Karlheinz Holzmann

TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome

To assess the frequency of TP53 alterations and their correlation with other genetic changes and outcome in acute myeloid leukemia with complex karyotype (CK-AML), we performed integrative analysis using TP53 mutational screening and array-based genomic profiling in 234 CK-AMLs. TP53 mutations were found in 141 of 234 (60%) and TP53 losses were identified in 94 of 234 (40%) CK-AMLs; in total, 164 of 234 (70%) cases had TP53 alterations. TP53-altered CK-AML were characterized by a higher degree of genomic complexity (aberrations per case, 14.30 vs 6.16; P < .0001) and by a higher frequency of specific copy number alterations, such as -5/5q-, -7/7q-, -16/16q-, -18/18q-, +1/+1p, and +11/+11q/amp11q13∼25; among CK-AMLs, TP53-altered more frequently exhibited a monosomal karyotype (MK). Patients with TP53 alterations were older and had significantly lower complete remission rates, inferior event-free, relapse-free, and overall survival. In multivariable analysis for overall survival, TP53 alterations, white blood cell counts, and age were the only significant factors. In conclusion, TP53 is the most frequently known altered gene in CK-AML. TP53 alterations are associated with older age, genomic complexity, specific DNA copy number alterations, MK, and dismal outcome. In multivariable analysis, TP53 alteration is the most important prognostic factor in CK-AML, outweighing all other variables, including the MK category.

Hypermethylation of the CDKN2/p16 Promoter During Neoplastic Progression in Barrett's Esophagus

BACKGROUND & AIMS Inactivation of the CDKN2/p16(INK4A) tumor-suppressor gene is one of the most frequent genetic alterations in human malignancies. In esophageal adenocarcinomas, mutations of the p16 gene or homozygous deletions of the gene locus 9p21 are rare. This study investigated whether p16 promoter hypermethylation is an alternative mechanism for p16 gene inactivation during neoplastic progression in Barretts esophagus. METHODS A methylation-specific polymerase chain reaction protocol was applied. A total of 95 specimens from 14 patients with Barretts esophagus were analyzed longitudinally. The p16 promoter status was compared with histomorphological findings. RESULTS p16 promoter hypermethylation was detected in 9 of the 10 patients who had displayed dysplasia at some time during surveillance, whereas none of the patients who had not displayed dysplasia during surveillance had p16 promoter hypermethylation. p16 promoter hypermethylation was detected in 3% (2 of 67) of the samples without dysplasia, 60% (3 of 5) of the samples with lesions indefinite for dysplasia, 55.6% (10 of 18) of the specimens with low-grade dysplasia, and 75% (3 of 4) of the specimens with high-grade dysplasia. CONCLUSIONS These data suggest that p16 promoter hypermethylation is a common mechanism of p16 gene inactivation during neoplastic progression in Barretts esophagus.

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Synergy between PI3K Signaling and MYC in Burkitt Lymphomagenesis

In Burkitt lymphoma (BL), a germinal center B-cell-derived tumor, the pro-apoptotic properties of c-MYC must be counterbalanced. Predicting that survival signals would be delivered by phosphoinositide-3-kinase (PI3K), a major survival determinant in mature B cells, we indeed found that combining constitutive c-MYC expression and PI3K activity in germinal center B cells of the mouse led to BL-like tumors, which fully phenocopy human BL with regard to histology, surface and other markers, and gene expression profile. The tumors also accumulate tertiary mutational events, some of which are recurrent in the human disease. These results and our finding of recurrent PI3K pathway activation in human BL indicate that deregulated c-MYC and PI3K activity cooperate in BL pathogenesis.

Reticular dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2

Human severe combined immunodeficiencies (SCID) are phenotypically and genotypically heterogeneous diseases. Reticular dysgenesis is the most severe form of inborn SCID. It is characterized by absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth. In bone marrow of individuals with reticular dysgenesis, myeloid differentiation is blocked at the promyelocytic stage, whereas erythro- and megakaryocytic maturation is generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelo-lymphoid lineages. The dramatic clinical course of reticular dysgenesis and its unique hematological phenotype have spurred interest in the unknown genetic basis of this syndrome. Here we show that the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) is mutated in individuals with reticular dysgenesis. Knockdown of zebrafish ak2 also leads to aberrant leukocyte development, stressing the evolutionarily conserved role of AK2. Our results provide in vivo evidence for AK2 selectivity in leukocyte differentiation. These observations suggest that reticular dysgenesis is the first example of a human immunodeficiency syndrome that is causally linked to energy metabolism and that can therefore be classified as a mitochondriopathy.

Genomic DNA-Chip Hybridization Reveals a Higher Incidence of Genomic Amplifications in Pancreatic Cancer than Conventional Comparative Genomic Hybridization and Leads to the Identification of Novel Candidate Genes

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.

High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations

To identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphism-array analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n = 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-of-heterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.21 (5% of patients) to a segment 486 kb proximal to the MYC locus. 13q deletions exhibited proximal and distal breakpoint cluster regions. Among the most common novel lesions were deletions at 15q15.1 (4% of patients), with the smallest deletion (70.48 kb) found in the MGA locus. Sequence analysis of MGA in 59 samples revealed a truncating mutation in one CLL patient lacking a 15q deletion. MNT at 17p13.3, which in addition to MGA and MYC encodes for the network of MAX-interacting proteins, was also deleted recurrently.

Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia

Mutations in the nucleophosmin 1 (NPM1) gene are considered a founder event in the pathogenesis of acute myeloid leukemia (AML). To address the role of clonal evolution in relapsed NPM1-mutated (NPM1mut) AML, we applied high-resolution, genome-wide, single-nucleotide polymorphism array profiling to detect copy number alterations (CNAs) and uniparental disomies (UPDs) and performed comprehensive gene mutation screening in 53 paired bone marrow/peripheral blood samples obtained at diagnosis and relapse. At diagnosis, 15 aberrations (CNAs, n = 10; UPDs, n = 5) were identified in 13 patients (25%), whereas at relapse, 56 genomic alterations (CNAs, n = 46; UPDs, n = 10) were detected in 29 patients (55%) indicating an increase in genomic complexity. Recurrent aberrations acquired at relapse included deletions affecting tumor suppressor genes (ETV6 [n = 3], TP53 [n = 2], NF1 [n = 2], WT1 [n = 3], FHIT [n = 2]) and homozygous FLT3 mutations acquired via UPD13q (n = 7). DNMT3A mutations (DNMT3Amut) showed the highest stability (97%). Persistence of DNMT3Amut in 5 patients who lost NPM1mut at relapse suggests that DNMT3Amut may precede NPM1mut in AML pathogenesis. Of note, all relapse samples shared at least 1 genetic aberration with the matched primary AML sample, implying common ancestral clones. In conclusion, our study reveals novel insights into clonal evolution in NPM1mut AML.

CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models

Background: CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic cancer, however, remains elusive. Methods and Results: Silencing of CD147 by RNA interference (RNAi) reduced the proliferation rate of MiaPaCa2 and Panc1 cells. CD147 is required for the function and expression of the monocarboxylate transporters MCT1 and MCT4 that are expressed in human PDAC cells as demonstrated by real-time reverse transcription-PCR (RT-PCR) as well as immunohistology. MCT1 and MCT4 are the natural transporters of lactate, and MiaPaCa2 cells exhibited a high rate of lactate production, which is characteristic for the Warburg effect, an early hallmark of cancer that confers a significant growth advantage. Further induction of lactate production by sodium azide in MiaPaCa2 cells increased MCT1 as well as MCT4 expression. CD147 silencing inhibited the expression and function of MCT1 and MCT4 and resulted in an increased intracellular lactate concentration. Addition of exogenous lactate inhibited cancer cell growth in a dose-dependent fashion. In vivo, knock-down of CD147 in MiaPaCa2 cells by inducible short hairpin RNA (shRNA)-mediated CD147 silencing reduced invasiveness through the chorioallantoic membrane of chick embryos (CAM assay) and inhibited tumourigenicity in a xenograft model in nude mice. Conclusion : The function of CD147 as an ancillary protein that is required to sustain the expression and function of MCT1 and MCT4 is involved in the association of CD147 expression with the malignant potential of pancreatic cancer cells exhibiting the Warburg effect.

Identification of acquired copy number alterations and uniparental disomies in cytogenetically normal acute myeloid leukemia using high-resolution single-nucleotide polymorphism analysis

Recent advances in genome-wide single-nucleotide polymorphism (SNP) analyses have revealed previously unrecognized microdeletions and uniparental disomy (UPD) in a broad spectrum of human cancers. As acute myeloid leukemia (AML) represents a genetically heterogeneous disease, this technology might prove helpful, especially for cytogenetically normal AML (CN-AML) cases. Thus, we performed high-resolution SNP analyses in 157 adult cases of CN-AML. Regions of acquired UPDs were identified in 12% of cases and in the most frequently affected chromosomes, 6p, 11p and 13q. Notably, acquired UPD was invariably associated with mutations in nucleophosmin 1 (NPM1) or CCAAT/enhancer binding protein-α (CEBPA) that impair hematopoietic differentiation (P=0.008), suggesting that UPDs may preferentially target genes that are essential for proliferation and survival of hematopoietic progenitors. Acquired copy number alterations (CNAs) were detected in 49% of cases with losses found in two or more cases affecting, for example, chromosome bands 3p13–p14.1 and 12p13. Furthermore, we identified two cases with a cryptic t(6;11) as well as several non-recurrent aberrations pointing to leukemia-relevant regions. With regard to clinical outcome, there seemed to be an association between UPD 11p and UPD 13q cases with overall survival. These data show the potential of high-resolution SNP analysis for identifying genomic regions of potential pathogenic and clinical relevance in AML.