Chromosome microarray proficiency testing and analysis of quality metric data trends through an external quality assessment program for Australasian laboratories.

Chromosome microarrays are an essential tool for investigation of copy number changes in children with congenital anomalies and intellectual deficit. Attempts to standardise microarray testing have focused on establishing technical and clinical quality criteria, however external quality assessment programs are still needed. We report on a microarray proficiency testing program for Australasian laboratories. Quality metrics evaluated included analytical accuracy, result interpretation, report completeness, and laboratory performance data: sample numbers, success and abnormality rate and reporting times. Between 2009 and 2014 nine samples were dispatched with variable results for analytical accuracy (30-100%), correct interpretation (32-96%), and report completeness (30-92%). Laboratory performance data (2007-2014) showed an overall mean success rate of 99.2% and abnormality rate of 23.6%. Reporting times decreased from >90 days to <30 days for normal results and from >102 days to <35 days for abnormal results. Data trends showed a positive correlation with improvement for all these quality metrics, however only 'report completeness' and reporting times reached statistical significance. Whether the overall improvement in laboratory performance was due to participation in this program, or from accumulated laboratory experience over time, is not clear. Either way, the outcome is likely to assist referring clinicians and improve patient care.

Novel BRD4-NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Overexpression and altered glycosylation of MUC1 in malignant mesothelioma.

Current interest in the MUC1/EMA mucin relates to its role in malignancy, and its potential as a therapeutic target. MUC1/EMA expression has been observed in the majority of epithelioid mesotheliomas. However, little is known of the characteristics of MUC1/EMA in mesothelioma. Herein, we studied the cell surface and soluble expression of the MUC1/EMA glycoprotein, and determined the mRNA and genomic expression profiles in mesothelioma. We found that the anti-MUC1 antibody, E29, was the most diagnostically useful of seven antibody clones examined with a sensitivity of 84% (16 out of 19 cases) and no false positive results. MUC1 mRNA expression was significantly higher in mesothelioma samples than in benign mesothelial cells. No amplification of the MUC1 gene was observed by FISH. Seven of 9 mesothelioma samples expressed MUC1-secreted mRNA isoform in addition to the archetypal MUC1/transmembrane form. CA15.3 (soluble MUC1) levels were significantly higher in the serum of mesothelioma patients than in healthy controls but were not significantly different to levels in patients with benign asbestos-related disease. CA15-3 in effusions could differentiate malignant from benign effusions but were not specific for mesothelioma. Thus, as in other cancers, alterations in MUC1 biology occur in mesothelioma and these results suggest that specific MUC1 characteristics may be useful for mesothelioma diagnosis and should also be investigated as a potential therapeutic target.

Cytogenetic analysis of embryos generated from in vitro matured mouse oocytes reveals an increase in micronuclei due to chromosome fragmentation.

PURPOSE: (i) To determine the prevalence of micronuclei in the cytoplasm of embryos generated from in vitro matured oocytes. (ii) Assess whether micronuclei presence are the result of chromosome fragmentation or the loss of whole chromosomes. METHODS: In vitro fertilization was performed on mature oocytes generated from superovulated mice (control) and in vitro matured mouse oocytes. Fertilized oocytes were cultured to the two-cell stage and fixed to slides. Micronuclei assessment was performed after staining with Giemsa. Centromere assessment was made using immunofluorescent staining (CREST) of the centromeric kinetochores. RESULTS: Micronuclei were observed in 2% (4/197) of control two-cell embryos and 36.2% (46/127) of two-cell embryos generated from in vitro matured oocytes (P < 0.02). Centromeres were not detected in micronuclei from either group. CONCLUSIONS: A significant increase in micronuclei was observed in embryos generated from in vitro matured oocytes. The lack of accompanyingcentromeres would suggest the micronuclei are the result of chromosome fragmentation.

Telomeric fusion is a major cytogenetic aberration of giant cell tumors of bone.

Giant cell tumor of bone (GCT) is regarded as a rare primary bone neoplasm derived from stromal cells, which have the ability to recruit and harbor macrophage and multinucleated osteoclast-like giant cells. Despite being often considered benign, GCT is a problematic neoplasm in that it is aggressive, unpredictable and difficult to treat effectively. Cytogenetically GCT is characterised by a high frequency of telomeric fusion, a process which has been implicated in the production of chromosome instability and tumorigenesis. To extend our knowledge of the significance of telomere association in GCT, the cytogenetics of cell lines derived from spindle-shaped stromal-like mononuclear cells (the tumor cells) of GCT was investigated. Cell lines from three different patients showed telomeric association in all passages. The rate of telomeric association varied from line to line and from passage to passage, but there was no particular pattern to the variations. Many other cytogenetic abnormalities were seen as well as telomeric association, but these were rarely clonal. The nature of most of the other abnormalities seen, such as deleted chromosomes and chromosomes with additional unidentifiable material, was consistent with their being formed as a result of breakage of the dicentric fused chromosomes at a telophase. Chromosomes 13, 14 and 21 were most commonly involved in telomeric fusion. It appears that telomeric association persists in long-term cultures of GCT and is responsible for the accumulation of other associated cytogenetic aberrations. Telomeric reduction and telomerase activity may act as oncogenic events, promoting and sustaining the transformed GCT phenotype.

Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermic man.

A man with globozoospermia was treated in our in-vitro fertilization-intracytoplasmic sperm injection (ICSI) programme. In the treatment cycle, 24 oocytes were collected from his wife. All the oocytes were at metaphase II stage. The semen sample produced on the day had a normal sperm count, good motility, but with 100% globozoospermia. All oocytes were injected with randomly selected spermatozoa and of these, two oocytes showed two pronuclei and another contained a single pronucleus. The remainder were unfertilized. The normally fertilized oocytes (two pronuclear) cleaved to the four-cell stage and were transferred to the patient. At 48 h after ICSI, the 21 unfertilized oocytes were processed for cytogenetic analysis. All oocytes contained a haploid chromosome set. The only abnormality seen was a chromosome fragment in one metaphase. Eighteen oocytes contained decondensed sperm nuclei and of these, 14 nuclei were beginning to show signs of premature chromatin condensation (PCC) and the other four showed strong signs of PCC. Thus it appears that in some forms of globozoospermia, arrest of nuclear decondensation and/or PCC are another cause of fertilization failure. The most likely cause for this is the absence or down-regulation of spermatozoa associated activating factor in round-headed spermatozoa.

Trophoblast antigen levels in the first trimester of a trisomy 22 pregnancy.

We report trophoblast antigen (pregnancy-associated plasma protein-A, PAPP-A; free beta-human chorionic gonadotrophin, F beta hCG) expression in a trimosy 22 pregnancy. Maternal concentrations of these antigens were depressed prior to detection of abnormalities by ultrasonography. Immunohistochemical findings were consistent with depressed marker expression.

Cytogenetic analysis of human oocytes and embryos in an in-vitro fertilization programme.

Oocytes (unfertilized and preovulatory) and embryos (normal and polypronuclear), which were donated to research by patients undergoing procedures of assisted reproductive treatment, were analysed for cytogenetic abnormalities. A total of 362 oocytes and embryos were analysed. The unfertilized oocytes with readable metaphases (53.4%) gave 25.2% chromosomal abnormality with diploidy being the main aberration observed. A high incidence of premature chromosome condensation (PCC) was observed and the incidence of PCC in oocytes exposed to colcemid was significantly higher (14/62, 22.6%) than in those not exposed to this treatment (3/41, 7.3%, P less than 0.05). When chromosomal anomalies and PCC in the unfertilized oocytes were correlated to various patient criteria such as stimulation regimen, number of human menopausal gonadotrophin ampoules, peak oestradiol levels, age of patient and number of previous attempts, none of the criteria tested had any significant relationship to the incidence of chromosomal abnormality. However a significant increase in the incidence of PCC was noted in the gonadotrophin-releasing hormone (GnRH) 'flare' group (6/15, 40.0%) compared to the GnRH 'down-regulation' group (11/88, 12.5%). The incidence of chromosomal abnormalities among preovulatory oocytes was 16.7% and diploidy was the only abnormality noted. For embryos arising from two-pronuclear oocytes, the chromosomal constitution related mainly to embryo quality. The rate of chromosomal abnormality for apparently good quality embryos was 23.5% and for poor or fragmented embryos 83.3%. The majority (77.3%) of the readable metaphase plates for polypronuclear 1-cell and cleaved embryos showed grossly abnormal chromosome complements but 19% of the cleaved embryos contained sets of normal diploid chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions.

Malignant mesothelioma (MM) is an aggressive tumour of the serosal cavities which is associated with exposure to asbestos. Studies of this tumour have been limited by a paucity of well-characterized human MM cell lines. In this study, 5 human MM cell lines were established from pleural effusions of patients with this malignancy. All 5 patients were males with known crocidolite asbestos exposure, who had received no treatment for their disease and in whom the diagnosis was confirmed by cytology, histology and electron microscopy (EM). These lines have been in culture from 11 to 25 months, and all of them for more than 18 passages. The appearance of the cells in culture was extremely varied; in 3 of the lines they were spindle-shaped with few vacuoles (JU77, LO68 and ONE58); in 1 line they had a thick, stellate shape with vacuoles (NO36) and in 1 they were very pleomorphic in both shape and size with irregular membranes and numerous vacuoles [DeH128 (M)]. Upon reaching confluence, cells in 3 of the 5 lines assumed the cobblestone-like pattern characteristic of epithelial-type cells, whereas in the other 2 (LO68 and ONE58) they remained spindle-shaped. All 5 lines demonstrated a loss of contact inhibition (i.e., piling) at confluence. Minimum doubling times varied significantly from 18 hr (JU77) to more than 30 hr [DeH128 (M)]. Cytological examination showed characteristic mesothelial/mesothelioma morphology, and epithelial membrane antigen (EMA) and cytokeratin were demonstrated in cells from all 5 lines. These cells lacked CEA and epithelial mucin. The presence of cell junctions, glycogen and numerous long, thin, branching microvilli was readily demonstrable by EM. All lines had abnormal karyotypes, with the modal chromosome number varying from 40 to 80. Variable chromosome numbers, numerous structural rearrangements and unrecognizable marker chromosomes were readily observed; however, the only consistent change seen was del 6q21 in 4 of the 5 lines. The establishment of these 5 cultured human MM cell lines now provides an opportunity for comparative study of several aspects of the biology of MM in vitro as well as screening new treatment modalities.

A cytogenetic study of subcutaneous murine macrophages and multinucleate giant cells.

Macrophages and multinucleate giant cells (MGC), collected by subcutaneous implantation of melinex discs into the dorsum of mice for 7 days, were examined cytogenetically. Two per cent of the metaphases seen were polyploid and were considered to represent dividing MGC. Twenty-two per cent of the diploid metaphases showed chromosomal damage. Seventy per cent of damage consisted of chromosomal gaps, but the remaining 30% consisted of breaks and chromosomal fragments which would lead to unbalanced karyotypes in the next generation. More than 50% of the polyploid metaphases analysed had chromosomal damage, including 16.6% which displayed premature chromatin condensation, the damage being more severe in polyploid than in diploid metaphases. The chromosomal damage parallels that previously reported in resident peritoneal macrophages and in cultured exudate macrophages. The cause of such damage is unclear, but it was concluded that, because of the extent of damage, mitosis is unlikely to play a major part in the maintenance of macrophages or MGC at inflammatory sites.

Improved chimaeric mouse model confirms that resident peritoneal macrophages are derived solely from bone marrow precursors.

The origin of resident peritoneal macrophages was studied in radiation mouse chimaeras with and without reconstitution of the peritoneum with viable isogeneic peritoneal cells. The selection of host and donor strains were such that the isoenzymes of glucose-6-phosphate isomerase could be used to distinguish host from donor bone marrow derived cells. It was found that the resident peritoneal macrophages were completely replaced by bone marrow donor derived cells within 5-6 weeks. There was little difference between the results from mice which had been additionally reconstituted with peritoneal cells and those which were not.

Direct evidence that inflammatory multinucleate giant cells form by fusion.

Multinucleate giant cells (MGC) are believed to be formed by fusion of macrophages. In a chimaeric mouse composed of two histoincompatible strains each homozygous for one of the two isoenzymic forms of glucose-6-phosphate isomerase it was found that hybrid enzyme was produced in MGC-rich leucocytic exudates. This hybrid can only occur if nuclei of the two different strains reside within a common syncytial cytoplasm, demonstrating unequivocally that macrophage fusion occurred between cells of the two strains. Since the two strains were histoincompatible it appears that no strain specific recognition is necessary for fusion to occur.

The kinetics of murine peritoneal macrophage replication.

The percentage of peritoneal macrophages synthesising DNA in the unstimulated mouse peritoneum was found to be 0.4 per cent. after a 30 min in-vivo pulse label of 3H Tdr. This percentage was found to rise to 4.0, 24 hr after stimulation of the cavity with thioglycollate. Using BrdU incorporation as an indicator of DNA synthesis it was found that in the unstimulated animals S + G2 lasted 12-15 hr while the same functions in stimulated animals took 7-10 hr. A small proportion of much more rapidly dividing cells was found in both stimulated an unstimulated animals. These findings possibly reflect differences between resident and exudate macrophages, or alterations in the kinetics of macrophage division as a result of inflammation. It was concluded that as the increase in the number of cells synthesising DNA and their rate of division was too low, the increase in the number of cells in the stimulated cavity could not be solely the result of cell division within the cavity.

A cytogenetic and cytophotometric analysis of resident and exudate murine macrophages.

Cytogenetic analysis and Feulgen--DNA cytophotometry were performed on murine resident and exudate peritoneal macrophages. A large proportion (14--36 per cent.) of metaphase profiles from cell populations rich in resident macrophages displayed chromosomal aberrations compared with 0--8 per cent. seen in metaphases from cell populations rich in exudate macrophages. The most common aberration observed was the presence of chromosomal gaps. It is suggested that should the resident macrophage represent a later, and perhaps terminal stage of macrophage development, the high frequency of aberrations found may be a result of cell ageing.

Cytogenetic survey of a hospital for the mentally retarded.

A cytogenetic survey of all 588 patients in Strathmont Training Centre, an Australian hospital for the mentally retarded, was carried out. Abnormal karyotypes were found in 90 (15.3%) patients, of whom 73 (12.4%) had clinical Down syndrome, 12 (2.04%) other autosomal abnormalities, and 5 (0.85%) sex chromosome abnormalities.