Integrated genome-wide genotyping and gene expression profiling reveals BCL11B as a putative oncogene in acute myeloid leukemia with 14q32 aberrations.

Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.

Addition of ß-mercaptoethanol is a prerequisite for high-quality RNA isolation using QIAsymphony technology as demonstrated by detection of molecular aberrations in hematologic malignancies.

The isolation of high-quality RNA and DNA from various specimens is essential to perform reliable molecular diagnostic assays. In routine diagnostics of hematologic malignancies isolation of high-quality RNA is a prerequisite. We used QIAsymphony technology (QST) using a customized RNA CT 800 V6 protocol for automated semi-high-throughput isolation of RNA from human specimens and compared the results for breakpoint cluster region-c-abl oncogene 1 (BCR-ABL1) quantification by real-time quantitative polymerase chain reaction (RQ-PCR) and detection of JAK2 V617F mutations by reverse-transcriptase PCR (RT-PCR) on QST RNA with RNA isolation performed with our routine manual method using RNA-Bee (RB). QST RNA was isolated with and without the addition of ß-mercaptoethanol (BME). Addition of BME to the lysis buffer RLT Plus resulted in consistently lower Ct values in analyses of the reference gene porphobilinogen deaminase (PBGD). Further, the BCR-ABL1 mRNA levels of the QST RNA isolation were highly consistent with RB RNA isolation, only when the lysis buffer RLT Plus in addition contained BME. Moreover, cases of myeloproliferative neoplasms (MPN) with low levels of JAK2 V617F mRNA were even missed in QST when lysis buffer RLT Plus was used, but they were readily detected after addition of BME.

SNPExpress: integrated visualization of genome-wide genotypes, copy numbers and gene expression levels.

BACKGROUND: Accurate analyses of comprehensive genome-wide SNP genotyping and gene expression data sets is challenging for many researchers. In fact, obtaining an integrated view of both large scale SNP genotyping and gene expression is currently complicated since only a limited number of appropriate software tools are available. RESULTS: We present SNPExpress, a software tool to accurately analyze Affymetrix and Illumina SNP genotype calls, copy numbers, polymorphic copy number variations (CNVs) and Affymetrix gene expression in a combinatorial and efficient way. In addition, SNPExpress allows concurrent interpretation of these items with Hidden-Markov Model (HMM) inferred Loss-of-Heterozygosity (LOH)- and copy number regions. CONCLUSION: The combined analyses with the easily accessible software tool SNPExpress will not only facilitate the recognition of recurrent genetic lesions, but also the identification of critical pathogenic genes.

Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias.

DNA from 110 adult de novo acute myeloid leukaemia (AML) patients exhibiting either inv(16) (n = 63) or t(8;21) (n = 47) was screened for mutations in the c-KIT (exon 8 and Asp816) and FLT3 (ITD and Asp835) genes. c-KIT exon 8 mutations were found in 15/63 (23.8%) inv(16) patients and 1/47 (2.1%) t(8;21) patients. c-KIT Asp816 mutations were present in 5/63 (7.9%) inv(16) AML and 5/47 (10.6%) t(8;21) AML. FLT3 mutations were identified in five patients (7.9%) with inv(16) and three patients (5.6%) with t(8;21) AML. All mutations were mutually exclusive; 40% of inv(16) AML patients possessed either a c-KIT or FLT3 mutation. c-KIT exon 8 mutations were shown to be a significant factor adversely affecting relapse rate.