Diagnostic utility of p57 immunohistochemistry and DNA ploidy analysis by fluorescence in situ hybridisation in hydatidiform moles.

INTRODUCTION: Hydatidiform moles (HMs) include complete and partial moles, are the result of abnormal fertilisation. The accurate classification of HMs and its distinction from non-molar specimens is utmost important for clinical management and risk assessment. It is diagnostically challenging if the distinction is based solely on histomorphology with poor interobserver reproducibility, especially in early gestations. This study aimed to investigate the diagnostic ability of combined p57 immunohistochemistry and DNA ploidy analysis to distinguish between complete moles, partial moles and non-molar abortus. MATERIALS AND METHODS: We included all HMs cases diagnosed in our centre over a six-year period. p57 immunohistochemistry stain was performed. Only nuclear immunoreactivity in >50% of cytotrophoblasts and villous stromal cells was regarded as positive for p57. DNA ploidy status was determined by fluorescence in situ hybridisation. A total of 250 cells from five chorionic villi were counted and were scored as diploid or triploid if more than 10% of nuclei demonstrated two or three signals, respectively. RESULTS: A total of 51 cases originally diagnosed by histomorphology as complete mole (n = 18), partial mole (n = 24) and non-molar abortus (n = 9) were recruited. The cases were reclassified based on the p57 immunostaining pattern and DNA ploidy status, into 27 complete moles (p57-/diploid), 9 partial moles (p57+/triploid) and 15 non-molar abortus (p57+/diploid). The diagnostic accuracy by histomorphological features alone in each category: complete moles, partial moles and non-molar abortus was 78.4%, 70.6% and 88.2% respectively. CONCLUSION: This study highlighted the importance of the utility of combined p57 immunostain and DNA ploidy analysis in arriving at an accurate diagnosis in HMs. An algorithmic approach utilising these ancillary techniques is advocated in routine diagnostic workup for a more refined diagnostic approach to HMs.

H396P mutation in chronic myeloid leukaemia patient on nilotinib - A case report.

INTRODUCTION: The advent of BCR-ABL1-targeted therapy with the tyrosine kinase inhibitor (TKI), for example, imatinib and nilotinib, marked a turning point in the therapy of chronic myeloid leukaemia (CML). However, a substantial proportion of patients experience primary or secondary disease resistance to TKI. There are multifactorial causes contributing to the treatment failure of which BCR-ABL1 kinase domain mutation being the most common. Here, we describe a case of a CML patient with H396P mutation following treatment with nilotinib. CASE: A 60-year-old woman presented with abdominal discomfort and hyperleukocytosis. She was diagnosed as CML in the chronic phase with positive BCR-ABL1 transcripts. Due to the failure to obtain an optimal response with imatinib treatment, it was switched to nilotinib. She responded well to nilotinib initially and achieved complete haematological and cytogenetic responses, with undetectable BCR-ABL1 transcripts. However, in 4 years she developed molecular relapse. Mutation analysis which was done 70 months after commencement of nilotinib showed the presence of BCRABL1 kinase domain mutation with nucleotide substitution at position 1187 from Histidine(H) to Proline(P) (H396P). Currently, she is on nilotinib 400mg twice daily. Her latest molecular analysis showed the presence of residual BCR-ABL1 transcripts at 0.22%. DISCUSSION/CONCLUSION: This case illustrates the importance of BCR-ABL1 mutation analysis in CML patients with persistent BCR-ABL1 positivity in spite of treatment. Early detection and identification of the type of BCRABL1 mutation are important to guide appropriate treatment options as different mutation will have different sensitivity to TKI.

Mixed phenotype acute leukaemia with t(9,22), BCR-ABL1: A case report.

INTRODUCTION: Mixed phenotype acute leukaemia (MPAL) is a rare entity of acute leukaemia. CASE REPORT: Here we report a case of a 39-year-old lady, with an incidental finding of hyperleukocytosis (white blood cells count: 139.2 x 109/L). Her peripheral blood film revealed 36% of blasts and a bone marrow aspiration showed 53% of blasts. Immunophenotyping showed a population of blasts exhibiting positivity of two lineages, myeloid lineage and B-lymphoid lineage with strong positivity of CD34 and terminal deoxynucleotidyl transferase (Tdt). A conventional karyotyping revealed the presence of Philadelphia chromosome. She was diagnosed with MPAL with t(9,22), BCR ABL1, which carried a poor prognosis. She was treated with acute lymphoblastic leukaemia (ALL) chemotherapy protocol coupled with a tyrosine kinase inhibitor and was planned for an allogeneic stem cells transplant. CONCLUSION: This MPAL case was diagnosed incidentally in an asymptomatic patient during medical check-up. We highlight this rare case report to raise the awareness about this rare disease. Understanding the pathogenesis of the disease with the underlying genes responsible for triggering the disease, uniform protocols for diagnosis and targeted treatment will help for proper management of these patients.

Genetic, epigenetic, and lineage-directed mechanisms in benzene-induced malignancies and hematotoxicity targeting hematopoietic stem cells niche.

Benzene is a known hematotoxic and leukemogenic agent with hematopoietic stem cells (HSCs) niche being the potential target. Occupational and environmental exposure to benzene has been linked to the incidences of hematological disorders and malignancies. Previous studies have shown that benzene may act via multiple modes of action targeting HSCs niche, which include induction of chromosomal and micro RNA aberrations, leading to genetic and epigenetic modification of stem cells and probable carcinogenesis. However, understanding the mechanism linking benzene to the HSCs niche dysregulation is challenging due to complexity of its microenvironment. The niche is known to comprise of cell populations accounted for HSCs and their committed progenitors of lymphoid, erythroid, and myeloid lineages. Thus, it is fundamental to address novel approaches via lineage-directed strategy to elucidate precise mechanism involved in benzene-induced toxicity targeting HSCs and progenitors of different lineages. Here, we review the key genetic and epigenetic factors that mediate hematotoxicological effects by benzene and its metabolites in targeting HSCs niche. Overall, the use of combined genetic, epigenetic, and lineage-directed strategies targeting the HSCs niche is fundamental to uncover the key mechanisms in benzene-induced hematological disorders and malignancies.

FISH versus real-time quantitative PCR for monitoring of minimal residual disease in chronic myeloid leukaemia patients on tyrosine kinase inhibitor therapy.

INTRODUCTION: BCR-ABL fusion gene, the oncogenic driver of CML, results from a translocation between short arms of chromosome 9 and 22. Monitoring of CML patients during treatment is essential, not only for tailoring the treatment but also to detect early relapse to enable timely intervention. Commonly used methods for detection of residual disease are conventional karyotyping, FISH and molecular methods. In this study, we compared FISH with QRT-PCR for detection of residual disease in CML. MATERIALS AND METHODS: CML patients on tyrosine kinase inhibitor (TKI) therapy and on regular follow up at University Kebangsaan Malaysia Medical Center (UKMMC) were selected. A comparative study was conducted between FISH and QRT-PCR for BCR-ABL transcripts at diagnosis and during follow-up. RESULTS: There was good correlation between FISH and QRT-PCR for BCR-ABL. At 6th month of follow-up post diagnosis, FISH had a sensitivity of 83.3% and specificity of 65.2% (k >0.339, p<0.033). At 12th month, the sensitivity of FISH was 83% and the specificity was 59.1% (k >0.286, p <0.065). Similarly, at the 24th month, FISH had a sensitivity of 100% and specificity of 68.8% (k >0.642, p<0.000). DISCUSSION: Early achievement of major molecular response (MMR) and complete cytogenetic remission (CCyR) were reliable predictors of long-term maintenance of molecular remission.

Effects of prolonged isolation in a confined space on status of oxidative stress and prothrombogenesis: In preparation for possible future manned space expedition to Mars.

INTRODUCTION: Apart from inflammation and endothelial dysfunction, other key components in the development of atherogenesis include prothrombogenesis and oxidative stress. The effects of long-term confinement and isolation, exposure to radiation and different gravity forces during space travel could potentially increase the long-term risk of atherosclerosis. To the best of our knowledge, this is the first study determining the status of prothrombogenesis and oxidative stress in six cosmonauts subjected to the longest duration of confined isolation period of 520 days in preparation for prospective undetermined manned space travel to Mars. MATERIALS AND METHODS: This collaborative research between the National Space Agency (ANGKASA), Universiti Teknologi MARA, Malaysia and Institute of Biomedical Problems (IBMP), Russia was conducted at the Russian Academy of Sciences IBMP, Moscow, Russia. Six multi-national cosmonauts were assigned to live in a ground-based confined module for 520 days. Standard exercise and diet regime were instituted throughout the isolation phase. Six age, ethnic and gender-matched healthy, free-living ground controls were recruited in parallel. Serial serum and whole blood were analysed for biomarkers of prothrombogenesis [plasminogen activator inhibitor-1 (PAI-1) and homocysteine] and oxidative stress [oxidised low-density lipoprotein (ox-LDL) and malondialdehyde (MDA)]. RESULTS: There were significantly lower concentrations of PAI-1 and homocysteine in cosmonauts during confinement compared to the controls. There were no significant differences seen in the concentrations of biomarkers of oxidative stress during confinement but there was a significant percentage change increment for serum MDA in cosmonauts. CONCLUSION: Long-term confinement decreased the risk of prothrombogenesis and this could be attributed to the exercise and diet regime which includes omega-3 fatty acids supplementation given to the crew members during their confinement period. However, oxidative damage could not be excluded and may be attributed to the influence of psychological stress during this prolonged confinement.

Myelodysplastic syndrome with fibrosis and complex karyotype arising in a patient with essential thrombocythaemia.

INTRODUCTION: Essential thrombocythaemia (ET) is a chronic myeloproliferative neoplasm (MPN) characterised by persistent thombocytosis. It is an indolent disorder but transformation to myelofibrosis (MF), acute myeloid leukaemia (AML) or myelodyplastic syndrome (MDS) has been reported. CASE REPORT: We described a patient with ET whose disease evolved into MDS with fibrosis and complex karyotype after 15 years of stable disease. She was asymptomatic and was on hydroxyurea (HU) treatment until recently when she presented with worsening anaemia. Physical examination showed mild splenomegaly. Full blood picture showed leukoerythroblastic picture with presence of 3% circulating blasts and background of dysplastic features such as hypogranular cytoplasm and nuclear hyposegmentation of neutrophils. The bone marrow aspiration was haemodiluted but revealed presence of 6% blast cells, trilineage dysplasia and predominant erythroid precursors (60%). Trephine biopsy showed no excess of blast cells and normal quantity of erythroid precursors, but there was increased in fibrosis (WHO grade 2) and presence of dysmegakaryopoeisis such as nuclear hypolobation, multinucleation and micromegakaryocytes. Cytogenetic study showed complex karyotype; monosomy of chromosome 2, chromosome 5, chromosome 18 and presence of a marker chromosome (42~44, XX,-2,-5,-18,+mar). Fluorescence in situ hybridisation (FISH) showed 5q deletion (CSF1R and EGR1). CONCLUSION: The findings were consistent with transformation of ET to MDS with fibrosis and complex karyotype. ET progression to MDS is considered rare. The presence of complex karyotype and fibrosis in MDS are associated with unfavourable outcome.

Duplication 17p11.2 (Potocki-Lupski Syndrome) in a child with developmental delay.

Potocki-Lupski syndrome (PTLS), also known as duplication 17p11.2 syndrome, trisomy 17p11.2 or dup(17)(p11.2p11.2) syndrome, is a developmental disorder and a rare contiguous gene syndrome affecting 1 in 20,000 live births. Among the key features of such patients are autism spectrum disorder, learning disabilities, developmental delay, attention-deficit disorder, infantile hypotonia and cardiovascular abnormalities. Previous studies using microarray identified variations in the size and extent of the duplicated region of chromosome 17p11.2. However, there are a few genes which are considered as candidates for PTLS which include RAI1, SREBF1, DRG2, LLGL1, SHMT1 and ZFP179. In this report, we investigated a case of a 3-year-old girl who has developmental delay. Her chromosome analysis showed a normal karyotype (46,XX). Analysis using array CGH (4X44 K, Agilent USA) identified an ~4.2 Mb de novo duplication in chromosome 17p11.2. The result was confirmed by fluorescence in situ hybridization (FISH) using probes in the critical PTLS region. This report demonstrates the importance of microarray and FISH in the diagnosis of PTLS.

A rare case of ambiguous genitalia.

Genes on the Y chromosome are essential for normal sex determination and sex differentiation of male genitalia. However, genes on the X chromosome and other autosomes have been shown to be anti-testes and have a detrimental effect on this process. Addition of X chromosomes to the 46,XY karyotype results in seminiferous tubules dysgenesis, hypogonadism and malformed genitalia. We report a term male newborn with 49,XXXXY syndrome presenting with ambiguous genitalia, multiple extra-gonadal anomalies, facial dysmorphism, and radioulnar synostosis.