Analysis of CUX1 and SH2B2 in the pathogenesis of myeloproliferative neoplasms

Myeloproliferative Neoplasien (MPN) sind klonale hämatologische Erkrankungen, welche zu den myeloischen Malignomen gehören. Laut der WHO Klassifizierung von 2008, gehören Polycythämia vera (PV), Essentielle Thrombozythämie (ET), Primäre Myelofibrose (PMF), Chronische myeloische Leukämie (CML), Chronische Neutrophilenleukämie (CNL), Chronische Eosinophilenleukämie (CEL), Systemische Mastozytose (SM) und unklassifizierbare Myeloproliferative Neoplasien zu den MPN. Das Hauptmerkmal von MPN ist die übermäßige Bildung von terminal differenzierten Blutzellen, die zu der myeloischen Reihe gehören. Die individuellen klinischen Merkmale der drei klassischen BCR-ABL1-negativen MPN sind Erythrozytose in PV, Thrombozythämie in ET und Fibrosierung des Knochenmarkgewebes in PMF. Eine Transformation zur akuten Leukämie ist, neben Thrombose, die größte Komplikation von MPN aufgrund von einer schlechten Prognose und einer voraussichtlichen Lebensdauer von 5 Monaten. Diese Studie konzentriert sich auf den drei klassischen BCR-ABL1 negativen MPN, PV, ET und PMF und deren Transformation zu AML. In 29 Patienten unserer Kohorte wurden Deletionen und uniparentale Disomien (UPD) von Chromosom 7q gefunden und in einer der Deletionen befand sich nur CUX1. Es wurde berichtet, dass Deletionen von Chromosom 7q mit der Transformation assoziiert sind, deswegen wurde eine Sequenzanalyse von CUX1 und SH2B2, das 1kb von CUX1 entfernt liegt, in Patienten, die akute Leukämie entwickelt haben, durchgeführt. Als Methode zur Aufklärung der Konsequenzen von CUX1 und SH2B2 Deletionen wurden shRNAs, die gegen die spezifischen Gene gerichtet waren, in der Baf3/EpoR Zelllinie getestet. Die Wachstumskinetik der transduzierten Zelllinien hat keinen Effekt von den shRNAs gezeigt. TP53 wurde in 24 Patienten, die eine Krankheitsprogression zu sMF oder AML erfahren haben, sequenziert. Es wurde berichtet, dass auch dieses Gen mit der Transformation von MPN Patienten assoziiert ist. In unserer Kohorte wurden 5 somatische Mutationen (21%) gefunden. Diese Resultate liefern Beweise zu der Komplexität von MPN. Die Identifikation dieser neuen genetischen Veränderungen in MPN Patienten könnte Auswirkungen auf bessere Möglichkeiten der Diagnose und Therapie haben

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL, MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized

Whole exome sequencing identifies novel MPL and JAK2 mutations in triple negative myeloproliferative neoplasms.

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR, exon 10 of MPL or JAK2-V617F in >90% of the cases, while the remaining cases are termed "triple negative". We aimed to identify the disease causing mutations in the triple negative cases of ET and PMF by applying whole exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5/8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3/8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis, analyzed by WES, we identified a somatic MPL-S204P and a germline MPL-V285E mutation, as well as a germline JAK2-G571S variant. Sequencing of entire coding region of MPL and JAK2 was performed in additional 62 and 49 triple negative cases of ET or PMF, respectively. We detected new somatic (T119I, S204F, E230G, Y591D) and one germline (R321W) MPL mutation in 5/62 cases. All the mutations were gain-of-function mutations when analyzed in functional assays. JAK2 variants were identified in 5/57 triple negative cases and 3 of them were germline. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target <i>CBL, MLL, DDB1</i> and <i>LMO2</i>

<div><p>Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of <i>CBL</i>. In one patient the 11qUPD amplified somatic mutations in both <i>CBL</i> and the DNA repair gene <i>DDB1</i>. A duplication within <i>MLL</i> exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with <i>de novo</i> acute myeloid leukemia (P=0.013). One patient with a deletion at the <i>LMO2</i> locus harbored an additional point mutation on the other allele indicating that <i>LMO2</i> might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.</p> </div

Summary of chromosome 11 aberrations.

<p>Large chromosomal aberrations are indicated with colored bars around the ideogram of chromosome 11. Green – gains; red – deletions; blue – uniparental disomies. The position of the bars relative to the chromosome ideogram indicates the position and size of the aberration. For the two patients of whom two samples were analyzed (UPN 23 and UPN 42 – see Table S1) recurrent aberrations are depicted only once. The positions of <i>CBL, MLL, EED, SF1</i>, <i>DDB1</i> and <i>LMO2</i> are indicated by vertical lines. Mutations in these genes are depicted by orange circles along these lines. Common deleted regions are indicated at the bottom of Figure 2 listing the genes they cover. The ideogram depicts G-banding pattern at ~850-band resolution level.</p

Mutations detected in <i>DDB1, MLL</i> and <i>LMO2</i>.

<p><b>A</b>: Sample 36 harbored an 11q UPD as indicated by the blue bar below the chromosome 11 ideogram. We found two somatic mutations in <i>DDB1</i> and <i>CBL</i>. As can be seen in the Sanger sequencing traces, both mutations are homozygous due to amplification by the UPD. <b>B</b>: In sample 50 a tandem duplication in <i>MLL</i> exon 3 was detected. The top graph shows whole exome coverage data across <i>MLL</i> exon 3. The data is plotted as the log₂ ratio of the normalized exome sequencing coverage in the patient sample divided by the median normalized coverage of 8 independent control samples at each genomic position (X-axis). The position of the duplication is indicated by the red bar. Sanger sequencing confirmed an in-frame tandem duplication of 171 amino acids as shown at the bottom. <b>C</b>: A common deleted region on chromosome 11p targets <i>LMO2</i>. All deletions in the analyzed cohort that span the <i>LMO2</i> locus are depicted next to the chromosome 11 ideogram. Red bars indicate deletions, green bars indicate gains. In sample 42, which harbored a deletion spanning the LMO2 locus, we also detected a point mutation in <i>LMO2</i>. The middle section shows a signal intensity plot measuring copy number from Affymetrix microarrays. The plot depicts signal intensity (log₂ scale) differences between the patient and a healthy control pool for each probe (as implemented in the Affymetrix Genotyping Console software). The deletion in sample 42 can be seen as the deviation from 0 for all probes in the deleted genomic region (X-axis). The point mutation in <i>LMO2</i> as identified by Sanger sequencing is depicted at the bottom of panel C. <b>A</b>, <b>B</b> and <b>C</b>: Depicted are the genomic (letters) as well as the respective amino acid (box chains) sequences. Numbers above the boxes indicate amino acid positions in the proteins. Amino acids substituted in the patient samples are indicated by red boxes. The red circle indicates a splice site mutation. Reference and mutant sequences are shown. The arrows indicate the site of mutations below the Sanger sequencing traces.</p