Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

<p>Abstract</p> <p>Background</p> <p>Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23.</p> <p>Conclusions</p> <p>Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future.</p

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Quantification of the Effectiveness of a Residency Program Using the Resident In-Service Examination

This study describes a quantitative tool in the assessment of residency programs, in which national ranking of residents after the resident in-service examination in postgraduate year 4 is compared to that in postgraduate year 1. The relationship between training and changes in ranking, resident in-service examination results before and after training in specific areas are also compared. To illustrate the use of this novel approach, data from a large residency program were analyzed. The 70 residents were ranked as a postgraduate year 1 group at the 50th national percentile. As postgraduate year 4 residents, they were ranked at the 59th percentile, a significant ( P < .003) improvement. There was moderate correlation between performance in postgraduate year 1 and that in postgraduate year 4 (0.61); however, initial ranking was no indication of the final ( R 2 = .34), with the exception of high performers. Training in specific areas improved ranking, demonstrating association between training and performance. In conclusion, the effectiveness of training provided by a residency program can be quantified using the resident in-service examination. This should provide a quantitative tool in the assessment of postgraduate programs

SNP Array Karyotyping Improves Detection Rate of Clonal Chromosomal Abnormalities in Refractory Anemia with Ringed Sideroblasts

Abstract Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more accurately diagnosed by characteristic pathomorphology. Clonal hematopoiesis and chromosomal abnormalities exemplify a close pathogenetic relationship to other forms of MDS. RARS shows considerable clinical variability even for patients (pts) with identical cytogenetic defects. Due to the low resolution of metaphase cytogenetics (MC) and its dependence on cell growth in vitro, this test is often non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions and copy-neutral loss of heterozygozity. We hypothesize that cryptic chromosomal (chr) aberrations exist in most, if not all, pts with RARS. Their detection may help to improve prognostication, distinguish distinct phenotypes and point towards unifying pathogenic defects. Initially, we analyzed the results of MC in pts with MDS and MDS/MPD (N=455) and in a sub-cohort of RARS, RCMD-RS, RARSt and other MDS subtypes with >15% RS. When we compared pts with/without RS, chr defects were found at comparable frequencies (∼50%). The most commonly occurring defects associated with RS, compared to other forms of MDS, included those of chr 5 (9% vs. 16%, 7 (8% vs. 12%) and 20 (3% vs. 8%). DNA was available for 36 pts with RS and was subjected to 250K SNP-A karyotyping. Pathologic lesions were defined upon exclusion of normal copy number polymorphisms identified in 81 controls (O’Keefe at al ASH 2007), as well as the Database of Genomic Variants (http://projects.tcag.ca/variation). By MC, a defective karyotype was present in 16/36 pts (44%). Deletions involving chr 5, 7 and complex MC were found in 3, 5, and 2pts, respectively. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), all aberrations found by MC were confirmed, but also new lesions were detected so that an abnormal karyotype was established in 62% of pts. Several previously cryptic/recurrent lesions included losses of a portion of chr. 2 (N=2; 2p16.2, 2p16.3), and deletions (N=4; 7p11.1–14.1, 7p21.3, 7q11.23–21.11, 7q21.12-qter) as well as gains (N=1; 7q33) on chr 7. We have also detected segmental uniparental disomy (UPD) in chr 1 (N=2; 1p21.3–22.2, 1p). This type of lesion cannot be detected using MC and provides an additional mechanism leading to LOH. When both bone marrow and blood of 5 RARS patient were tested using SNP-A, blood analysis had 100% accuracy rate as compared to marrow; all defects seen in the marrow were also found in blood. We conclude that chromosomal defects are present in a majority of RARS patients and arrays with higher resolution will identify defects in most, if not all of the patients. Our study also demonstrates testing of peripheral blood by SNP-A can complement marrow MC, especially in cases in which marrow is not available. Detection of clonal marker aberrations in blood of RARS patients suggests that mostly clonal dysplastic progenitor cells contribute to blood production rather than residual “normal” progenitors

Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML

Using metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with myelodysplastic syndrome (MDS). We hypothesized that with new precise methods more cryptic karyotypic lesions can be uncovered that may show important clinical implications. We have applied 250K single nucleotide polymorphisms (SNP) arrays (SNP-A) to study chromosomal lesions in samples from 174 patients (94 MDS, 33 secondary acute myeloid leukemia [sAML], and 47 myelodysplastic/myeloproliferative disease [MDS/MPD]) and 76 controls. Using SNP-A, aberrations were found in around three-fourths of MDS, MDS/MPD, and sAML (vs 59%, 37%, 53% by MC; in 8% of patients MC was unsuccessful). Previously unrecognized lesions were detected in patients with normal MC and in those with known lesions. Moreover, segmental uniparental disomy (UPD) was found in 20% of MDS, 23% of sAML, and 35% of MDS/MPD patients, a lesion resulting in copy-neutral loss of heterozygosity undetectable by MC. The potential clinical significance of abnormalities detected by SNP-A, but not seen on MC, was demonstrated by their impact on overall survival. UPD involving chromosomes frequently affected by deletions may have prognostic implications similar to the deletions visible by MC. SNP-A–based karyotyping shows superior resolution for chromosomal defects, including UPD. This technique further complements MC to improve clinical prognosis and targeted therapies

Presence of JAK2 Mutations in MDS/MPD-u WHO Classified Patients and Not Other Forms of MDS Suggests Their Derivation from Classical Myeloproliferative Syndrome

Abstract The WHO classification of myeloid neoplasms recognizes a category of myeloid disorders that overlaps traditional myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD) as the entity MDS/MPD disease. This disease category includes CMML, JMML, atypical CML and MDS/MPD unclassifiable (MDS/MPD-u). The provisional entity termed RARS associated with marked thrombocytosis (RARS-t) is currently best classified as MDS/MPD-u until further information is available regarding its pathogenesis. Recently a JAK2 mutation V617F (G→T) was identified as a pathogenetic lesion in typical myeloproliferative disorders (Kralovics et al, NEJM, 2005). Subsequent studies of the JAK2 mutation concentrated on more proliferative forms of MDS or MDS/MPD such as CMML and CNL (Steensma et al, Blood, 2005). These studies demonstrated that homo- and heterozygous JAK2 mutants are present in a rather small proportion of these patients. Based on similar clinical features, we theorized that JAK2 mutants might be also found in patients with MDS/MPD-u. We have collected a cohort of these patients (N=202) and analyzed them for the presence of JAK2 mutation using a molecular allele-specific PCR assay. Positive cases were confirmed by sequencing with a sensitivity of about 20% of mutated cells. A group of patients with PV, MF and ET served as positive controls (N=66). In agreement with previous reports, the detection rate of JAK2 mutants for PV, MF and ET was 92%, 55% and 55%, respectively. Our experimental group included 104 patients with MDS or MDS/MPD (53 RA/RS, 14 RAEB, 22 RAEB-t/sAML, and 15 CMML). Most significantly, the RA/RS group contained 13 patients with WHO-defined MDS/MPD overlap and 3 with RARS-t (among these patients, 3 were JAK2 mutants; all of them were heterozygous - 18.8%). Within 16 patients with CMML1 or 2 we found only 2 heterozygous for JAK2 mutation (12.5%). The remaining cohort of 73 patients in MDS categories revealed only 1 patient to be heterozygous for JAK2 mutation (RARS, 1.4%). As expected MDS/MPD patients with JAK2 mutation showed various degrees of BM fibrosis, splenomegaly and less pronounced cytopenias. Except for one patient, JAK2 mutants had normal cytogenetics. All had normal MCV and ANC. In 4/5 increased megakaryocytes with/or without atypia was seen. One CMML patient with abnormal cytogenetics showed an unusual translocation t(8;9)(q22;p24). JAK2 is located at 9p24 so it is possible that the JAK2 gene was involved in the translocation generating a novel fusion protein in addition to an activating JAK2 mutant. Our results showed that JAK2 mutations are rarely found in typical cases of MDS or CMML. However, further analysis of JAK2 mutational status in patients with MDS/MPD-u is warranted. The reported detection rate may suggest that the pathogenesis of these entities is more akin to myeloproliferative than myelodysplastic syndromes