesis in Asian populations.

Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms.

BACKGROUND: MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis. METHODS: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs. RESULTS: Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation. CONCLUSION: This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.

Genetic susceptibility for chronic lymphocytic leukemia among Chinese in Hong Kong.

The genetic basis of chronic lymphocytic leukemia (CLL) has not been fully elucidated to date. Although it is the most common haematological malignancy in Caucasians, it is uncommon among Asians. A recent genome-wide scan of CLL in Caucasians, which was carried out in the UK, identified six variants showing strong association. We attempted to replicate these findings in 71 patients with CLL and 1273 controls in Hong Kong Chinese. Three of the six variants were significantly associated with CLL. The rs872071 variant (Odds Ratio (95% Confidence Interval) = 1.78 (1.25-2.53), P = 0.0013) in the IRF4 gene region showed the strongest association, similar to that reported in the UK study. Polymorphisms in SP140 and ACOXL were also associated with risk of CLL. Further, the mean allele frequencies of the six variants were moderately (59%) to extremely (0.5%) lower in the Chinese population compared with Caucasians. These results suggest that variants in three loci may contribute to risk of CLL among Chinese.

Essential thrombocythemia with deleted 5q--a genetic and morphologic hybrid?

A 66-year-old man who presented with progressive and marked thrombocytosis but normal hemoglobin was diagnosed to have essential thrombocythemia upon the demonstration of JAK2 V617F mutation. Bone marrow examination, however, showed the presence of monolobulated megakaryocytes and conventional cytogenetic analysis revealed an isolated interstitial deletion of the long arm of chromosome 5, characteristic of 5q- syndrome. A literature review indicated that isolated deletion of 5q is uncommon in essential thrombocythemia but that, when this isolated deletion is present, the disease often shows mixed features of both essential thrombocythemia and 5q- syndrome.

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.

BACKGROUND: We 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. METHODS: BCR-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. RESULTS: Using 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). CONCLUSION: Although 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.

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.