Multicolor Fluorescence In Situ Hybridization Research Articles

Molecular Cytogenetic Characterization of Novel Wheat-rye T1RS.1BL Translocation Lines with High Resistance to Diseases and Great Agronomic Traits.

Rye has been used worldwide as a source for the genetic improvement of wheat. In this study, two stable wheat-rye primary T1RS.1BL translocation lines were selected from the progeny of the crossing of the wheat cultivar Mianyang11-1 and a Chinese local rye variety, Weining. These two novel translocation lines were identified by molecular cytogenetic analysis. PCR results, multi-color fluorescence in situ hybridization (MC-FISH), and acid polyacrylamide gel electrophoresis (A-PAGE) indicated that both new translocation lines harbor a pair of T1RS.1BL translocation chromosomes, and have been named RT828-10 and RT828-11, respectively. The cytogenetic results also indicated that the pSc119.2 signals of 5AL were absent in both lines along with the pSc119.2 signals of 4AL of RT828-11. When inoculated with different stripe rust and powdery mildew isolates, both lines expressed high resistance to Puccinia striiformis f. sp. tritici and Blumeria graminis f. sp. tritici pathotypes, which are prevalent in China and are virulent on Yr9 and Pm8. The line RT828-11 also exhibited excellent agronomic traits in the field. The present study indicates that this rye variety may carry untapped variations that could potentially be used for wheat improvement.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the ‘hot spots’ and ‘cold spots’ of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

Interdependency Between TP53 Mutations, Cytogenetics, Genetic Instability and Prognosis in MDS and Secondary AML

Introduction: Alterations of TP53 (cytogenetic 17p13.1 deletions and molecular TP53 mutations) were reported to be frequent in pts with myelodysplastic syndromes (MDS) and complex abnormalities (≥3 clonal cytogenetic aberrations, CA) that represent around 15% of all MDS cases. It was suggested that pts with MDS and complex abnormalities may be further prognostically subdivided by the molecular TP53 mutation status (Bejar et al, ASH, 2014, abstract #532). In this study we investigated the frequency of different types of TP53 alterations, their cytogenetic profile and their clinical impact in the adverse cytogenetic MDS subgroup of complex abnormalities. We performed comprehensive cytogenetic and molecular genetic analysis focusing as well on the extent of cytogenetic instability.Methods: We included 105 pts (57 m/48 f; median 71 yrs, range, 47-95 yrs) with MDS (n=86) and sAML after MDS (n=19) with complex abnormalities in our study. A total of 56/89 (62.9%) pts had received azacitidine. Survival was censored at allogeneic stem cell transplantation (26/83; 31.3%). Pts were characterized by chromosome banding analysis, interphasefluorescence in situ hybridization (FISH) with a panel including a 17p13/TP53-covering probe, multicolor FISH (mFISH), Sanger sequencing of TP53 and SNP-array analysis (SNP-A). The extent of genetic imbalances was objectified by counting the number of CA, the number of cytogenetic fusions as shown by mFISH and the size of total genomic aberrations (TGA) measured by SNP-A in megabases (Mb).Results: A molecular TP53 mutation was found in 46/105 (43.8%) pts; a cytogenetic TP53 deletion in 38/105 (36.2%) pts. TP53 was not affected by a molecular mutation or a cytogenetic deletion in 44/105 (42.2%) pts, 23/105 (21.9%) pts were affected by combined TP53 alterations (molecular mutation and cytogenetic deletion), 23/105 (21.9%) pts by a molecular mutation only and 15/105 (14.3%) pts by a cytogenetic deletion only. The median number of CA was 6 (range, 3-41) in the entire cohort. Median overall survival for the entire cohort was 17 months.The degree of genomic imbalances was higher in pts with any TP53 alteration (molecular mutation and/or cytogenetic deletion) as compared to those without: The median number of CA was 8 (range, 3-41) vs. 4 (3-20) (P<0.001), the median number of fusions was 5 (0-13) vs. 2 (0-9) (P<0.001), and the median TGA size was 327 (97-511) vs. 105 (64-226) Mb (P<0.001). The extent of genomic imbalances was higher in pts with a TP53 deletion only compared to pts with a TP53 mutation only: The median number of CA was 10 (range, 4-21) vs. 8 (3-25) (P=0.093; n.s.); the median number of fusions was 7 (2-13) vs. 3 (0-10) (P=0.031).By univariate analysis, presence of ≥5 CA as compared to 3-4 CA increased the hazard ratio (HR) to 3.34 (P=0.017). When we limited the analysis to the subgroup of pts without evidence of a molecular TP53 mutation, presence of a cytogenetic TP53 deletion resulted in a HR of 5.67 (P=0.029). When the analysis was restricted to pts without a TP53 deletion, presence of a molecular TP53 mutation did not significantly change the HR (1.58, P=0.448; n.s.).Multivariate analysis that considered molecular TP53 mutation status, cytogenetic TP53 deletion status, the number of CA and treatment with azacitidine identified presence of a cytogenetic TP53 deletion as the most significant prognostic marker for OS (HR 15.1, P=0.010). HR was increased for pts with ≥5 CA compared to pts with 3-4 CA (HR 5.9, P=0.012). The molecular TP53 mutation status showed no significant impact on HR in our cohort.Conclusion: Presence of a cytogenetic TP53 deletion and a higher number of cytogenetic aberrations (≥5) showed a negative prognostic impact even within the unfavorable cytogenetic subgroup of MDS with complex abnormalities. In contrast, we found no strong prognostic impact for a molecular TP53 mutation in our cohort of MDS with complex abnormalities. The lower impact of TP53 mutations compared to TP53 deletions might be due to the lower degree of cytogenetic imbalances in pts with TP53 mutations only compared to pts with TP53 deletions only. However, molecular as well as cytogenetic TP53 aberrations were associated with a greater extent of chromosomal imbalances and displayed clear interdependencies. Our data suggest that TP53 alterations (molecular mutations and/or cytogenetic deletions) may not have an independent prognostic impact in the MDS subgroup with complex abnormalities. [Display omitted] [Display omitted] [Display omitted] DisclosuresPlatzbecker:Amgen: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding.

Sorting of chromosomes on FACSAria(TM) SORP for the preparation of painting probes.

High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx, which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment, low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However, whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here, we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50mW for 355nm and 20mW for 444nm excitation. Furthermore, the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably, human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype, and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition, multicolor FISH (mFISH) with human chromosome painting probes to 13,18, 21, and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together, our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover, a FISH analysis confirmed high purity of the sorted chromosomes. Additionally, in contrast to centromeric satellite probes, chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations, such as deletions and translocations, in prenatal diagnosis. © 2016 International Society for Advancement of Cytometry.

Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

BackgroundMarker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assays and array comparative genomic hybridization (array CGH).ResultsIn a series of 2871 patients for whom cytogenetic analysis was requested, 14 cases with small supernumerary marker chromosomes (sSMCs) were identified. Nine sSMCs were mosaic, and five nonmosaic. Of the nine cases with known parental origins, four were identified as de novo, and four and one were maternally and paternally inherited, respectively. Six sSMCs were identified by FISH using centromeric probes; three sSMCs were derived from chromosome 15, including two heterochromatic sSMC(15)s and a large sSMC(15) spanning 15q11.1q13.1, and three sSMCs originated from chromosome 14 or 22. Array CGH revealed two cases with derivatives of chromosome 2 and whole chromosome painting multicolor-FISH (M-FISH) identified three cases with derivatives of chromosome 6, 16, and 19, respectively. One maker chromosome in Turner syndrome was characterized as sSMC(X) by preferential application of a centromeric probe for X-chromosome. In addition, one sSMC composed of genomic materials from chromosomes 12 and 18 was identified in parallel with parental karyotype analysis that revealed the reciprocal balanced translocation.ConclusionsThis report is the largest study on sSMCs in Korea and expands the spectrum of sSMCs that are molecularly characterized.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0273-5) contains supplementary material, which is available to authorized users.

Karyotyping and identifying all of the chromosomes of allopolyploid Brassica juncea using multicolor FISH

Chromosome identification and karyotype using fluorescence in situ hybridization (FISH) provides a technical platform for genome and cytogenetic studies. Brassica juncea (brown mustard, 2n=4×=36; genome AABB) is an allopolyploid species that originated from a spontaneous hybridization of Brassica rapa and Brassica nigra and contains many valuable traits. In this study, a multicolor FISH procedure allowing the identification of all 18 chromosomal pairs was developed by two-step hybridizations with probes on the same metaphase chromosomes. The distribution patterns and chromosomal localizations of six repeat sequences (satellite repeat pBrSTR, 5S rDNA, 45S rDNA, B genome-specific repeat pBNBH35, and centromeric satellite repeats CentBr1 and CentBr2) on B. juncea chromosomes were characterized. Comparative karyotype analyses showed that the genome is relatively stable in comparison with its diploid progenitor species and revealed intraspecific karyotypic diversity among three accessions of B. juncea. This study provides valuable information about the genome evolution of B. juncea and a toolkit that will be helpful for chromosome identification.

Molecular characterization of KMT2A fusion partner genes in 13 cases of pediatric leukemia with complex or cryptic karyotypes.

In pediatric acute leukemias, reciprocal chromosomal translocations frequently cause gene fusions involving the lysine (K)-specific methyltransferase 2A gene (KMT2A, also known as MLL). Specific KMT2A fusion partners are associated with the disease phenotype (lymphoblastic vs. myeloid), and the type of KMT2A rearrangement also has prognostic implications. However, the KMT2A partner gene cannot always be identified by banding karyotyping. We sought to identify such partner genes in 13 cases of childhood leukemia with uninformative karyotypes by combining molecular techniques, including multicolor banding FISH, reverse-transcriptase PCR, and long-distance inverse PCR. Of the KMT2A fusion partner genes, MLLT3 was present in five patients, all with acute lymphoblastic leukemia, MLLT1 in two patients, and MLLT10, MLLT4, MLLT11, and AFF1 in one patient each. Reciprocal reading by long-distance inverse PCR also disclosed KMT2A fusions with PITPNA in one patient, with LOC100132273 in another patient, and with DNA sequences not compatible with any gene in three patients. The most common KMT2A breakpoint region was intron/exon 9 (3/8 patients), followed by intron/exon 11 and 10. Finally, multicolor banding revealed breakpoints in other chromosomes whose biological and prognostic implications remain to be determined. We conclude that the combination of molecular techniques used in this study can efficiently identify KMT2A fusion partners in complex pediatric acute leukemia karyotypes. Copyright © 2016 John Wiley & Sons, Ltd.

Active centromere and chromosome identification in fixed cell lines.

BackgroundThe centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells.ResultsA panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric.ConclusionsWe propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.

Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system.

Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus.

Chromosome Painting by GISH and Multicolor FISH.

Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique for identifying chromosomes and mapping specific genes and DNA sequences on individual chromosomes. Genomic in situ hybridization (GISH) and multicolor FISH (mc-FISH) represent two special types of FISH techniques. Both GISH and mc-FISH experiments have general steps and features of FISH, including chromosome preparation, probe labeling, blocking DNA preparation, target-probe DNA hybridization, post-hybridization washes, and hybridization signal detection. Specifically, GISH uses total genomic DNA from two species as probe and blocking DNA, respectively, and it can differentiate chromosomes from different genomes. The mc-FISH takes advantage of simultaneous hybridization of several DNA probes labeled by different fluorochromes to different targets on the same chromosome sample. Hybridization signals from different probes are detected using different fluorescence filter sets. Multicolor FISH can provide more structural details for target chromosomes than single-color FISH. In this chapter, we present the general experimental procedures for these two techniques with specific details in the critical steps we have modified in our laboratories.

Physical mapping of black spot disease resistance/susceptibility-related genome regions in Japanese pear (Pyrus pyrifolia) by BAC-FISH.

Black spot disease, caused by Alternaria alternata Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. In the present study, the locations of black spot disease resistance/susceptibility-related genome regions were studied by fluorescence in situ hybridization using BAC clone (BAC-FISH) on Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai) chromosomes. Root tips of self-pollinated seedlings of ‘Osa Gold’ were used as materials. Chromosome samples were prepared by the enzymatic maceration and air-drying method. The BAC clone adjacent to the black spot disease-related gene was labeled as a probe for FISH analysis. Black spot disease-related genome regions were detected in telomeric positions of two medium size chromosomes. These two sites and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH. The effectiveness of the physical mapping of useful genes on pear chromosomes achieved by the BAC-FISH method was unequivocally demonstrated.

Potential Pitfalls of Serum Free Light Chain Analysis to Assess Treatment Response for Multiple Myeloma

BackgroundThe serum free light chain (FLC test) allows measurement of low concentrations of FLC. Post-transplantation, especially after tandem autologous transplants and consolidation therapy, the immune system is often extremely suppressed and its recovery is disorganized.Patients and MethodsThis study was limited to patients with multiple myeloma, plasma cell leukemia and amyloidosis, who received autologous transplantation and consolidation therapy at the University of Iowa Hospitals and clinics. Most of these patients had already received some form of induction therapy with IMIDs or proteasome inhibitors or in combination. They then proceeded with a cycle of D-PACE followed by one or two autologous transplants. The preparative regimen for virtually all patients was VDT-MEL. Post-transplantation, most of the patients received consolidation therapy with VTD the first year and either VCD or Revlimid/dexamethasone for second year. Thereafter, all myeloma therapy was halted. Serum free light chains were measured with polyclonal FLC antisera according to Freelite, Binding site, UK. The kappa/ lambda ratio was calculated.ResultsIn total 142 patients were evaluated; 12 % (17/142) of patients were found to have abnormal light chains ratio but no other evidence of active disease, including negative serum M-protein, serum IFE, urine M-protein and urine IFE. In addition, bone marrows showed no evidence of clonal plasma cells by both 10-color flow cytometry and FISH analysis of CD138 selected plasma cells; both methods have a sensitivity of ≥ 10-4. The κ/λ ratio was abnormal due to increase/decrease in the same light chain as the M- protein in 11/17 patients; 6/17 patients had abnormal κ/λ ratio due to increase in the opposite light chain as the M- protein (Table 1)Table 1Patient with abnormal light chains due changes in Involved light chain levelsAgeGenderM protein/Light chainTransplantM proteinAbnormal light chainImmunofixationElevated or decreasedDuration of abnormal ratio64MaleIgG KappaTandem0.0KappaNegativeElevated2 weeks58FemaleIgA KappaTandem0.0KappaNegativeElevated4 weeks46FemaleKappaTandem0.0KappaNegativeElevated4 weeks58MaleIgG KappaTandem0.0KappaNegativeElevated20 weeks55MaleIgG KappaTandem0.0KappaNegativeElevated4 weeks60MaleIgG KappaTandem0.0KappaNegativeElevatedOne week60FemaleIgA KappaSingle0.0KappaNegativeElevated8 weeks68FemaleIgG KappaSingle0.0KappaNegativeElevated26 weeks42MalekappaTandem0.0KappaNegativeElevatedOne week67MaleKappaSingle0.0KappaNegativeDecreased3 weeks68MaleIgG KappaSingle0.0KappaNegativeElevated8 weeksPatient with abnormal light chain ratio due to changes in the opposite light chain levels53FemaleIgG LambdaTandem0.0KappaNegativeElevated2 weeks62FemaleIgA lambdaTandem0.0KappaNegativeElevated6 weeks50MaleLambdaSingle0.0KappaNegativeElevatedOne week60MaleIgG LambdaTandem0.0KappaNegativeElevated100 weeks68FemaleIgA LambdaSingle0.0KappaNegativeElevated8 weeks68MalelambdaSingle0.0KappaNegativeElevated4 weeksConclusions According to the IMWG uniform response criteria, patients achieving CR for whom the involved FLC reduced sufficiently to normalize the FLC ratio (range, 0.26 to 1.65) in the absence of monoclonal BMPCs as assessed by immunohistochemistry or immunofluorescence are considered to have achieved stringent CR. However, patients can be in stringent complete remission with abnormal k/l ratios if 1) the ratio is abnormal because the non-involved free light chain is elevated while the involved free light chain is normal; 2) the ratio is abnormal because involved light chain is elevated, with no other evidence of disease, including multicolor flow cytometry and FISH analysis on selected plasma cells of the bone marrow and imaging by MRI and/or PET-CT scan. This occurred in > 10% of patients. It should be noted that the FLC causing the abnormal k/l ratio was always kappa. The IMWG criteria should be adjusted these potential pitfalls. DisclosuresNo relevant conflicts of interest to declare.

Pathological Roles of p18 Deficiency Toward B Cell Malignances

The cyclin-dependent kinases inhibitor CDKN2C (p18INK4C, p18) is a member of the INK4 family that specifically blocks the activity of CDK4/6 in the G1 phase of cell cycle. In the hematopoietic system, deletion of p18 was indicated to be associated with T cell malignancies in mice and B cell malignancies in humans. Moreover, p18 deficiency is a poor prognosis factor for the patients with multiple myeloma (MM). However, a formal investigation on the pathological roles of p18 deficiency in hematopoietic malignancies, especially B cell malignancies is lacking.In this study, we first obtained direct clinical relevance of p18 deficiency with hematopoietic malignancies. Based on the Oncomine data set analysis, low expression of p18 was found in the patients with B-Cell Acute Lymphoblastic Leukemia (54 out of 80). In addition, by Gene expression Profile (GEP) analysis (n=361) and multi-color FISH analysis (n=265) of first-visit MM patients, there were 11% MM patients showed low expression and 9.06% biallelic deletion of p18 gene respectively, which was correlated with poor prognosis. Further analysis indicated higher expression of c-Myc, Bcl-2 and TRAF3 in p18-deleted MM patients or MM cell lines. We then focused on the impact of p18 deletion on B cell development with the mice deficient in p18 (p18-/-). The frequency and absolute number of B220+ B cell were significantly decreased in the bone marrow (21.075±0.168% vs 13.956±1.613%, n=5) or spleen (49.320±1.773 vs 35.35±1.673, n=5) of p18-/- mice. Secretion of immunoglobulin (Ig) from plasma cells was also impaired. Furthermore, p18-/- BM or enriched hematopoietic stem cell (LSK+) transplantation also recaptured the deficiency of mature B cells in the recipients despite higher repopulation in the p18-/- group. Ectopic over-expression of p18 in the hematopoietic stem and progenitor cells (HSPCs) via retroviral transduction could partially correct the abnormality of p18-/- B cells in the transplant recipients. These results suggested that the defect of B cell development in the absence of p18 was intrinsic to the hematopoietic cells, rather than extrinsic (via micro-environmental). To further define the effects of p18 deficiency on HSPCs prior to B cell commitment, we enumerated the frequencies of LT-HSC, MPP, CMP, GMP, MEP, Lin-IL-7R+ and CLP cell populations in p18-/- or control mice. There was no significant difference in the frequency or absolute number of CMP, GMP, MEP, or CLP between p18-/- and control groups. Notably however, the colony-forming cells of pre-B cells in p18-/- BM were significantly increased (24.4±2.1 vs 32.6±1.8, n=5). Moreover, we also examined the B cells at different developmental stages including pre-pro-B cell, pre-B, immature-B and mature B cells in BM, as well as transitional stage 1(T1), transitional stage 2 (T2) and mature B cells in the spleen. Our data showed an accumulation of the cells at pre-B cell stage in the absence of p18, while dramatically decreased at mature B cells stage. To further explore the molecular basis, single cell RT qPCR analysis was performed and revealed that the transcription factors including Foxo1, Rag2, E2A, EBF1and Pax5 were significantly higher inCLP, pro-B, pre-B, immature-B subpopulations of p18-/- group. However, lamada 5, which is necessary for B cells maturation, was remarkably decreased in p18-/- immature B cells compared with control group. Taken together, our study provides definitive evidence for the disruption of B cell development due to p18 deficiency and this new evidence underlies the pathological contributions of p18 down-regulation or deletion to B cell malignancies in humans. DisclosuresNo relevant conflicts of interest to declare.

Clonal Evolution Paths Significantly Impact the Outcome of Myeloma

BackgroundMultiple myeloma is a malignancy of clone plasma cells that were composed of various multisteps development and had different molecular genetical variation in different phases in the course of disease. Clonal evolution promotes the disease development from prophase to advanced stage. We investigated the paths of clonal evolution in a cohort myeloma patients by a unique multicolor FISH (mFISH) to identify the clonal architecture of different time points, and first reported the survival and prognostic value of various clonal evolution paths.MethodsTotally 85 myeloma patients with CD138+ cells at diagnosis versous relapse or former relapse versous latter relapse were enrolled in this study between January 2004 and December 2014. Firstly, the clonal evolution were analyzed by conventional iFISH (cFISH) with a panel probes of 1q21, RB-1 and TP53. The clonal architecture of different time points were detected by mFISH in 62 samples from different time points of selected 25 patients among the 85 patients. The survival significance and prognostic value of 3 different clonal evolution paths identified by mFISH were analyzed.Results: 1). Clonal evolution were detected in 34/85(40%) cases by cFISH analysis. The survival analysis showed that patients with clonal evolution had significant shorter over survival (OS) than those with clonal stabilization. The meadian OS of these 2 groups were 42.67 (95% CI:32.15-53.19) and 71.46 (95%CI:58.99-83.93) months respectively, the difference between these 2 groups was significant (p=0.010). Comparing the survival since post relapse/clonal evolution, significant difference (p=0.003) also existed in these 2 groups with a median survival of 14.79 (95% CI:10.01-19.56) months and 37.61 (95%CI:27.30-47.92) months respectively.2). Three clonal evolution paths were identified by mFISH in different phase samples from 25 myeloma patients. 36% (9/25) of them were clonal stabilization, 32% (8/25) were linear clonal evolution, 32% (8/25) were branching clonal evolution. mFISH had more powerful efficacy of clonal evolution detection than cFISH. Of these 25 cases, 14 cases were clonal stabilization and 11 were clonal evolution identified by cFISH. 7/14 (50%) with clonal stabilization by cFISH were identified into clonal evolution by mFISH, 1 with linear evolution and 6 with branching evolution. The 11 cases with clonal evolution identified by cFISH were further classified into 7 with linear evolution and 4 with branching evolution by mFISH detection.3). Our results indicated that clonal evolution type had significant impact on survival of myeloma patients. The median OS of patients with clonal stabilization, linear evolution and branching evolution were 87.00 (95%CI:74.53-99.47), 24.99 (95% CI:16.58-33.39) and 35.70 (95% CI:27.10-44.30) months respectively. There were significant difference among these 3 groups (p=0.004). The median survival post relapse/clonal evolution in these 3 groups were 34.00 (95%CI:20.14-47.86), 8.06 (95% CI:4.91-11.22) and 22.75 (95% CI:13.49-32.01) months, the difference among them was significant (p=0.007).ConclusionsmFISH is a simple and clinical practicable tool for clonal detection and analysis of myeloma. Our results firstly demonstrated that the clonal evolution type had significant impact on survival of patients with myeloma. According to the 3 different clonal evolution paths, individualized treatment strategy would be required for different patients with myeloma. DisclosuresNo relevant conflicts of interest to declare.

RAS/RAF Pathway Mutations Define a Distinct Subset of Chronic Lymphocytic Leukemia

Widespread use of next-generation sequencing (NGS) based mutational analysis has identified recurrent mutations of prognostic significance in chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL). In addition to novel genes such as NOTCH1 and SF3B1, targetable mutations in genes involved in RAS-MAPK pathway such as KRAS and BRAF including V600E have been reported. However, the detailed clinicopathologic, cytogenetic and molecular genetic features of KRAS/NRAS/BRAF mutated CLL/SLL (CLLrasmut) are not known. We describe the clinicopathologic findings in 44 cases of CLLrasmut.NGS analysis targeting 53 cancer-related genes was performed on Miseq Illumina on CLL/SLL cases on bone marrow (BM) and/or peripheral blood (PB) samples. ZAP70 expression was assessed by immunohistochemistry/ flow cytometry. Somatic hypermutation was performed by Sanger sequencing. Deviation from the germline above 2% was considered mutated. Interphase multi-color FISH studies were performed using probes targeting regions 11q22.3 (ATM), centromeric region of chromosome 12, 13q14.3 (D13S319 locus),13q34 (LAMP1) and 17p13.1 (TP53), Clinical data were correlated with molecular and cytogenetic findings.We reviewed 503 CLL cases in our laboratory database that underwent next-generation sequencing based multi-gene profiling. 44 (8.8%) patients had mutations including BRAF alone (n=18), KRAS alone (n=14), NRAS alone (n=8) and dual BRAF +KRAS/NRAS (n=4), respectively. We compared these 44 cases with 75 consecutive CLL/SLL cases without mutations in KRAS/NRAS/BRAF (CLLraswt). CLLrasmut showed a male predilection compared to the CLLraswt (M:F=3.9:1 versus 1.2:1, p =0.01) with a median age of 62 years (range, 42-80). There were no significant differences between CLLrasmut and CLLraswt in the median age of disease onset, PB counts, PB/BM tumor burden. Studies for somatic hypermutation showed unmutated IGHV in 79.4% of CLLrasmutversus 55.4% in CLLraswt (p =0.03). ZAP70 was positive in 73% of CLLrasmut compared to 42.9% in CLLraswt (0.05). TP53 was the most frequent concurrently mutated gene, noted in 22.7% (10/44) of CLLrasmut cases. CLLrasmut showed higher frequency of trisomy 12 (54.6% versus 30.7%, p =0.0001) and lower frequencies of D13S319 deletions (31.8% vs. 82.7%, p =0.0001) compared to CLLraswt. In a significant proportion of cases, the mutant allelic frequencies of RAS/RAF mutations were disproportionately lower than predicted based on the tumor burden. Interestingly, 15 out of 22 (69%) BRAF mutations involved codons other than V600.We conclude that BRAF/KRAS/NRAS mutations are seen in about 9% of CLL and could represent a distinct subset of CLL due to their frequent association with male gender, trisomy 12 and unmutated IGHV. These findings may provide additional insight into oncogenesis and guide design of potential targeted therapies in CLL. DisclosuresKeating:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy.

Abstract 4911: Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH

Abstract Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridization (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data was also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirms that RTK/RAS components are mutually exclusively amplified in GC, and demonstrates the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient, before a definitive diagnosis can be attained. Citation Format: Kakoli Das, Bavani Gunasegaran, Iain Beehuat Tan, Niantao Deng, Kiat Hon Lim, Patrick Tan. Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2015-4911

Abstract 3869: Exploration of mechanisms for chromothripsis by irradiation

Abstract Chromothripsis is the massive but highly localized chromosome rearrangements within a one-step catastrophic event rather than a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and it is thought to be associated with increasing malignancy and oncogenesis. However the causes and consequences of chromothripsis remain unclear. Therefore, to explore mechanisms of chromothripsis, we investigated whether chromothripsis was artificially induced by irradiation. We elicited DNA double-strand breaks in the oral cancer cell line HOC313, or its highly metastatic subline HOC313-LM by The Single Particle Irradiation system to Cell (SPICE), a focused vertical micro-beam system designed to irradiate the nuclei of adhesive cells. We established irradiated monoclonal sublines and analyzed the copy number variations (CNVs) and SNPs using SNP array. Chromosomal alterations, induced by irradiation including CNVs, were detected in some monoclonal sublines. Interestingly SNP array analysis showed that one subline named LM200-#25 had multiple chromosomal alterations which happened locally in chromosome 7. Moreover, we detected several chromosomal alterations such as t(12;7;11) translocation in LM200-#25 subline by Multi-Color FISH analysis. These results suggested that chromothripsis might be induced by irradiation in LM200-#25 subline. Citation Format: Maki Morishita, Tomoki Muramatsu, Shin Hayashi, Momoki Hirai, Yumiko Suto, Teruaki Konishi, Keiji Moriyama, Johji Inazawa. Exploration of mechanisms for chromothripsis by irradiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2015-3869

MBAND and mFISH analysis of chromosomal aberrations and breakpoint distribution in chromosome 1 of AG01522 human fibroblasts that were exposed to radiation of different qualities

High-resolution multicolour banding FISH (mBAND) and multiplex FISH (mFISH) were used to analyse the aberrations of chromosome 1 in irradiated-AG01522 human primary fibroblasts. The cells were exposed to 1Gy of a panel of radiation of different qualities, such as X-rays, low-energy protons (28keV/μm), helium-ions (62keV/μm) and carbon-ions (96 and 252keV/μm). mBAND and mFISH analysis in calyculin-A G2-condensed chromosome spreads allowed us to detect intra- and interchromosome aberrations involving chromosome 1, including simple and complex-type exchanges, inversions (both para- and pericentric ones), deletions and rings. The data indicate that the induction of chromosomal exchanges was influenced by both Linear energy transfer (LET) and particle types. Moreover, the complex-to-simple exchanges ratio (C-ratio) and interchromosome to intrachromosome exchanges ratio (F-ratio) were evaluated by mFISH and mBAND techniques, respectively. Our results indicate that the C-ratio is a more reliable marker of radiation quality, with values that increased linearly in an LET-dependent manner.In addition, by means of mBAND analysis, the distribution of radiation-induced breakpoints along chromosome 1 was analyzed and compared with the expected distributions of the breaks. The expected values were calculated assuming a random distribution of the breakpoints. The data indicate that, irrespective of the radiation that was used, the breakpoints were non-randomly distributed along chromosome 1. In particular, breaks in the pericentromeric region were encountered at a higher frequency than expected. A deeper analysis revealed that breaks were not located in the constitutive heterochromatin (G-bands 1p11/1q11 and 1q12), but rather in a region comprised between 1p11.2 and 1p22.1, which includes G-light and G-dark bands.

Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

A novel acquired cryptic three-way translocation t(2;11;5)(p21.3;q13.5;q23.2) with a submicroscopic deletion at 11p14.3 in an adult with hypereosinophilic syndrome

Hypereosinophilic syndrome (HES) is a clinically and pathologically heterogeneous disease entity. It is characterized by persistent eosinophilia and organ damage after excluding other causes. Clonal eosinophilia is distinguished from idiopathic eosinophilia by the presence of histologic, cytogenetic, or molecular evidence of an underlying malignancy. There are two distinct subcategories of clonal eosinophilia: chronic eosinophilic leukemia, not otherwise specified and myeloid/lymphoid neoplasms with eosinophilia and mutations involving platelet-derived growth factor receptor α/β or fibroblast growth factor receptor 1. More than 50% of HES are without knowledge of underlying pathogenic molecular pathways.Here we examined a HES patient by oligo-based aCGH analysis and molecular cytogenetic methods. Examination for the common eosinophilia-related cytogenetic abnormalities involving the genes PDGFRA, PDGFRB, and FGFR1 together with BCR-ABL fusion gene was negative. Cytogenetic analysis and multi-color FISH analysis revealed a novel cryptic three-way translocation t(2;11;5)(p21.3;q13.5;q23.2). By oaCGH analysis we could not find any copy number changes related to the cytogenetic breakpoints but instead detected a 0.9Mb submicroscopic deletion at 11p14.3. The deleted region involved the 5′-upstream sequences and exons 1-4 of the LUZP2 gene, which encodes a leucine zipper protein. Analysis of surrogate germ-line cells revealed a normal result showing that the detected chromosomal aberrations were acquired.This is the first report on a HES patient associated with a novel complex three-way translocation t(2;11;5)(p21.3;q13.5;q23.2) and a submicroscopic deletion in chromosome band 11p14.3. The study also demonstrates the benefits of oligo-based aCGH analysis in detecting hidden disease related chromosomal abnormalities. The present findings provide additional clues to unravel important molecular pathways in HES to obtain the full spectrum of acquired chromosomal and genomic aberrations in this heterogeneous disease entity. As more cases become characterized this may eventually improve on classification and treatment options.