FISHing for 1p19q codel in oligodendroglioma

@#Together with isocitrate dehydrogenase (IDH) mutation, co-deletion of 1p19q (1p19q codel) is a prerequisite for diagnosis of oligodendroglioma, making it imperative that histopathology laboratories introduce testing for 1p19q codel. To date there is still no consensus reference range and cut-offs that confirm deletion of 1p or 19q. We embarked on determining our reference range in 11 formalinfixed, paraffin-embedded non-neoplastic brain tissue using fluorescence in situ hybridisation (FISH) with the Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Abbott Molecular Inc., USA). At same time we attempted to validate our methodology in 13 histologically-confirmed IDH-mutant oligodendrogliomas. For 1p, percentage cells with deletion (range=8-23%; mean±SD = 15.73±5.50%) and target: control (1p36:1q25) ratio (range = 0.89-0.96; mean±SD = 0.92±0.03) in non-neoplastic brain, differed significantly (p<0.000) from oligodendroglioma (percentage cells with deletion: range = 49-100%; mean±SD = 82.46±15.21%; target:control ratio range:0.50-0.76; mean±SD = 0.59±0.08). For 19q, percentage cells with deletion (range = 7-20%; mean±SD = 12.00±3.49%) and target:control (19q13/19p13) ratio (range:0.90-0.97; mean±SD = 0.94±0.02) in non-neoplastic brain also differed significantly from oligodendroglioma (percentage cells with deletion: range = 45-100%; mean±SD = 82.62±18.13%; target:control ratio range:0.50-0.78; mean±SD = 0.59±0.09). Using recommended calculation method, for diagnosis of 1p deletion, percentage of cells showing deletion should be >32-33% and/or target:control ratio <0.83. For 19q, percentage of cells showing deletion should be >22% and target:control ratio <0.88. Using these cut-offs all 13 oligodendroglioma demonstrated 1p19q codel.

Early relapse after complete remission of primary plasma cell leukaemia manifesting clonal evolution: A case report

@#Introduction: Plasma cell leukaemia (PCL) is a rare variant of multiple myeloma. We report a case of PCL to demonstrate the clonal evolution, resulting in disease relapse after achieving complete remission, and its aggressive nature of the disease, leading to poor clinical outcome. Case Report: A 69-year-old man presented with a three-day-history of worsening generalized body weakness, poor oral intake, nausea, significant loss of weight and lower back pain. He was diagnosed as primary PCL, based on hypercalcaemia, renal insufficiency, anaemia, thrombocytopenia, lytic bone lesions, 24% abnormal plasma cells in peripheral blood, immunophenotype of clonal plasma cells which were positive for CD38, CD138 and CD56 markers with kappa light chain restriction, 49% abnormal plasma cells in bone marrow, monoclonal paraprotein (IgG kappa) in serum and urine, and positive IGH rearrangement (Fluorescence in-situ hybridisation, FISH). He achieved complete remission after four cycles of Bortezomib-based therapy. There was a plan for high-dose therapy plus autologous haematopoietic cell transplantation. A month later, the disease relapsed, as evidenced by 94% abnormal plasma cells in his bone marrow aspirate, complex karyotype and abnormal FISH results. He passed away a few days later, from severe septicaemia. Time-to-progression of disease was 1 month and overall survival was 5 months. Discussion: This case report illustrates the clonal evolution and aggressive nature of primary PCL with older age at presentation, leading to a shorter duration of remission and overall survival.

Mycobacterium tuberculosis promotes genomic instability in macrophages

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.

Cryptic Insertion of the BCR Gene at 9q34 in Philadelphia-Negative Chronic Myelogenous Leukaemia

Chronic myelogenous leukaemia (CML) is a myeloproliferative neoplasm that is almost always characterised by the presence of t(9;22)(q34;q11.2). Approximately 5% to 10% of CML patients lack cytogenetic evidence of t(9;22)(q34;q11.2) but have the breakpoint cluster region (BCR)/ABL1 fusion, as revealed by fl uorescence in situ hybridisation (FISH) or the reverse transcription-polymerase chain reaction (RT-PCR). We present a case of Philadelphia-negative CML with a cryptic insertion of BCR at 9q34. A 22-year-old woman incidentally presented with marked leucocytosis and anaemia. Her complete blood count results were as follows: white blood cells, 238.61x10(9)/L; haemoglobin, 9.6 g/dL; platelets, 395x10(9)/L. A peripheral blood smear showed leucocytosis with neutrophilia, basophilia, left-shifted neutrophils, and circulating blasts comprising 2% of the total leucocytes. The bone marrow showed a striking increase in megakaryocytes and granulocytic precursors. The myeloid/erythroid ratio was 7.4:1, and blasts comprised up to 1.8% of all nucleated cells. Bone marrow sections revealed active megakaryopoiesis and granulopoiesis with 100% cellularity. Chromosomal analysis revealed a normal karyotype. However, interphase FISH using a dual-colour BCR/ABL1 fusion probe showed an atypical pattern consisting of one red, two green, and one fusion (1R2G1F) signal in 97.5% of the 200 analysed cells. Metaphase FISH revealed a single BCR/ABL1 fusion signal on chromosome 9. RT-PCR was positive for BCR/ABL1 (b3a2). Quantitative PCR revealed a normalised copy number of 15.32. The patient started her treatment with imatinib, reached a complete molecular response eight months afterwards, and has been coping well without any adverse events.

Identification of Y Chromosomal Material in Turner Syndrome by Fluorescence In Situ Hybridisation (FISH)

Turner syndrome is one of the most common chromosomal abnormalities affecting newborn females. More than half of patients with Turner syndrome have a 45X karyotype. The rest of the patients may have structurally abnormal sex chromosomes or are mosaics with normal or abnormal sex chromosomes. Mosaicism with a second X sex chromosome is not usually of clinical significance. However, Turner syndrome patients having a second Y chromosome or Y chromosomal material are at risk of developing gonadoblastoma later in life. The aim of this study is to compare the results of conventional (karyotyping) and molecular cytogenetics (FISH), and discuss the advantages and limitations in the diagnosis of Turner syndrome. We also aim to compare the degree of mosaicism identified using conventional cytogenetics and FISH techniques. Conventional cytogenetics and FISH analyses were performed on eight peripheral blood samples of patients with Turner syndrome collected between 2004 and 2006. From this study, two out of eight patients with Turner syndrome were found to have the sex determining region on the Y chromosome (SRY) gene by FISH analysis. Our results showed that the rate of detection of mosaic cases in Turner syndrome was also increased to 88% after using the FISH technique. We concluded that FISH is more superior to conventional cytogenetics in the detection of the Y chromosomal material. FISH is also a quick and cost effective method in diagnosing Turner syndrome and assessing the degree of mosaicism.