Multicolor Fluorescence In Situ Hybridization Research Articles

A High Incidence of Adjacent-1 Meiotic Segregation Pattern, Revealed by Multicolor Sperm FISH, in a Carrier Boar of a New Reciprocal Translocation t(6;16)(p13;q23)

Reciprocal translocations pose a serious problem in pig breeding due to the reduced fertility of the carriers. This paper presents a new reciprocal translocation in a phenotypically normal, but hypoprolific (20% reduction) boar. Chromosome banding as well as the FISH technique with the use of BAC and telomeric probes was applied for a detailed characterization of this chromosome rearrangement. The karyotype of the studied boar was described as 38,XY,t(6;16)(p13;q23). The meiotic segregation of the quadrivalent was studied in 1,071 sperms by multicolor FISH. The most frequent segregation patterns were alternate (47.5%) and adjacent 1 (41.9%), while adjacent 2 and 3:1 were less frequent at 1.2 and 9.2%, respectively. Surprisingly, the frequency of the adjacent-1 segregation appeared to be relatively high, when compared with human and pig reciprocal translocations studied by sperm FISH. Our study, along with a review of the literature, shows that a reduction of fertility in the carriers and the incidence of different segregation patterns of the quadrivalent may vary within a broad range, and both aspects seem to be unrelated. A need for obligatory karyotype screening programs of artificial insemination boars is emphasized.

Deletion 11q Abnormality in Patients With Chronic Lymphocytic Leukemia (CLL) May Not Have Poor Clinical Outcomes and Bulky Disease (Clinical and Radiological) at Presentation – Clinical Characteristics of (n=172) Previously Untreated Patients With CLL and del11q Cytogenetic Abnormality

Abstract 2890 Introduction: Del11q aberration is universally considered as an adverse prognostic marker in patients with CLL. However, large studies describing the clinical characteristics of del11q in CLL patients (pts) have never been reported. We studied the clinical features, outcomes and treatment responses among (172) previously untreated pts with CLL and del11q. Methods: This is a retrospective analysis of CLL pts (n=172) with del11q, who presented at our institution between 2003–2012. All pts signed an informed consent as per the declaration of Helsinki and IRB approval. Del11q was determined from bone marrow (BM) aspirate or blood samples by FISH (Fluorescent in-situ hybridization) technique. Detection of the 11q22.3 (ATM gene) on chromosome 11 used a multi-color FISH technique. Pts were divided into five categories – One – 57; del11q alone, Two – 105; del11q and del13q, others – 10 (4; del11q with +12, 1; del11q with del17p and 5; del11q, del13q with +12). Baseline bulky lymphadenopathy by CT scan or physical exam (PE) was also analysed. The probabilities of OS, PFS and TTFT (time to first treatment) were estimated using KM plots (log rank). PFS was assessed in 121 treated pts, from date of starting therapy to date of progression or death on therapy. The Cox proportional hazards regression model was used to assess the association between pt characteristics and OS, PFS or TTFT. Results: At the baseline, majority of pts with del11q were young (median age IGHV unmutated (n=112; 89.6%), CD38 high (n=88; 51.8%), Zap-70 high (n=100; 75.8%). Status of bulky disease was assessed by PE and baseline CT scans. Bulky disease by PE was observed in n=11 (6.4%) only. Baseline CT scans were performed for 108 patients out of whom only 16 patients (14.8%) had bulky disease. Thus, majority of del11q pts did not have bulky disease by PE or by CT scanning. Analysis of time dependent variables was performed. Among the total 172 pts, 19 (11%) have died. and median time to death has not been reached (NR) - OS. 48/125 (38%) pts who were treated died or progressed and median PFS time was 49.2 months. Overall, 123 (71%) patients received therapy after initial presentation at MDACC. The median TTFT was 10.6 months. FCR based therapies were given in (91; 73 %) pts. Median PFS on FCR based therapy was 32 mo (Figure -1). CR was achieved in (70%; n=64) and overall response was 89%. Pts who received fludarabine based therapy had a significantly shorter TTFT as compared to non fludarabine based therapy (P To address whether del11q has any influence over presence of deletion 13 q abnormality we compared (del11q alone; n=57) vs (del11 and del13q; n=105) patients. Patients with sole del11q had significant proportions of younger patients, lesser WBC count, and ALC and β2 microglobulin. Furthermore, there was no significant difference in OS, PFS and TTFT among the two categories. In the multivariate analysis for OS and PFS among the two categories, β2 microglobulin was significantly predictive of increased risk of death or progression among the two categories and FISH categories were not significantly predictive of OS or PFS or TTFT. Bulky disease by PE (and not by CT scan) was significantly associated with a shorter TTFT (HR=2.93; 95%CI -1.5–5.69 and P=0.002). Furthermore, a separate cohort of (n=500) patients with sole del13q was compared with del11q alone (n=57) and del11q with del13q (n=105). There was no significant difference in OS but the TTFT was significantly shorter in del11q alone (data not shown). Conclusions: We describe the clinical features of del11q in CLL patients. This study suggests that occurrence of del11q is frequent with del13q abnormality. The majority of patients with del11q did not have bulky disease as assessed by PE or by CT scan. Presence of bulky disease by PE predicted for a shorter TTFT. β2 microglobulin level significantly predicted for OS and PFS. FCR based therapy has good responses. Del11q is similar to del13q in terms of OS, PFS and TTFT. Del11q may not have very poor adverse outcomes in patients with CLL inspite of a shorter TTFT. Disclosures: Burger: Pharmacyclics: Consultancy, Research Funding. Wierda: Abbott: Research Funding; Genentech: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; AmGen: Research Funding; Merck: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Genzyme: Consultancy. Kantarjian: Genzyme: Research Funding.

There is no interchromosomal effect in the embryos derived from young carriers of reciprocal and robertsonian translocations

This study aimed to analyse ICE in human preimplantation embryos derived from both reciprocal and Robertsonian translocation carriers who were undergoing preimplantation genetic diagnosis (PGD). Eleven cycles of PGD for Robertsonian translocation and twelve cycles of PGD for reciprocal translocation outcomes were analysed retrospectively from August 2010 to February 2011 in Bahceci Assisted Reproductive Technology Centres. PGD was performed for 200 cleavage stage embryos derived from translocation carriers in younger women (average maternal age of 33 for reciprocal and 30 for Robertsonian translocation carriers) using multicolour fluorescent in situ hybridisation (FISH). An additional aneuploidy screening was performed by multicolour FISH for nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y). A further 214 cleavage stage embryos (average maternal age of 32) with no structural chromosomal abnormalities were analysed as a control group. PGD was performed for 86 cleavage stage embryos derived from 10 couples carrying Robertsonian translocations and 11 couples carrying reciprocal translocations. This study showed that the female carriers of both Robertsonian and reciprocal translocations have a similar chance of producing a balanced embryo with normal copy number of the seven chromosomes analysed (13, 15, 16, 17, 18, 21, 22, X and Y). The risk of having an aneuploidy in one of these nine chromosomes was higher in embryos carrying either Robertsonian and reciprocal translocations. This study shows that there is ICE in the unbalanced embryos derived from carriers of both reciprocal and Robertsonian translocation.

The Breakage-Fusion-Bridge Cycle ProducingMLLAmplification in a Case of Myelodysplastic Syndrome

Telomere loss may lead to chromosomal instability via the breakage-fusion-bridge (BFB) cycle which can result in genetic amplification and the formation of ring and dicentric chromosomes. This cycle continues until stable chromosomes are formed. The case of a 72-year-old female with refractory anaemia with excess blasts type 2 illustrates these events. Conventional cytogenetics produced a complex karyotype which included unstable abnormalities of chromosomes 11, 12, and 15. Fluorescence in situ hybridization (FISH) analyses including multicolor-FISH (M-FISH) and multicolor-banding (M-BAND) revealed multiple clonal populations with 5 copies ofMLLon either a ring chromosome composed entirely of chromosome 11 material or a derivative chromosome composed of chromosomes 11, 12, and 15. The FISH results also clarified the likely evolution of the karyotypic complexity. The simplest cell line contained a dic(12;15) in addition to copy number aberrations that are typical of MDS or AML. As the disease progressed, a ring 11 was formed. Subsequently, the ring 11 appears to have unwound and inserted itself into the dic(12;15) chromosome followed by an inversion of the derivative chromosome, producing a der(11;15;12). Telomeric loss and BFB cycles appear to have played an important role in the chromosomal rearrangements and clonal evolution demonstrated in the karyotype.

Chromothripsis under the microscope: a cytogenetic perspective of two cases of AML with catastrophic chromosome rearrangement

Chromothripsis is a recently described phenomenon identified in cancer cells that produces catastrophic chromosome reorganization of one or a small number of chromosomes. It has been proposed that the multiple breakage events occur at a single point in time. Here we introduce the term anachromosome to describe an abnormal chromosome produced by chromothripsis. We report two cases of acute myeloid leukemia matching the description of chromothripsis that illustrate different aspects of this phenomenon from a cytogenetic perspective. Fluorescence in situ hybridization (FISH) analyses, including multicolor FISH and FISH for repeat elements that are not present on microarrays and that are resistant to sequencing, helped interpret the rearrangements but did not reveal their level of complexity. The anachromosomes conformed to the normal constraints of chromosome structure by including segments that provide two telomeres and a centromere. In patient samples, there are mixtures of cells with and without deletions. The deletion B allele frequencies for heterozygous loci in a mixture of cells with and without the deletions create a distinctive array pattern that is consistent with all the deletions in the anachromosomes having occurred concurrently. This evidence supporting the single-event hypothesis for chromothripsis has not previously been highlighted, to our knowledge. In the context of exploring mechanisms for chromosome shattering, we discuss a possible connection between chromosome pulverization and fragile sites. Understanding chromothripsis in the context of chromosome biology will help us identify its causes and consequences.

Multicolor microRNA FISH effectively differentiates tumor types.

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

RNA-guided diagnostics and therapeutics for next-generation individualized nanomedicine

The absence of reliable quantitative laboratory tests for measurements of microRNAs and other classes of small noncoding RNAs in archived, formalin- fixed, paraffin-embedded human samples with sufficient specificity and sensitivity has significantly limited the development of clinically relevant noncoding RNA–based diagnostic and therapeutic applications. A report by Renwick et al. in this issue of the JCI presents a significant technical and methodological advance toward the development of reliable clinical laboratory- compatible multicolor RNA FISH methodology for molecular diagnostic applications and the near-term prospect of introduction of microRNA- based biomarkers into clinical practice. Further, this work is likely to advance the development of RNA-based therapeutics and next-generation individualized nanomedicine.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

A myeloma cell line established from a patient refractory to thalidomide therapy revealed high-risk cytogenetic abnormalities and produced vascular endothelial growth factor

A myeloma cell line established from a patient refractory to thalidomide therapy revealed high-risk cytogenetic abnormalities and produced vascular endothelial growth factor

Enhancing the efficiency of wide hybridization mediated chromosome engineering for high precision crop improvement with special reference to wheat × Imperata cylindrica system

Traditional plant breeding has been revolutionized by the chromosome engineers during the last two decades by inventing novel approaches having far reaching implications in accelerating the crop improvement programmes with enhanced precision. Chromosome elimination- a dynamic process occurring during wide hybridization in wheat with various genera of Poaceae, has opened a new sphere in accelerating the genetic up-gradation endeavours. Since the invention of bulbosum approach, some other potential systems leading to selective chromosome elimination viz., wheat × maize and wheat × Imperata cylindrica have further enhanced the opportunities to increase the doubled haploid (DH) production efficiency in wheat. The I. cylindrica- mediated approach has emerged as an efficient alternative to the widely used maize- mediated system in terms of natural coincidence for flowering commensurate with the wheat, abundant pollen supply for longer span and developing significantly higher frequency of doubled haploids from winter and spring wheat ecotypes. The I. cylindrica- mediated system has evinced appreciable performance for the triticale × wheat derivatives where maize - mediated system remained unsuccessful. Furthermore, precision of the introgressive hybridization programmes in wheat and other crops can further be enhanced through physical mapping of the alien introgression with minimum linkage drag following novel molecular cytogenetic tools like fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), multicolour FISH (McFISH) and extended DNA fiber mapping (EDF). Combination of DH breeding and molecular cytogenetics has opened new horizon for undertaking cutting edge research to enhance the efficiency and precision of wheat genetic improvement endeavours. Likewise, these novel approaches can be of immense significance for accelerating the targeted and precised improvement of other crop plants.

Multicolor FISH methods in current clinical diagnostics

Multicolor FISH (mFISH) assays are currently indispensable for a precise description of derivative chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting probes in multiplex-FISH and spectral karyotyping. Since then, multiple approaches for chromosomal differentiation based on multicolor-FISH (MFISH) assays have been developed. Predominantly, they are applied to characterize marker or derivative chromosomes identified in conventional banding analysis. Since the introduction of array-based comparative genomic hybridization (aCGH), mFISH is also applied to verify and further delineate aCGH-detected aberrations. For the latter, it is important to consider the fact that aCGH cannot detect or characterize balanced rearrangements, which are important to be resolved in detail in infertility diagnostics. In addition, mFISH is necessary to distinguish different imbalanced situations detectable in aCGH; small supernumerary marker chromosomes have to be differentiated from insertions or unbalanced translocations. This review presents an overview on the available mFISH methods and their applications in pre- and post-natal clinical genetics.

297 ESTABLISHMENT OF A RENAL CELL CARCINOMA CELL LINE ASSOCIATED WITH TFE3 GENE FUSIONS

INTRODUCTION AND OBJECTIVES: Xp11.2 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. It has been recently recognized as a subtype of renal cell carcinoma primarily described in the 2004 World Health Organization classification of renal tumors, and various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome cause the tumor; however, little is known about the tumor characteristics. We report the cytogenetic and biological characteristics of a renal cell carcinoma cell line with TFE3 gene fusion established from a young female with locally advanced renal cell carcinoma. METHODS: Tumor cells were obtained from a renal tumor in a 16-year-old Japanese female. This primary tumor was pathologically diagnosed as renal cell carcinoma with Xp11 translocations. After cultured in vitro, cell suspension was inoculated into the nude mice to establish xenograft. TFE3 immunostaining and other immunohistochemistries were performed. Molecular and cytogenetic methods such as RT-PCR, karyotype analysis and FISH were added. RESULTS: The established cell line and its xenograft showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis, and ASPLTFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Multicolor FISH analysis showed more complicated rearrangements on the chromosome than expected. CONCLUSIONS: Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. This cell line will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma.

Multi-Color Spectral Transcript Analysis (SPECTRA) for Phenotypic Characterization of Tumor Cells.

Many human tumors show significant changes in their signal transduction pathways and, thus, the way the cells interact with their environment. Often caused by chromosomal rearrangements, including gene amplifications, translocations or deletions, the altered levels of gene expression may provide a tumor-specific signature that can be exploited for diagnostic or therapeutic purposes. We investigated the utility of multiplexed fluorescence in situ hybridization (FISH) using non-isotopically labeled cDNA probes detected by Spectral Imaging as a sensitive and rapid procedure to measure tumor-specific gene expression signatures. We used a commercially available system to acquire and analyze multicolor FISH images. Initial investigations used panels of fluorescent calibration standards to evaluate the system. These experiments were followed by hybridization of five-to-six differently labeled cDNA probes, which target the transcripts of tyrosine kinase genes known to be differently expressed in normal cells and tumors of the breast or thyroid gland. The relatively simple, yet efficient, molecular cytogenetic method presented here may find many applications in characterization of solid tumors or disseminated tumor cells. Addressing tumor heterogeneity by means of multi-parameter single cell analyses is expected to enable a wide range of investigations in the areas of tumor stem cells, tumor clonality and disease progression.

Comparison between two FISH techniques in the in vitro study of cytogenetic markers for low-dose X-ray exposure in human primary fibroblasts

This work is about the setup of an in vitro system to report low-dose of X-rays as measured as cytogenetic damage. Q- and multicolor FISH (m-FISH), for telomere length and chromosome instability analysis, respectively, were compared to evaluate their sensitivity in the low-dose range in human primary fibroblasts. No telomere length modulation was observed up to 1 Gy in cycling fibroblasts, though reported for high doses, by that frustrating the purpose of using it as a low-exposure marker. To date the m-FISH is the best setup for the assessment of the chromosome structural damage: it allows stable and instable aberrations to be detected all over the karyotype. Stable ones such as balanced translocations, are not eliminated due to cell-cycle as unstable ones, so they are considered transmissible markers for retrospective dosimetry. The induction of chromosome damage showed a clear dependence on dose delivered; unstable aberrations were demonstrated after doses of 0.1 Gy, and stable aberrations after doses higher than 0.5 Gy. Summarizing, q-FISH is unfit to report low exposures while m-FISH provides better results: unstable aberrations are sensible short-term reporters, while stable ones long report exposures but with a higher induction threshold.

In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

BackgroundThe freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge.ResultsWe show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts.ConclusionThe modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.

Lentivirally Transduced Human Cord Blood CD34+FLT3-ITD+ Cells Induce Murine Acute Leukemia in the NOD/SCID Transplantation Model.

AbstractAbstract 2984Targeting constitutively activated FLT3 (FLT3-ITD) by tyrosine kinase inhibition (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells (LSC). We have previously shown that interaction of CD34+FLT3-ITD+ LSC with stromal niche cells mimicking the bone marrow environment specifically protects these cells from the effects of TKI and confers a growth advantage to FLT3-ITD+ leukemic stem/progenitor cells over normal ones (Parmar et al, Cancer Research 2011). To study human FLT3-ITD+ LSC in vivo in the context of the bone marrow niche, we aimed to establish a xenogeneic NOD/SCID mouse model of human FLT3-ITD+AML.Human CD34+ enriched cord blood cells were transduced with a pWPI lentivirus containing a VSV-pseudotyped SIN/LTR vector with eGFP and full length human FLT3 cDNA harboring a 30 bp length internal tandem duplication (FLT3-ITD) or empty vector control. Transduction efficiency ranged between 1–4.4% for FLT3-ITD and 1.3–18% for vector control. Sub-lethally irradiated NOD/SCID mice were then transplanted with 1 × 104 – 7 × 104 unselected or GFP-sorted CD34+FLT3-ITD+ cells. Acute leukemia developed in 7/9 animals after a median latency of 85 days (range 70–168), with involvement of peripheral blood, bone marrow, spleen and liver. Three mice developed acute lymphoblastic leukemia (ALL) whereas the remaining mice showed signs of AML. In contrast, mice receiving empty pWPI vector-transduced human CD34+ cord blood cells (n = 8) all remained healthy during the observation period of 28 weeks and, in 4/8 animals, normal human CD34+cells could be recovered from the bone marrow (human engraftment range 0.3–35.5%). Leukemic mice exhibited hepatomegaly and splenomegaly with an average 10-fold increase in spleen weight, 2-fold increase in spleen length and 2.7-fold increase in liver weight compared to control mice. In mice that developed ALL, lymph node enlargement was also noted. Whole bone marrow, spleen and liver cells from primary mice were re-transplanted and were able to reproduce acute leukemia in all secondary (n=10/10) and tertiary mice (n=11/11) with a median latency of 25 and 20 days, respectively (p<0.01).Surprisingly, detailed immunophenotypical and immunohistochemical analysis revealed all leukemias to be of murine origin. Leukemic cells stained positively for murine CD45.1 antigen but negatively for human CD45. However, a small population of human CD34+CD45+ cells (range 1–7%) was continuously detectable in the bone marrow of primary, secondary and tertiary transplanted leukemic mice. Accordingly, human FLT3-ITD was detectable by PCR specific for human FLT3 up to the third serial transplantation. Viral integration site analysis by LM-PCR on genomic DNA isolated from spleens of leukemic mice revealed lentiviral integration into the human genome, excluding the possibility of in vivo viral shuttling from human cord blood CD34+ cells to mouse hematopoietic stem cells. Moreover, multicolor fluorescent in situ hybridization (M-FISH) on metaphases generated from peripheral blood lymphocytes revealed only murine chromosomes, also ruling out the possibility of fusion between human and mouse cells. To further characterize these murine leukemias, we performed array CGH on murine spleen gDNA of four immunophenotypically different mice. All mice showed recurrent clonal chromosomal aberrations frequently found in AML. Surprisingly, we have no evidence for the presence of FLT3-ITD in these murine leukemias, suggesting that CD34+FLT3-ITD+ stem cells can trigger development of acute leukemia. We propose that leukemogenesis may mechanistically be related to the host microenvironment and that the bone marrow niche in NOD/SCID mice is susceptible to modulation by the FLT3-ITD oncogene. Disclosures:No relevant conflicts of interest to declare.

Deletion 11q Abnormality in Patients with Chronic Lymphocytic Leukemia (CLL) May Not Have Poor Clinical Outcomes and Bulky Disease (clinical and radiological) At Presentation – Clinical Characteristics of (n=172) Previously Untreated Patients with CLL and del11q Cytogenetic Abnormality.

AbstractAbstract 2890 Introduction:Del11q aberration is universally considered as an adverse prognostic marker in patients with CLL. However, large studies describing the clinical characteristics of del11q in CLL patients (pts) have never been reported. We studied the clinical features, outcomes and treatment responses among (172) previously untreated pts with CLL and del11q. Methods:This is a retrospective analysis of CLL pts (n=172) with del11q, who presented at our institution between 2003–2012. All pts signed an informed consent as per the declaration of Helsinki and IRB approval. Del11q was determined from bone marrow (BM) aspirate or blood samples by FISH (Fluorescent in-situ hybridization) technique. Detection of the 11q22.3 (ATM gene) on chromosome 11 used a multi-color FISH technique. Pts were divided into five categories – One – 57; del11q alone, Two – 105; del11q and del13q, others – 10 (4; del11q with +12, 1; del11q with del17p and 5; del11q, del13q with +12). Baseline bulky lymphadenopathy by CT scan or physical exam (PE) was also analysed. The probabilities of OS, PFS and TTFT (time to first treatment) were estimated using KM plots (log rank). PFS was assessed in 121 treated pts, from date of starting therapy to date of progression or death on therapy. The Cox proportional hazards regression model was used to assess the association between pt characteristics and OS, PFS or TTFT. Results:At the baseline, majority of pts with del11q were young (median age <60 years), males (81.4%) without very high white cell count (median 33.8 K/UL), absolute lymphocyte count – (median 27.1 K/UL) and β2 microglobulin (3 mg/L). Only 16.7% (n=28) patients had advanced Rai stage (3, 4). Majority were IGHV unmutated (n=112; 89.6%), CD38 high (n=88; 51.8%), Zap-70 high (n=100; 75.8%). Status of bulky disease was assessed by PE and baseline CT scans. Bulky disease by PE was observed in n=11 (6.4%) only. Baseline CT scans were performed for 108 patients out of whom only 16 patients (14.8%) had bulky disease. Thus, majority of del11q pts did not have bulky disease by PE or by CT scanning.Analysis of time dependent variables was performed. Among the total 172 pts, 19 (11%) have died. and median time to death has not been reached (NR) - OS. 48/125 (38%) pts who were treated died or progressed and median PFS time was 49.2 months. Overall, 123 (71%) patients received therapy after initial presentation at MDACC. The median TTFT was 10.6 months. FCR based therapies were given in (91; 73 %) pts. Median PFS on FCR based therapy was 32 mo (Figure -1). CR was achieved in (70%; n=64) and overall response was 89%. Pts who received fludarabine based therapy had a significantly shorter TTFT as compared to non fludarabine based therapy (P<0.0001). Other pts (32) were treated with single agent rituximab and lenalidomide protocols.To address whether del11q has any influence over presence of deletion 13 q abnormality we compared (del11q alone; n=57) vs (del11 and del13q; n=105) patients. Patients with sole del11q had significant proportions of younger patients, lesser WBC count, and ALC and β2 microglobulin. Furthermore, there was no significant difference in OS, PFS and TTFT among the two categories. In the multivariate analysis for OS and PFS among the two categories, β2 microglobulin was significantly predictive of increased risk of death or progression among the two categories and FISH categories were not significantly predictive of OS or PFS or TTFT. Bulky disease by PE (and not by CT scan) was significantly associated with a shorter TTFT (HR=2.93; 95%CI -1.5–5.69 and P=0.002).Furthermore, a separate cohort of (n=500) patients with sole del13q was compared with del11q alone (n=57) and del11q with del13q (n=105). There was no significant difference in OS but the TTFT was significantly shorter in del11q alone (data not shown). Conclusions:We describe the clinical features of del11q in CLL patients. This study suggests that occurrence of del11q is frequent with del13q abnormality. The majority of patients with del11q did not have bulky disease as assessed by PE or by CT scan. Presence of bulky disease by PE predicted for a shorter TTFT. β2 microglobulin level significantly predicted for OS and PFS. FCR based therapy has good responses. Del11q is similar to del13q in terms of OS, PFS and TTFT. Del11q may not have very poor adverse outcomes in patients with CLL inspite of a shorter TTFT. [Display omitted] Disclosures:Burger:Pharmacyclics: Consultancy, Research Funding. Wierda:Abbott: Research Funding; Genentech: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; AmGen: Research Funding; Merck: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Genzyme: Consultancy. Kantarjian:Genzyme: Research Funding.

Translocations in Spleen Cells from Adult Mice Irradiated as Fetuses are Infrequent, but Often Clonal in Nature

We previously reported that mouse fetuses or neonates exposed to 2 Gy of X rays showed an unexpectedly low incidence of chromosome damage in lymphocytes, bone marrow, and spleen cells when the mice were subsequently examined at 20 weeks of age. However, cells bearing translocations were occasionally observed that, on the basis of 2-color whole chromosome painting appeared to be clonal descendants. Unfortunately, this approach typically did not permit unequivocal confirmation of their clonality. To overcome this problem, multi-color FISH (mFISH) was employed, which assigns all 21 individual chromosome types of the mouse a unique color. After mFISH analyses of the same cell samples studied previously, it was confirmed that spleen cells of 20-week-old mice irradiated either as 15.5-day fetuses or as 3- to 4-day-old neonates showed translocation frequencies close to zero. Translocations previously suspected as being clonal in nature were confirmed as such by mFISH, which also revealed the presence of an additional clone not previously detected or suspected. Since no evidence of clonality was observed in the irradiated mother, we concluded that in both fetuses and neonates, there exists a small fraction of stem cells that are distinct from the bulk of the stem cell compartment in terms of their ability to acquire and transmit radiation-induced chromosome damage through clonal expansion.

Patterns of genomic aberrations suggest that Burkitt lymphomas with complex karyotype are distinct from other aggressive B‐cell lymphomas with MYC rearrangement

We previously showed that complex karyotypes (CK) and chromosome 13q abnormalities have an adverse prognostic impact in childhood Burkitt lymphomas/leukemias (BL) and diffuse large B-cell lymphomas (DLBCL). The aim of our study was to identify recurrent alterations associated with MYC rearrangements in aggressive B-cell lymphomas with CK. Multicolor fluorescence in situ hybridization (M-FISH) was performed in 84 patient samples (59 adults and 25 children), including 37 BL (13 lymphomas and 24 acute leukemias), 12 DLBCL, 28 B-cell lymphomas with intermediate features (DLBCL/BL), 4 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), and 3 unclassifiable B-cell lymphomas. New (cytogenetically undetected) abnormalities were identified in 80% of patients. We also refined one-third of the chromosomal aberrations detected by karyotyping. M-FISH proved to be more useful in identifying chromosomal partners involved in unbalanced translocations and in revealing greater complexity of 13q rearrangements. Most of the newly identified or refined recurrent alterations involved 1q, 13q and 3q (gains/losses), 7q and 18q (gains), or 6q (losses), suggesting that these secondary aberrations may play a role in lymphomagenesis. Several patterns of genomic aberrations were identified: 1q gains in BL, trisomies 7 in DLBCL, and 18q-translocations in adult non-BL. BCP-ALL usually displayed an 18q21 rearrangement. BL karyotypes were less complex and aneuploid than those of other MYC-rearranged lymphomas. BCP-ALL and DLBCL/BL were associated with a higher rate of early death than BL and DLBCL. These findings support the categorization of DLBCL/BL as a distinct entity and suggest that BL with CK are indeed different from other aggressive MYC-rearranged lymphomas, which usually show greater genetic complexity. © 2012 Wiley Periodicals, Inc.

The Role of Fish in Hematologic Cancer

SUMMARY Cytogenetic analysis is now considered a mandatory investigation in the diagnostic work-up of hematologic malignancies. Recurring structural aberrations serve as powerful markers not only for diagnosis and prognosis of these conditions, but also guide the selection of targeted drugs for personalized oncology. The FISH approach is established as an indispensable tool to complement conventional cytogenetics, in addition to basic and clinical research applications. FISH is used to identify specific chromosomal aberrations through the detection of target DNA sequences by fluorescently labeled DNA probes. Multicolor FISH analysis allows the accurate identification of recurring translocations in neoplastic cells by means of genomic probes that flank the breakpoints. This review summarizes the panel of FISH probes for selection and the current utilization of these FISH techniques in unraveling chromosomal aberrations. The niche of FISH analysis is also highlighted. Variant signal patterns of the clinically useful FISH probes for hematologic oncology illustrated here provide useful interpretative reference for molecular pathology laboratories. In addition, the recent application of FISH tests in contributing information on drug targets at the genomic level to support personalized oncology will also be discussed.