A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots.

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.

Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

Complex karyotype in a polycythemia vera patient with a novel SETD1B/GTF2H3 fusion gene.

The patient had been diagnosed with polycythemia vera (PV) in 1999, at the age of 61, according to the criteria of the Polycythemia Vera Study Group (PVSG) on the basis of the increased red cell mass by isotope determination, normal oxygen saturation, low plasma erythropoietin level, presence of endogenous erythroid colonies (EEC), and splenomegaly. Histopathology of bone marrow biopsy was also consistent with polycythemia vera with no evidence of increased reticulin fibrosis. A karyotype analysis was not performed at that time. He had been treated initially with phlebotomies and then with hydroxyurea with the aim to obtain a better control of hematocrit; he was under low-dose aspirin. In 2009, 10 years after the diagnosis, while the patient was still being treated with hydroxyurea and phlebotomies, he noticed worsening of general conditions and fatigue, and the appearance of night sweats; he also reported that his spleen volume had increased rapidly in the past few months. He complained of severe pruritus especially after (but not limited to) a shower. He was referred to our center for further evaluation. At presentation, his blood counts were as follows: hemoglobin 157 g/L, hematocrit 54.7%, leukocytes 13.1 × 109 /L, platelets 238 × 109 /L, LDH 856 U/L (normal upper limit, 250 U/L). Blood film examination showed neutrophilia (8.9 × 109 /L) but immature myeloid cells and nucleated erythroblasts were absent. The spleen was 14 cm below the left costal margin, the liver was at 4 cm below the right costal margin. He was found to harbor the JAK2V617F mutation with an allele burden of 85% and the circulating CD34⁺ cell count was 14 × 106 /L. A bone marrow biopsy showed the presence of hyperplasia of myeloid and erythroid lineages, increased number of scattered megakarocytes without overt morphologic abnormalities; reticulin fibrosis was grade 1 according to the European classification. On these basis, we considered the patient as presenting the features of PV according to the 2008 WHO classification of myeloid neoplasms associated with grade 1 reticulin fibrosis.

Visualization of the spatial positioning of the SNRPN, UBE3A, and GABRB3 genes in the normal human nucleus by three-color 3D fluorescence in situ hybridization.

The three-dimensional (3D) structure of the genome is organized non-randomly and plays a role in genomic function via epigenetic mechanisms in the eukaryotic nucleus. Here, we analyzed the spatial positioning of three target regions; the SNRPN, UBE3A, and GABRB3 genes on human chromosome 15q11.2-q12, a representative cluster of imprinted regions, in the interphase nuclei of B lymphoblastoid cell lines, peripheral blood cells, and skin fibroblasts derived from normal individuals to look for evidence of genomic organization and function. The positions of these genes were simultaneously visualized, and all inter-gene distances were calculated for each homologous chromosome in each nucleus after three-color 3D fluorescence in situ hybridization. None of the target genes were arranged linearly in most cells analyzed, and GABRB3 was positioned closer to SNRPN than UBE3A in a high proportion of cells in all cell types. This was in contrast to the genomic map in which GABRB3 was positioned closer to UBE3A than SNRPN. We compared the distances from SNRPN to UBE3A (SU) and from UBE3A to GABRB3 (UG) between alleles in each nucleus, 50 cells per subject. The results revealed that the gene-to-gene distance of one allele was longer than that of the other and that the SU ratio (longer/shorter SU distance between alleles) was larger than the UG ratio (longer/shorter UG distance between alleles). The UG distance was relatively stable between alleles; in contrast, the SU distance of one allele was obviously longer than the distance indicated by the genome size. The results therefore indicate that SNRPN, UBE3A, and GABRB3 have non-linear and non-random curved spatial positioning in the normal nucleus, with differences in the SU distance between alleles possibly representing epigenetic evidence of nuclear organization and gene expression.

Centromere activity in dicentric small supernumerary marker chromosomes.

Twenty-five dicentric small supernumerary marker chromosomes (sSMC) derived from #13/21, #14, #15, #18, and #22 were studied by immunohistochemistry for their centromeric activity. Centromere protein (CENP)-B was applied as marker for all centromeres and CENP-C to label the active ones. Three different 'predominant' activation patterns could be observed, i.e., centric fusion or either only one or all two centromeres were active. In one inherited case, the same activation pattern was found in mother and son. In acrocentric-derived sSMC, all three activation patterns could be present. In contrary, in chromosome 18-derived sSMC, only the fusion type was observed. In concordance with previous studies a certain centromeric plasticity was observed in up to 13% of the cells of an individual case. Surprisingly, the obtained data suggests a possible influence of the sSMC carrier's gender on the implementation of the predominant activation pattern; especially, only one active centromere was found more frequently in female than in male carriers. Also, it might be suggested that dicentric sSMC with one active centromere could be less stable than such with two active ones-centromeric plasticity might have an influence here, as well. Also, centromere activity in acrocentric-derived dicentrics could be influenced by heteromorphisms of the corresponding short arms. Finally, evidence is provided that the closer the centromeres of a dicentric are and if they are not fused, the more likely it was that both of them became active. In concordance and refinement with previous studies, a distance of 1.4 Mb up to about 13 Mb the two active centromere state was favored, while centromeric distance of over approximately 15 Mb lead to inactivation of one centromere. Overall, here, the first and largest ever undertaken study in dicentric sSMC is presented, providing evidence that the centromeric activation pattern is, and parental origin may be of interest for their biology. Influence of mechanisms similar or identical to meiotic imprinting in the centromeric regions of human chromosomes might be present. Furthermore, centromeric activation pattern could be at least in parts meaningful for the clinical outcome of dicentric sSMC, as sSMC stability and mosaicism can make the difference between clinically normal and abnormal phenotypes.

7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

Homologous association of oppositely imprinted chromosomal domains.

Human chromosome 15q11-q13 encompasses the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) loci, which are subject to parental imprinting, a process that marks the parental origin of certain chromosomal subregions. A temporal and spatial association between maternal and paternal chromosomes 15 was observed in human T lymphocytes by three-dimensional fluorescence in situ hybridization. This association occurred specifically at the imprinted 15q11-q13 regions only during the late S phase of the cell cycle. Cells from PWS and AS patients were deficient in association, which suggests that normal imprinting involves mutual recognition and preferential association of maternal and paternal chromosomes 15.

Immunohistochemical and biogenetic features of diffuse-type tenosynovial giant cell tumors: the potential roles of cyclin A, P53, and deletion of 15q in sarcomatous transformation.

PURPOSE: Diffuse-type tenosynovial giant cell tumor (D-TSGCT) is an aggressive proliferation of synovial-like mononuclear cells with inflammatory infiltrates. Despite the COL6A3-CSF1 gene fusion discovered in benign lesions, molecular aberrations of malignant D-TSGCTs remain unidentified. EXPERIMENTAL DESIGN: We used fluorescent in situ hybridization and in situ hybridization to evaluate CSF1 translocation and mRNA expression in six malignant D-TSGCTs, which were further immunohistochemically compared with 24 benign cases for cell cycle regulators involving G(1) phase and G(1)-S transition. Comparative genomic hybridization, real-time reverse transcription-PCR, and a combination of laser microdissection and sequencing were adopted to assess chromosomal imbalances, cyclin A expression, and TP53 gene, respectively. RESULTS: Five of six malignant D-TSGCTs displayed CSF1 mRNA expression by in situ hybridization, despite only one having CSF1 translocation. Cyclin A (P = 0.008) and P53 (P < 0.001) could distinguish malignant from benign lesions without overlaps in labeling indices. Cyclin A transcripts were more abundant in malignant D-TSGCTs (P < 0.001). All malignant cases revealed a wild-type TP53 gene, which was validated by an antibody specifically against wild-type P53 protein. Chromosomal imbalances were only detected in malignant D-TSGCTs, with DNA losses predominating over gains. Notably, -15q was recurrently identified in five malignant D-TSGCTs, four of which showed a minimal overlapping deletion at 15q22-24. CONCLUSIONS: Deregulated CFS1 overexpression is frequent in malignant D-TSGCTs. The sarcomatous transformation involves aberrations of cyclin A, P53, and chromosome arm 15q. Cyclin A mRNA is up-regulated in malignant D-TSGCTs. Non-random losses at 15q22-24 suggest candidate tumor suppressor gene(s) in this region. However, P53 overexpression is likely caused by alternative mechanisms rather than mutations in hotspot exons.

Acquired acute myelogenous leukemia after therapy for acute promyelocytic leukemia with t(15;17): a case report and review of the literature.

Therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myelogenous leukemia (t-AML) in patients with acute promyelocytic leukemia (APL) are rare events. The cumulative exposure to chemotherapy with alkylating agents and topoisomerase II inhibitors is associated with t-AML that may develop any time after the completion of the treatment. We report the case of an acquired AML who previously received therapy for APL, after two years of being diagnosed. The diagnosis was established by morphologic findings, membrane markers, cytogenetic studies, and fluorescence in situ hybridization (FISH). To our knowledge this is the first documented case in Puerto Rico of a patient with APL that developed a t-AML without the characteristic chromosomal and morphologic findings of APL.

Sensorineural deafness and male infertility: a contiguous gene deletion syndrome.

BACKGROUND: Syndromic hearing loss that results from contiguous gene deletions is uncommon. Deafness-infertility syndrome (DIS) is caused by large contiguous gene deletions at 15q15.3. METHODS: Three families with a novel syndrome characterised by deafness and infertility are described. These three families do not share a common ancestor and do not share identical deletions. Linkage was established by completing a genome-wide scan and candidate genes in the linked region were screened by direct sequencing. RESULTS: The deleted region is about 100 kb long and involves four genes (KIAA0377, CKMT1B, STRC and CATSPER2), each of which has a telomeric duplicate. This genomic architecture underlies the mechanism by which these deletions occur. CATSPER2 and STRC are expressed in the sperm and inner ear, respectively, consistent with the phenotype in persons homozygous for this deletion. A deletion of this region has been reported in one other family segregating male infertility and sensorineural deafness, although congenital dyserythropoietic anaemia type I (CDAI) was also present, presumably due to a second deletion in another genomic region. CONCLUSION: We have identified three families segregating an autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility. This new syndrome is caused by the deletion of contiguous genes at 15q15.3.

Distribution of the D15Z1 copy number polymorphism.

Using fluorescent in situ hybridization (FISH) with the probe p15 (D15Z1), we investigated the distribution of the polymorphic 15p signal which has been reported to occur on acrocentric chromosomes in addition to chromosome 15. The short arm of chromosome 15 has a characteristic signal pattern when hybridized with the FISH probe D15Z1. However, the D15Z1 signal can occasionally be seen on the short arm of other acrocentric chromosomes. We studied the distribution of the D15Z1 probe in 1657 patients consisting both of individuals with a normal karyotype and those with a variety of chromosome abnormalities involving the acrocentric chromosomes. Our results show that one in six individuals, regardless of their patient ascertainment category or karyotypic status, had one or more additional D15Z1 signals, and that there were no significant differences in the distribution of extra signals among the patient groups.

Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies.

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.

Favourable outcome in an APL patient with PLZF/RARalpha fusion gene: quantitative real-time RT-PCR confirms molecular response.

Rare cases of acute promyelocytic leukemia (APL) are associated with a t(11;17) translocation and a PLZF-RARalpha fusion transcript. Because of molecular specificities of the fusion protein, ATRA efficiency is often reduced in these cases. We present herein the case of an 83-year old patient which has been successfully treated by ATRA and Daunorubicin. The described quantitative RT-PCR method allowed successful monitoring and confirmation of the molecular response.

Midline carcinoma with t(15;19) and BRD4-NUT fusion oncogene in a 30-year-old female with response to docetaxel and radiotherapy.

BACKGROUND: Poorly differentiated midline carcinoma with a translocation between chromosomes 15 and 19, i.e. t(15;19), has been recognized as a distinct clinical entity for over a decade. This tumor affects young individuals, shows a rapidly fatal clinical course despite intensive therapy. The t(15;19) results in the fusion oncogene BRD4-NUT. Information concerning treatment of this rare disorder is scarce. CASE PRESENTATION: A 30-year-old woman was admitted with a rapidly progressing tumor in the mediastinum, cervical lymph nodes, vertebral column and the epidural space. Pathological, cytogenetic, FISH and PCR analysis revealed a glycogenated carcinoma rarely expressing cytokeratins and showing t(15;19) and BRD4-NUT gene rearrangement. The patient was initially treated with a Ewing sarcoma chemotherapy regimen, but had rapid progression after two cycles. She then received docetaxel and radiotherapy, which resulted in almost complete disappearance of the tumor. CONCLUSION: Docetaxel may be considered for initial chemotherapy in young patients presenting with a midline carcinoma with bone marrow involvement and cytogenetic and molecular genetic finding of a t(15;19)/BRD4-NUT-rearrangement. We herein describe, in detail, the laboratory methods by which the BRD4-NUT -rearrangement can be detected.

A complex, four-way variant t(15;17) in acute promyelocytic leukemia.

Complex variant 15;17 translocations are increasingly recognized in acute promyelocytic leukemia (APL). We report the case of a 47-year-old woman with APL harboring a novel four-way translocation. She presented with persistent bleeding after a tooth extraction. Blood cell counts at admission were hemoglobin at 9.0 g/dL, platelets at 15 x 10(9)/L, and white blood cells at 0.460 x 10(9)/L with abnormal promyelocytes. Most nucleated cells of bone marrow aspirates were abnormal promyelocytes with Auer rods. Chromosome analysis of unstimulated bone marrow cell cultures revealed a variant t(15;17) in the form of t(10;17;15;22)(q22;q21;q22;q11.2). Fluorescence in situ hybridization with a PML/RARA dual-color DNA probe showed the fusion signals on der(15) and the residual PML signals on der(22). RT-PCR showed long-form PML/RARA fusion transcripts. A complete remission was attained with a course of conventional chemotherapy including ATRA. A literature review revealed that our case is one of the very rare four-way translocations and the first report of the involvement of chromosomes 10 and 22 in a variant t(15;17).

A t(11;15) fuses MLL to two different genes, AF15q14 and a novel gene MPFYVE on chromosome 15.

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation.

Characterization of the MLL partner gene AF15q14 involved in t(11;15)(q23;q14).

Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.