Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8x10-4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches

Genomic characterization of pediatric B‐lymphoblastic lymphoma and B‐lymphoblastic leukemia using formalin‐fixed tissues

BackgroundRecurrent genomic changes in B‐lymphoblastic leukemia (B‐ALL) identified by genome‐wide single‐nucleotide polymorphism (SNP) microarray analysis provide important prognostic information, but gene copy number analysis of its rare lymphoma counterpart, B‐lymphoblastic lymphoma (B‐LBL), is limited by the low incidence and lack of fresh tissue for genomic testing.ProcedureWe used molecular inversion probe (MIP) technology to analyze and compare copy number alterations (CNAs) in archival formalin‐fixed paraffin‐embedded pediatric B‐LBL (n = 23) and B‐ALL (n = 55).ResultsSimilar to B‐ALL, CDKN2A/B deletions were the most common alteration identified in 6/23 (26%) B‐LBL cases. Eleven of 23 (48%) B‐LBL patients were hyperdiploid, but none showed triple trisomies (chromosomes 4, 10, and 17) characteristic of B‐ALL. IKZF1 and PAX5 deletions were observed in 13 and 17% of B‐LBL, respectively, which was similar to the reported frequency in B‐ALL. Immunoglobulin light chain lambda (IGL) locus deletions consistent with normal light chain rearrangement were observed in 5/23 (22%) B‐LBL cases, compared with only 1% in B‐ALL samples. None of the B‐LBL cases showed abnormal, isolated VPREB1 deletion adjacent to IGL locus, which we identified in 25% of B‐ALL.ConclusionsOur study demonstrates that the copy number profile of B‐LBL is distinct from B‐ALL, suggesting possible differences in pathogenesis between these closely related diseases.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137353/1/pbc26363.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137353/2/pbc26363_am.pd

How confident are young adult cancer survivors in managing their survivorship care? A report from the LIVESTRONG™ Survivorship Center of Excellence Network

This study examined the association between sociodemographic, cancer treatment, and care delivery factors on young adult cancer survivors’ confidence in managing their survivorship care

Constitutive function of the Ikaros transcription factor in primary leukemia cells from pediatric newly diagnosed high-risk and relapsed B-precursor ALL patients.

We examined the constitutive function of the Ikaros (IK) transcription factor in blast cells from pediatric B-precursor acute lymphoblastic leukemia (BPL) patients using multiple assay platforms and bioinformatics tools. We found no evidence of diminished IK expression or function for primary cells from high-risk BPL patients including a Philadelphia chromosome (Ph)(+) subset. Relapse clones as well as very aggressive in vivo clonogenic leukemic B-cell precursors isolated from spleens of xenografted NOD/SCID mice that developed overt leukemia after inoculation with primary leukemic cells of patients with BPL invariably and abundantly expressed intact IK protein. These results demonstrate that a lost or diminished IK function is not a characteristic feature of leukemic cells in Ph(+) or Ph(-) high-risk BPL

Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8x10-4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches

Transcript Levels of Ikaros Target Genes in Primary Leukemia Cells from Pediatric Ph^- and Ph⁺ BPL Patients.

<p>Expression levels of IK target genes were compared for primary leukemic cells from 155 pediatric Ph^-/BCR-ABL^- BPL patients and 20 Ph ⁺ /BCR-ABL ⁺ /BPL patients on the Mullighan study (GSE12995). Transcript signal values were obtained from hybridization onto the Affymetrix Human Genome U133A genechip arrays. Heat map depicts up and down regulated transcripts ranging from red to green respectively for mean centered log₁₀ transformed expression values and clustered according to average distance metric (<b>A</b>). Rank ordered difference in standard deviation units for Ph⁺ samples (N=20) compared to other samples (N=155) in the Mullighan study (GSE12995) were processed for enrichment of IK target genes <b>(B1)</b> and IK-regulated lymphoid priming genes <b>(B2)</b> using a supervised approach implemented in GSEA2.08 (Broad institute). Enrichment scores for calculated for the ranked members of the gene sets and normalized to the gene set size (NES) for which the P-value was calculated using 1000 permutations of the pre-ranked gene list and the FDR corrected for testing 2 gene sets. There was a significant enrichment of IK target genes (NES = 1.43, P = 0.046) for Ph⁺ patients that included the leading edge subset comprised of TSPAN13<i>, GSN</i>, MDFIC<i>, ITGA4, TREML2, RNF125</i>, IQGAP2, LAMC1, TES, DHRS3<i>, S100A10</i>, IL12RB1, ADD3<i>, GRAMD3, ATRNL1</i>, RUNX2<i>, MCOLN3</i>, ATP1B1, and CALCRL. Likewise, there was a significant enrichment of lymphoid priming genes (NES = 1.51, P = 0.039) for Ph⁺ patients that included the leading edge subset comprised of <i>IGJ</i>, CNN3, CD52, DNTT<i>, CSF1R</i>, SATB1<i>, LTB, PTGER2</i>, MEF2C, RUNX2 (Figure <b>1B.2</b>). There was a significant increase in the multivariate mean for 45 transcripts in the Ph⁺ subset of specimens compared to the pooled mean of the other subsets (MANOVA, F_1,173 = 11.29, P=0.001). The mean level of expression for each transcript in each BPL subset is illustrated in the heat map organized using a two-way hierarchical clustering method (average distance metric) to group expression profiles of transcripts and specimen subsets (<b>C</b>).</p

Evaluation of Primary Leukemic Cells from High-Risk Pediatric BPL Patients for IKZF1 Deletions Involving Exons 4 or 5 Using Real-time Quantitative PCR.

<p><b>[A]</b> Depicted are the Box- and Whisker plots of the mean Ct values from 2 independent technical replicates of exon-specific IKZF1 qPCR performed on genomic DNA of primary leukemic cells from 32 BPL patients and 2 normal control bone marrow specimens. In each PCR reaction, the input of the template was standardized by using 105 ng of genomic DNA. In addition, a reference primer pair for Exon 7 was included for normalization of the qPCR data for Exon 4 and Exon 5. There were no outliers for Ct values for IKZF1 Exons 4, 5 or 7. No BPL case was identified by interpatient comparisons or comparisons between leukemic and normal samples that had a significantly higher Ct value suggestive of an IKZF1 deletion. <b>[B]</b> The Q2 (Median), Q1 (25^th Percentile/Lower quartile) and Q3 (75^th Percentile/Upper quartile) values for the box plots shown in [A]. <b>[C]</b> Bar graphs of Ct data from exon-specific IKZF1 qPCR for 32 BPL patients averaged across 2 independent experiments and then compared with Ct data for normal hematopoietic cells from 2 normal bone marrow specimens averaged from 3 independent experiments in which they were included as controls for side-by-side comparison with leukemic specimens from BPL patients (2 experiments) or BPL xenograft cells (one experiment). The inset shows the mean±SE values. No statistically significant differences were observed between normal vs. BPL specimens as documented by the P-values shown. <b>[D]</b> Bar graphs of Ct data from [C] for Exon 4-specific qPCR and Exon 5-specific qPCR after normalization by referencing the raw data to the Ct values from Exon 7-specific qPCR. The data normalization employed the method reported by Weksberg et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B15" target="_blank">15</a>] and Moody et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B16" target="_blank">16</a>] (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#pone.0080732.s001" target="_blank">File <b>S1</b></a>). <b>[E-H]</b> Depicted are representative cycle number vs. Log (∆Rn) (fluorescence signal) amplification plots obtained using the IKZF1 Exon 4-specific test primers and IKZF1 Exon 7-specific reference primers for the normal hematopoietic cells and randomly picked 5 BPL cases.</p

Exon-specific Detection of IKZF1 Transcripts in Normal Hematopoietic Cells and Primary Leukemic Cells from Pediatric Ph⁺ vs. Ph^- BPL Patients.

<p>IKZF1 transcript levels, as measured by 5 IKZF1 probe sets specific for IKZF1 Exons 1-4, were compared between primary leukemic cells from 123 pediatric Ph⁺ BPL patients, 327 pediatric Ph^- BPL patients and non-leukemic hematopoietic cells from 74 normal bone marrow specimens analyzed in 2 independent studies (viz.: GSE13159 and GSE13351). Depicted in (<b>A</b>) are bar charts of RMA-normalized transcript levels for the 5 IKZF1 probe sets, including 2 probe sets exhibiting Exon 4 specificity to test for reduction in signal due to an intragenic IKZF1 deletion involving a region within Exons 4-7 or Exons 2-7. No significant reduction in expression levels were observed for these 2 Exon 4 probesets (<b>B</b>) and significant increases in expression were observed for both comparisons of Ph⁺ with Normal and Ph^- samples for the two Exon 3 probesets. Heat map depicts up- and down-regulated transcripts ranging from red to green respectively for RMA-normalized IKZF1 transcript levels of leukemic cells mean centered to normal hematopoietic cells and clustered according to the average distance metric (<b>C</b>).</p

Pediatric Ph⁺ BPL is not Characterized by IK-deficiency.

<p><b>[A1-A4] Genomic PCR</b>. <b>Analysis of the Human <i>Ikaros</i>/<i>IKFZ1</i> Gene Exons E4-E7 in High-Risk BPL</b>. We performed exon-specific IKZF1 PCR with DNA sequencing on purified genomic DNA samples from 3 pediatric patients with Ph⁺ high-risk BPL. Exons E4-E7 and their intron-exon junctions were PCR amplified using the PCR primers in Table <b>S3</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#pone.0080732.s001" target="_blank">File <b>S1</b></a>. Depicted are agarose gels documenting that normal size PCR products (E4: 577-bp, E5: 646-bp, E6: 530-bp, E7:785-bp) for each of the 4 IKZF1 exons were obtained in each of the 3 patients, providing strong evidence against the existence of homozygous deletions of the entire IKZF1 locus or within. IKZF1 exons E4-E7. <b>[A5] Western Blot Analysis of Ikaros Expression in Ph⁺ High-Risk</b>. <b>BPL</b>. Depicted is an anti-IK Western blot of whole cell lysates of primary leukemia cells from 3. Ph⁺ BPL patients. Similar to RAJI cells and primary cells from an MLL-AF4⁺ ALL case that were included as controls, Ph⁺ ALL cells from each of the 3 cases expressed an intact 57 kDa IK1 protein. <b>[B] Nuclear Expression of Intact Ikaros Protein and Its Regulators in Ph⁺ and Ph^- High-Risk BPL</b>. Depicted are Western blots of nuclear protein extracts (NE) from primary leukemic cells of 8 high-risk pediatric BPL patients, including 4 Ph⁺ ALL cases. NE were examined for presence of IK (B1), Ku70 (B2), SYK (B3) and BTK (B4). See text for discussion. <b>[C & D] Assessment of Sequence-Specific DNA Binding Function of Nuclear Ikaros in Ph⁺ and Ph^- High-Risk BPL</b>. Electrophorectic mobility shift assays (EMSA) were performed on nuclear extracts from BPL cells using the Thermo Scientific LightShift Chemiluminescent EMSA Kit and the <i>IK-BS1</i> test probe containing a high-affinity IK1 binding site and the <i>IK</i>-<i>BS5</i> negative control probe that has a single base pair (G>A) substitution at position 3 within the core consensus and does not bind IK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B10" target="_blank">10</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B12" target="_blank">12</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B13" target="_blank">13</a>]. The nuclear protein extracts from each of the 4 Ph⁺ and 4 Ph^- BPL cases showed significant gel retardation of the IK1-specific IK-BS1 probe (but not the control IK-BS5 probe) (<b>C</b>). Supershift assays were performed in 3 Ph⁺ and 4 Ph^- BPL cases with an anti-IK monoclonal antibody (2 µg/sample) to confirm the presence of IK in the retarded DNA-binding protein complexes, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B12" target="_blank">12</a>] (<b>D</b>). Positions of the retarded and supershifted bands are indicated with arrowheads. In each case, the retarded complex of the IK-BS1 probe was supershifted with the anti-IK antibody.</p

Transcript Levels of Ikaros Target Genes in Primary Leukemia Cells from Pediatric Ph^-/BCR-ABL^- and Ph ⁺ /BCR-ABL⁺ BPL Patients on the MILE Study.

<p>Expression levels of IK target genes were compared between primary leukemic cells (GSE13159) from 122 pediatric BPL patients with t(9;22) translocation (Ph ⁺ /BCR-ABL⁺) and 237 pediatric BPL patients without t(9;22) translocation (Ph^-/BCR-ABL^-). Heat map depicts up and down regulated transcripts ranging from red to green respectively for standardized expression values and clustered according to average distance metric (<b>A</b>). 25 transcripts representing 16 IK target genes were expressed at significantly higher levels in leukemia cells from Ph⁺ patients, no significant differences were observed for 10 transcripts representing 9 IK target genes and 7 transcripts representing 5 IK target genes were significantly down regulated (<b>Table S2</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#pone.0080732.s001" target="_blank">File <b>S1</b></a>). Hierarchical cluster analysis identified MDFIC (0.69 SD units, P = 8.8 x 10^-12), DHRS3 (0.6 SD units, P = 3.7 x 10^-11), <i>GSN</i> (0.49 SD units, P = 3.4 x 10^-10), <i>ITGA4</i> (0.53 SD units, P = 2.6 x 10^-9) and TSPAN13 (0.2 SD units, P = 1.2 x 10^-7) as the most significantly up-regulated genes in the 122 Ph⁺ patient samples. Rank ordered difference in standard deviation units for Ph⁺ samples compared to Ph^- samples for enrichment of IK target genes <b>(B1)</b> as well as lymphoid priming genes <b>(B2)</b> using a supervised approach implemented in GSEA2.08 (Broad institute). Enrichment scores were calculated for the ranked members of the gene sets and normalized to the gene set size (NES) for which the p-value was calculated using 1000 permutations of the pre-ranked gene list and the FDR corrected for testing 2 gene sets. There was a significant enrichment of IK target genes (NES = 2.04, P < 0.001) and lymphoid priming genes (NES = 1.62, P = 0.013) that included the leading edge subsets MDFIC<i>, EIF4E3</i>, DHRS3<i>, ITGA4</i>, PTK2<i>, GSN, S100A10, GRAMD3</i>, F13A1, CALCRL, ATP1B1, LAMC1, TES, ADD3<i>, TCTEX1D1, ATRNL1</i>, IL12RB1<i>, RNF125, MCOLN3</i>, RUNX2, PDLIM2<i>, TSGA10, TREML2</i> for IK targets and <i>IGJ</i>, CNN3<i>, CSF1R, PTGER2, LTB</i>, DNTT, CD52, MEF2C, and RUNX2 for lymphoid priming genes. There was a significant increase in the multivariate mean for 45 transcripts in the Ph⁺ subset of specimens (MANOVA, F_1,357 = 30.65, P<0.0001). The mean level of expression for each transcript in each BPL subset is illustrated in the heat map organized using a two-way hierarchical clustering method (average distance metric) to group expression profiles of transcripts and BPL subsets (<b>C</b>).</p