Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and advances in its clinical and laboratory biology have grown exponentially over the last few decades. Treatment outcome has improved steadily with over 90% of patients surviving 5 years from initial diagnosis. This success can be attributed in part to the development of a risk stratification approach to identify those subsets of patients with an outstanding outcome that might qualify for a reduction in therapy associated with fewer short and long term side effects. Likewise, recognition of patients with an inferior prognosis allows for augmentation of therapy, which has been shown to improve outcome. Among the clinical and biological variables known to impact prognosis, the kinetics of the reduction in tumor burden during initial therapy has emerged as the most important prognostic variable. Specifically, various methods have been used to detect minimal residual disease (MRD) with flow cytometric and molecular detection of antigen receptor gene rearrangements being the most common. However, many questions remain as to the optimal timing of these assays, their sensitivity, integration with other variables and role in treatment allocation of various ALL subgroups. Importantly, the emergence of next generation sequencing assays is likely to broaden the use of these assays to track disease evolution. This review will discuss the biological basis for utilizing MRD in risk assessment, the technical approaches and limitations of MRD detection and its emerging applications

Transcriptional and Epigenetic Regulation of <em>KIAA1199</em> Gene Expression in Human Breast Cancer

<div><p>Emerging evidence has demonstrated that upregulated expression of <em>KIAA1199</em> in human cancer bodes for poor survival. The regulatory mechanism controlling <em>KIAA1199</em> expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human <em>KIAA1199</em> promoter in terms of regulation of <em>KIAA1199</em> gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the <em>KIAA1199</em> gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic <em>KIAA1199</em> promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human <em>KIAA1199</em> gene. Bioinformatics analysis suggested that the 1.4 kb <em>KIAA1199</em> promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for <em>KIAA1199</em> gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these <em>cis</em>- and <em>trans</em>-acting elements in controlling <em>KIAA1199</em> gene expression. Finally, we found that upregulated <em>KIAA1199</em> expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of <em>KIAA1199</em> expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling <em>KIAA1199</em> gene expression in human cancer.</p> </div

Characterization of NF-κB as a regulatory element in distal part of promoter.

<p><b>A)</b> Luciferase reporter gene assay for the effect of NF-κB and Twist-1 on the activity of <i>KIAA1199</i> promoter: Lysates of COS-1 cells transfected with cDNAs as indicated were examined by Dual Luciferase activity assay. Wild type and substitute mutation (pro-1.4 kb swap NF-κB^4th) at distal region of NF-κB binding site were used. <b>B)</b> ChIP assay for identification of the interaction between NF-κB (p65) and <i>KIAA1199</i> promoter. Top panel: A schematic diagram of four putative NF-κB binding sites relative to +1 site. Primer sets A, B, and C were designed for NF-κB binding site IV and I + II, and an area of the first <i>KIAA1199</i> intron, respectively. Middle and low panels: ChIP PCR in HeLa cells transfected with GFP control and P65 cDNAs. Anti-p-65 antibody and normal IgG (control) were used for immunoprecipitation. Results were calculated according to the bound/input ratio.</p

Sequence alignment of the <i>KIAA1199</i> promoter between human and mouse genomes and putative transcription factor-binding sites.

<p>Putative transcription factor-binding motifs are underlined and TATA/GC boxes and transcription start site are shown in the box. The asterisks mark the fully conserved sequences across the species.</p

Elevated expression of <i>KIAA1199</i> in human cancers.

<p>A) By mining Oncomine and GEO databases, <i>KIAA1199</i> expression pattern in more than 40 microarray data sets shows significant alteration (P<0.01). Representative data are presented. High <i>KIAA1199</i> expression in various human cancers.1-breast cancer n:27, normal n:7, p = 6.97E-4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Radvanyi1" target="_blank">[41]</a>; 2-colon cancer n:36, normal n:24, p = 1.7E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Skrzypczak1" target="_blank">[42]</a>; 3-gastric cancer n:26, normal n:31, p = 3.69E-13<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-DErrico1" target="_blank">[43]</a>; 4-lung cancer n:45, normal n:65, p = 8.59E-9<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Hou1" target="_blank">[44]</a>; 5-head&neck cancer n:41, normal n:13, p = 2.35E-7<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Ginos1" target="_blank">[45]</a>; 6-ovarian cancer n:6, normal n:4, p = 5.92E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Adib1" target="_blank">[46]</a>; 7-pancreatic cancer n:11, normal n:11, p = 0.001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Grutzmann1" target="_blank">[47]</a>. <b>B)</b> Expression of <i>KIAA1199</i> in human cancer cell lines: Human prostate (LNCaP and Du145), and breast cancer (MCF-7 and MDA-MB-231) cell lines were examined by real time RT-PCR using <i>KIAA1199</i> specific primers. The expression of <i>KIAA1199</i> was normalized by house-keeping genes (HPRT-1 and GAPDH). The relative levels of genes were determined using the ΔΔCt method. Each bar represents the mean ± S.E (*<0.05).</p

Requirement of AP-1 binding element in the <i>KIAA1199</i> promoter.

<p><b>A)</b> A schematic diagram of mutations at the AP-1 binding site: A site-directed mutagenesis was carried out to generate either a deletion mutant by removing the AP-1 consensus sequence (GAGT) or a substitute mutation within the pro-1.4 promoter construct. The relative promoter activities of the mutations (ratio of firefly luciferase over Renilla luciferase) were then compared to the activity of the wild type pro-1.4 promoter construct (defined as arbitrary value of 100) in COS-1, MDA-MB-231, and MCF-7 cells. <i>Error bars indicate</i> mean +/− S.E. <b>B)</b> Binding of nuclear proteins to the AP-1 site in the <i>KIAA1199</i> promoter: EMSA was carried out using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, binding was competed with 50–200 fold excess amounts of unlabeled probe. DNA-protein complexes formed are indicated as Shift-1 and Shift-2. <b>C)</b> Determination of specific binding between AP1 and the <i>KIAA1199</i> promoter: EMSA was performed using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, biotinylated probe along with anti-C-Jun antibody or biotinylated probe containing mutated site of AP-1 consensus sequence were incubated with nuclear extracts from MDA-MB-231 cells. <b>D)</b> ChIP assay for analysis of association between endogenous AP-1 and the KIAA199 promoter sequence: A strong relation between AP-1 and DNA sequence was shown by 20% bound/input ratio as compared to the unrelated intron region. Normal rabbit IgG was used as a negative control. Results were calculated according to the bound/input ratio.</p

Analysis of the transcriptional activity of the <i>KIAA1199</i> promoter.

<p><b>A</b>) <i>Left panel</i>: A schematic description of the <i>KIAA1199</i> reporter constructs containing fragments of three different lengths cloned into the pGL3-basic vector. The numbers in the names of the constructs indicate their respective lengths in nucleotides relative to the main transcription start site. <i>Right three panels</i>: Normalized firefly luciferase activity from each construct: Lysates from cells transfected with the different reporter gene constructs together with R<i>enilla luciferase reporter were examined for luciferase activities.</i> The relative promoter activities (ratio of firefly luciferase over Renilla luciferase) were compared to the activity of the pro-1.4 promoter construct (defined as arbitrary value of 100). <i>Error bars indicate</i> mean +/− S.E. <b>B</b>) <i>Top panel</i>: A ladder of PCR fragments for generating deletion mutants from the pro-1.4 promoter. <i>Left lower panel</i>: A schematic description of the pro-1.4 kb and truncations of the <i>KIAA1199</i> reporter constructs cloned in pGL3-basic vector. <i>Right lower three panels</i>: Normalized firefly luciferase activity from each construct: Lysates from cells transfected with the different reporter gene constructs together with R<i>enilla luciferase reporter were examined for luciferase activities. The firefly luciferase value of each sample has been normalized to its renilla luciferase value. Error bars indicate</i> mean +/− S.E.</p