Development of a dual-color, double fusion FISH assay to detect RPN1/EVI1 gene fusion associated with inv(3), t(3;3), and ins(3;3) in patients with myelodysplasia and acute myeloid leukemia.

Approximately 2-3% of adult patients with acute myeloid leukemia harbor a rearrangement of RPN1 (at 3q21) and EVI1 (at 3q26.2) as inv(3)(q21q26.2), t(3;3)(q21;q26.2), or ins(3;3)(q26.2;q21q26.2). The most recent World Health Organization (WHO) classification has designated AML with inv(3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. We have created a dual color, double fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the RPN1 and EVI1 genes. A blinded investigation was performed using 30 normal bone marrow samples and 51 bone marrow samples from 17 patients with inv(3)(q21q26.2), 11 patients with t(3;3)(q21;q26.2), and one patient with ins(3;3)(q26.2;q21q26.2) previously defined by chromosome analysis. The unblinded results indicated abnormal RPN1/EVI1 fusion results in 30 (97%) of 31 samples from the inv(3)(q21q26.2) group including seven bone marrow samples for which chromosome analysis was unsuccessful or failed to detect an inv(3)(q21q26.2). Abnormal FISH results were detected in 14 (88%) of 16 samples with t(3;3)(q21;q26.2) and in the sole sample with an ins(3;3)(q26.2;q21q26.2). All 30 negative controls were normal and were used to establish a normal cutoff of 0.6% for the typical abnormal D-FISH signal pattern. Overall, this D-FISH assay was more accurate than chromosome analysis and based on the normal cutoff of 0.6%, this assay can be used for minimal residual disease detection and disease monitoring in patients with RPN1/EVI1 fusion.

Identification of novel HMGA2 fusion sequences in lipoma: evidence that deletion of let-7 miRNA consensus binding site 1 in the HMGA2 3' UTR is not critical for HMGA2 transcriptional upregulation.

Lipoma is a benign tumor composed of mature adipocytes and one of the most common mesenchymal tumors seen in adults. Rearrangement of HMGA2 in chromosome band 12q15 has been found in approximately 60-70% of ordinary lipomas with cytogenetic abnormalities. Herein, we report two novel HMGA2 fusion sequences in lipomas with chromosome 12 rearrangements. Cytogenetic studies showed 12q abnormalities in both cases, and fluorescence in situ hybridization (FISH) confirmed the involvement of HMGA2 in each instance. Rapid amplification of cDNA ends (RACE) PCR experiments revealed that one lipoma contained a fusion of the HMGA2 3' untranslated region (UTR) to a genomic area downstream of the DYRK2 locus on 12q15; the second lipoma showed a fusion of the HMGA2 3' UTR to a genomic sequence upstream of the DCN locus on 12q21. In both instances the breakpoint on HMGA2 occurred downstream to let-7 miRNA (microRNA) consensus binding site (CBS) 1. These two and several other previously reported tumors containing HMGA2 3' UTR rearrangements show breakpoints after let-7 miRNA CBS 1, which suggests that the elimination of this miRNA binding site is not critical for driving HMGA2 transcriptional upregulation.

Splenic small B-cell lymphoma with IGH/BCL3 translocation.

Isolated chromosomal translocations are important defining features of many non-Hodgkin lymphomas, especially of B-cell type. In contrast to some other translocations, the significance of IGH/BCL3 translocations is not well defined. Although often considered a feature of the ill-defined entity atypical chronic lymphocytic leukemia, very few cases are reported in which involvement of BCL3 and the precise B-cell neoplasm are both well documented. For this reason, we report a splenic-based CD5(-), CD10(-), CD43(-), CD23(-), CD103(-), FMC7(+), CD25(+) small B-cell lymphoma associated with epithelioid histiocyte clusters and a t(14;19)(q32;q13) representing an IGH/BCL3 translocation based on classical cytogenetic studies, chromosomal painting, and fluorescence in situ hybridization studies. The previously reported neoplasms with t(14;19)(q32;q13) or IGH/BCL3 translocations are also reviewed. The present case did not fall into any of the classic B-cell lymphoma categories and clearly did not represent chronic lymphocytic leukemia/small lymphocytic lymphoma. This case suggests that the IGH/BCL3 translocation may help to define a new clinicopathologic entity.

Fluorescent-labeled DNA probes applied to novel biological aspects of B-cell chronic lymphocytic leukemia.

Fluorescent-labeled DNA probes were used to study 52 chronic lymphocytic leukemia (B-CLL) patients for (1) disease progression, (2) angiogenesis genes, (3) T-cell leukemia 1 gene (TCL1), (4) immunoglobulin heavy chain variable region (IGHv) and (5) chromosome 6q. Compared to stable disease, more patients with progressive disease had > or =2 anomalies and a high percentage of neoplastic nuclei. Anomalies of genes for basic fibroblast growth factor, interleukin 4, vascular endothelial growth factor or TCL1 were not detected. Deletions in IGHv occurred in 25% of patients and correlated with IGHv gene expression. Probes for 6q23 detected more deletions in 6q than probes for 6q21.

Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia.

PURPOSE: Retrospective studies suggest cytogenetic abnormalities detected by interphase fluorescent in situ hybridization (FISH) can identify patients with chronic lymphocytic leukemia (CLL) who will experience a more aggressive disease course. Other studies suggest that patients may acquire chromosome abnormalities during the course of their disease. There are minimal prospective data on the clinical utility of the widely used hierarchical FISH prognostic categories in patients with newly diagnosed early-stage CLL or the frequency of clonal evolution as determined by interphase FISH. PATIENTS AND METHODS: Between 1994 and 2002, we enrolled 159 patients with previously untreated CLL (83% Rai stage 0/I) on a prospective trial evaluating clonal evolution by FISH. Patients provided baseline and follow-up specimens for FISH testing during 2 to 12 years. RESULTS: Chromosomal abnormalities detected by FISH at study entry predicted overall survival. Eighteen patients experienced clonal evolution during follow-up. The rate of clonal evolution increased with duration of follow-up with only one occurrence in the first 2 years (n = 71; 1.4%) but 17 occurrences (n = 63; 27%) among patients tested after 5+ years. Clonal evolution occurred among 10% of ZAP-70-negative and 42% of ZAP-70-positive patients at 5+ years (P = .008). CONCLUSION: This clinical trial confirms prospectively that cytogenetic abnormalities detected by FISH can predict overall survival for CLL patients at the time of diagnosis, but also suggests that many patients acquire new abnormalities during the course of their disease. Patients with higher ZAP-70 expression may be more likely to experience such clonal evolution. These findings have important implications for both clinical management and trials of early treatment for patients with high-risk, early-stage CLL.

Interphase FISH to detect PBX1/E2A fusion resulting from the der(19)t(1;19)(q23;p13.3) or t(1;19)(q23;p13.3) in paediatric patients with acute lymphoblastic leukaemia.

Approximately 6% of paediatric patients with precursor B-cell acute lymphoblastic leukaemia (B-ALL) harbour a rearrangement involving the gene regions of PBX1 (1q23) and E2A (19p13.3) which is visualized cytogenetically either as a der(19)t(1;19)(q23;p13.3) or the less common balanced t(1;19)(q23;p13.3). Unfortunately, no commercial dual-colour, double fusion fluorescence in situ hybridization (D-FISH) strategies are available to detect this recurrent anomaly. Therefore, we have created a D-FISH assay to detect these translocations and monitor minimal residual disease. This probe set was created using four bacterial artificial chromosomes (BACs) corresponding to the PBX1 gene region at 1q23 and four BACs corresponding to the E2A gene region at 19p13.3. We analysed 30 negative bone marrow controls and 20 diagnostic and post-treatment specimens from 13 paediatric B-ALL patients with a cytogenetically defined 1;19 translocation. Once unblinded, the results demonstrated that our D-FISH method effectively identified all diagnostic samples as abnormal and identified disease in four post-treatment samples that were previously considered to be normal by conventional cytogenetic analysis. The development of this FISH strategy for the detection of der(19)t(1;19)(q23;p13.3) and t(1;19)(q23;p13.3) proved to be an effective technique, allowing both the detection of disease in diagnostic samples and in post-treatment samples.

Familial 22q11.2 deletions in DiGeorge/velocardiofacial syndrome are predominantly smaller than the commonly observed 3Mb.

PURPOSE: DiGeorge/velocardiofacial syndrome (DG/VCFS) is the most common cytogenetically characterized microdeletion of 22q11.2 region. In approximately 90% of patients, the deletion size is 3 Mb, whereas the remaining range from 1.5 to 2.5 Mb. The purpose of this study was to test the hypothesis that small deletions may be more easily tolerated in a familial fashion than larger deletions, especially for this syndrome. METHOD: Sixteen FISH probes designed from bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) mapped to 22q11.2 were used to determine the deletion sizes in 22 individuals from ten families with familial 22q11.2 deletion detected by standard FISH tests. RESULT: Seven families had deletions of < 3 Mb ( approximately 1.5 Mb) in size and 3 families had the common 3-Mb deletion. The 70% frequency of smaller sized deletions among this group of patients with familial del(22)(q11.2) is significantly higher than that reported among unselected group of patients with del(22)(q11.2) (P < 0.0001, Fisher exact test). CONCLUSION: Familial del(22)(q11.2) are predominantly smaller than the common deletion size of 3 Mb, indicating that there may be some underlying mechanisms that favor parent-to-child transmission of smaller deletions in individuals with del(22)(q11.2), therefore, underscoring the need to exclude a familial basis in cases of del(22)(q11.2) smaller than 3 Mb.

Human SULT1A3 pharmacogenetics: gene duplication and functional genomic studies.

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines. Inheritance is an important factor responsible for individual variation in SULT1A3 activity, and gene resequencing studies have shown the presence of one functionally significant SULT1A3 nonsynonymous cSNP. However, following completion of the Human Genome Project, it appeared that SULT1A3 might be duplicated. We used specific PCR-based assays and fluorescence in situ hybridization to verify that 2 SULT1A3 genes-SULT1A3 and SULT1A4-were present on chromosome 16 in all human DNA samples studied. Furthermore, reanalysis of previous gene resequencing data confirmed the presence of the SULT1A3 SNPs identified previously, but also revealed 11 novel polymorphisms, including 3 nonsynonymous cSNPs. Functional genomic studies showed that two of those cSNPs, C302T, and C302A, resulted in decreased enzyme activity without striking changes in substrate kinetics but with parallel changes in levels of immunoreactive protein. In addition, RT-PCR revealed that both SULT1A3 and SULT1A4 can be transcriptionally active. The duplication of SULT1A3 will have to be taken into account in future efforts to understand individual variation in SULT1A3 activity or properties.

FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia.

A novel oncogenic mutation (FIP1L1-PDGFRA), which results in a constitutively activated platelet-derived growth factor receptor-alpha (PDGFRA), has been invariably associated with a primary eosinophilic disorder. The current study examines both the prevalence and the associated clinicopathologic features of this mutation in a cohort of 89 adult patients presenting with an absolute eosinophil count (AEC) of higher than 1.5 x 10(9)/L. A fluorescence in situ hybridization (FISH)-based strategy was used to detect FIP1L1-PDGFRA in bone marrow cells. None of 8 patients with reactive eosinophilia displayed the abnormality, whereas the incidence of FIP1L1-PDGFRA in the remaining 81 patients with primary eosinophilia was 14% (11 patients). None (0%) of 57 patients with the hypereosinophilic syndrome (HES) but 10 (56%) of 19 patients with systemic mast cell disease associated with eosinophilia (SMCD-eos) carried the specific mutation. The bone marrow mast cell infiltration pattern in FIP1L1-PDGFRA(+) SMCD-eos was distinctly diffuse with loose tumoral aggregates. Treatment with low-dose imatinib (100 mg/d) produced complete and durable responses in all 8 FIP1L1-PDGFRA(+) cases treated. In contrast, only 40% partial response rate was seen in 10 HES cases. FIP1L1-PDGFRA is a relatively infrequent but treatment-relevant mutation in primary eosinophilia that is indicative of an underlying systemic mastocytosis.

Microduplication 22q11.2, an emerging syndrome: clinical, cytogenetic, and molecular analysis of thirteen patients.

Chromosome 22, particularly band 22q11.2, is predisposed to rearrangements due to misalignments of low-copy repeats (LCRs). DiGeorge/velocardiofacial syndrome (DG/VCFS) is a common disorder resulting from microdeletion within the same band. Although both deletion and duplication are expected to occur in equal proportions as reciprocal events caused by LCR-mediated rearrangements, very few microduplications have been identified. We have identified 13 cases of microduplication 22q11.2, primarily by interphase fluorescence in situ hybridization (FISH). The size of the duplications, determined by FISH probes from bacterial artificial chromosomes and P(1) artificial chromosomes, range from 3-4 Mb to 6 Mb, and the exchange points seem to involve an LCR. Molecular analysis based on 15 short tandem repeats confirmed the size of the duplications and indicated that at least 1 of 15 loci has three alleles present. The patients' phenotypes ranged from mild to severe, sharing a tendency for velopharyngeal insufficiency with DG/VCFS but having other distinctive characteristics, as well. Although the present series of patients was ascertained because of some overlapping features with DG/VCF syndromes, the microduplication of 22q11.2 appears to be a new syndrome.

CHIC2 deletion, a surrogate for FIP1L1-PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy.

Imatinib mesylate is effective in the treatment of hematologic malignancies that are characterized by either abl- or PDGFR beta- activating mutations. The drug is also active in a subset of patients with eosinophilic disorders and systemic mast cell disease (SMCD). Recently, a novel tyrosine kinase that is generated from fusion of the Fip1-like 1 (FIP1L1) and PDGFR alpha (PDGFRA) genes has been identified as a therapeutic target for imatinib mesylate in hypereosinophilic syndrome (HES). We used fluorescence in situ hybridization (FISH) to detect deletion of the CHIC2 locus at 4q12 as a surrogate for the FIP1L1-PDGFRA fusion. CHIC2 deletion was observed in bone marrow cells for 3 of 5 patients with SMCD associated with eosinophilia. Deletion of this locus and expression of the FIP1L1-platelet-derived growth factor receptor alpha (PDGFRA) fusion was also documented in enriched eosinophils, neutrophils, or mononuclear cells by both FISH and reverse transcriptase-polymerase chain reaction (RT-PCR) for one patient. While all 3 patients with the FIP1L1-PDGFRA rearrangement achieved a sustained complete response with imatinib mesylate therapy, the other two, both carrying the c-kit Asp816 to Val (Asp816Val) mutation, did not. These observations suggest that the FIP1L1-PDGFRA rearrangement occurs in an early hematopoietic progenitor and suggests that the molecular pathogenesis for a subset of SMCD patients is similar to that of HES. Screening for the FIP1L1-PDGFRA rearrangement and Asp816Val mutation will advance rational therapy decisions in SMCD.