Detection, Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation-Dependent Probe Amplification in Chronic Leukemia

Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD) are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05), and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01), respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001) from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19%) were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory

Significant association between polymorphism of the erythropoietin gene promoter and myelodysplastic syndrome

<p>Abstract</p> <p>Background</p> <p>Myelodysplastic syndrome (MDS) may be induced by certain mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP) rs1617640 in the erythropoietin (<it>EPO</it>) promoter has been shown to be associated with decreased EPO expression. We examined the association of rs1617640 genotype with MDS.</p> <p>Methods</p> <p>We genotyped the EPO rS1617640 SNP in 189 patients with MDS, 257 with acute myeloid leukemia (AML), 106 with acute lymphoblastic leukemia, 97 with chronic lymphocytic leukemia, 353 with chronic myeloid leukemia, and 95 healthy controls.</p> <p>Results</p> <p>The G/G genotype was significantly more common in MDS patients (47/187; 25.1%) than in controls (6/95; 6.3%) or in patients with other leukemias (101/813; 12.4%) (all <it>P </it>< 0.001). Individuals with the G/G genotype were more likely than those with other genotypes to have MDS (odd ratio = 4.98; 95% CI = 2.04-12.13). Clinical and follow up data were available for 112 MDS patients and 186 AML patients. There was no correlation between EPO promoter genotype and response to therapy or overall survival in MDS or AML. In the MDS group, the GG genotype was significantly associated with shorter complete remission duration, as compared with the TT genotype (<it>P </it>= 0.03). Time to neutrophils recovery after therapy was significantly longer in MDS patients with the G/G genotype (<it>P </it>= 0.02).</p> <p>Conclusions</p> <p>These findings suggest a strong association between the rs1617640 G/G genotype and MDS. Further studies are warranted to investigate the utility of screening for this marker in individuals exposed to environmental toxins or chemotherapy.</p

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P &lt; 0.001) and PARP inhibitor therapy (P &lt; 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P &lt; 0.018) and WEE1 inhibitor (P &lt; 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P &lt; 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Representative MLPA analysis on CLL patients.

<p>(<b>a</b>) Normalized ratio plot (relative copy number, RCN) from a CLL patient with trisomy 12 and 19, and 13q14 deletions. The data were normalized to those of normal controls. The reference ranges of 2 SD (Mean ± 2SD, 95% CI, <i>P</i> = 0.05) and 3 SD (Mean ± 3SD, 99% CI, <i>P</i> = 0.01) for each probe are shown by diamonds and squares, respectively. (<b>b</b>) MLPA analysis of copy number changes on multiple CLL samples are called and highlighted on a heatmap as blue (gain, ≥3SD), light blue (gain, ≥2SD), red (loss, ≤3SD) and pink (loss, ≤2SD) blocks that lie outside of reference ranges of each probe. Note that the high resolution of MLPA in several 13q- and 11q- cases can pinpoint deletion region down to a single gene level.</p

Correlation between MLPA and FISH analyses on 13q14- samples.

<p>(<b>a</b>) Normalized ratios by MLPA were grouped by the percentage of FISH-identified leukemia clones carrying 13q14 deletion. The distribution of MLPA ratios showed that 13q14- cells must represent at least 20% (if using 2SD as cutoff) or 40% (if using 3SD as cutoff) of the total population in a sample to be called a statistically significant copy number change. (<b>b</b>) Mixing study using a homozygous 13q- DNA spiked into normal control DNA to create samples containing 0, 10%, 20%, 50%, 75% or 100% alterations. This study also showed a 20% detection limit on 13q deletion by MLPA.</p

Concordance of MLPA with FISH on 94 suspected CLL specimens.

<p>*All comparisons used two-tailed Fisher's exact test.</p