Ulrike Gamerdinger

Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma.

PIK3CA, encoding the catalytic subunit p110α of phosphatidylinositol 3‐kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well‐defined regions of formalin‐fixed tissue sections and to compare these data with protein and mRNA expression of p110α. The mRNA and protein levels of p110α were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low‐to‐moderate dysplasias (17%) and in seven of nine high‐grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high‐grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110α mRNA and protein expression, whereas only two cases (18%) (one high‐grade dysplasia and one carcinoma) with no gain or low‐level gain displayed increased p110α protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110α mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events. Copyright

Cryptic chromosomal aberrations leading to an AML1/ETO rearrangement are frequently caused by small insertions

The translocation t(8;21)(q22;q22), which results in the fusion of the AML1 (RUNX1) and ETO (CBFA2T1) genes, is a recurrent aberration in acute myeloid leukemia (AML), preferentially correlated with FAB M2, and has the highest incidence in childhood AML. Because of the favorable prognosis, the evidence of the t(8;21) or the AML1/ETO fusion gene is mandatory in most of the therapy trials, allowing the stratification of the patients to the correct risk group in terms of treatment. Here we present six out of 59 children with AML who were positive for AML1/ETO by RT‐PCR, but showed no evidence of the classical t(8;21)(q22;q22) by conventional cytogenetics. Because of the discrepancies between molecular and cytogenetic analyses, these six patients were further investigated by fluorescence in situ hybridization analysis. Small hidden interstitial insertions resulting in an AML1/ETO rearrangement were detected in five (8.5%) of the 59 patients, whereas the sixth patient showed a cryptic three‐way translocation. The insertions could be characterized as ins(21;8) in three patients and ins(8;21) in the remaining two. Additionally, three of the patients showed secondary chromosome aberrations leading to a higher complexity of the karyotype. In conclusion, the combination of more than one standard technique in the analysis of AML1/ETO is useful to reveal the overall frequency of cryptic chromosome rearrangements and permits a better understanding of the mechanisms involved in the generation of this fusion gene.

A Recurrent Activating PLCG1 Mutation in Cardiac Angiosarcomas Increases Apoptosis Resistance and Invasiveness of Endothelial Cells

Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies.

Visualization of Postmortem Chondrocyte Damage by Vital Staining and Confocal Laser Scanning 3D Microscopy

The present study was designed to investigate whether the combination of vital dyes [calcein acetomethyl ester and ethidium homodimer (LIVE/DEAD Viability/Cytoxicity Kit)] together with confocal laser scanning 3D microscopy was a suitable process to detect postmortem chondrocyte damage, and whether this process could be used to establish postmortem interval. Human knee cartilage from 13 autopsies (postmortem interval from 1 day to 2.5 months) was incubated with the two dyes. The chondrocytes revealed intense staining according to their vitality. For those cases that were stored mainly at 4 degrees C there was a vitality of approximately 88 to 96% within the first 4.5 days, which decreased to 58% after 6 days and to 9% after 1.5 months. After 2 days and 14 days at summer temperatures there were 70% and 8% vital chondrocytes respectively. Three of the 13 cases showed that altered body and storage conditions limited the efficacy of the method. Initial data suggested a time and temperature dependent increase in cell breakdown. Under stable cooling conditions the use of vital dyes and confocal laser scanning 3D microscopy to measure chondrocyte loss may be a valuable tool for estimating the postmortem interval.

Mosaic tetrasomy 14pter-q13 due to a supernumerary isodicentric derivate of proximal chromosome 14q

Tetrasomy of proximal 14q is an extremely rare condition and has never been reported to be associated with survival. We here report on the first case of mosaic tetrasomy of 14pter‐q13 due to a de‐novo supernumerary pseudoisodicentric chromosome in a 2‐year‐old boy with multiple dysmorphisms and malformations. The marker was detectable in nearly 25% of lymphocytes as well as in cells from buccal mucosa. Detailed fluorescence in situ hybridization (FISH) analyses allowed the characterization of the marker to entirely consist of proximal 14q material and to be symmetric. The pattern of clinical features in our patient only slightly correspond to that of patients with trisomy of proximal 14q, but further cases are needed to define whether tetrasomy of proximal 14q is a separate entity.

Rare interstitial deletion 9q31.2 to q33.1 de novo: Longitudinal study in a patient over a period of more than 20 years

The female carrier of a de novo interstitial deletion 9q [karyotype 46,XX,del(9)(q31.2q33.1)] was followed up over a period of more than 20 years. She shows facial dysmorphisms and significant growth retardation. Motor abilities are restricted by muscular hypotonia and malposition of the feet. She has mental retardation. There was no speech development and phases of autism were reported. By analyses with FISH and short tandem repeat markers, the interstitial deletion was confirmed and characterized to span 9q31.2q33.1, comprising at least 7.07 Mb. The aberration is of paternal origin.

Detection of an activated JAK3 variant and a Xq26.3 microdeletion causing loss of PHF6 and miR-424 expression in myelodysplastic syndromes by combined targeted next generation sequencing and SNP array analysis

Myelodysplastic syndromes (MDS) are hematopoietic disorders characterized by ineffective hematopoiesis and progression to acute leukemia. In patients ineligible for hematopoietic stem cell transplantation, azacitidine is the only treatment shown to prolong survival. However, with the availability of a growing compendium of cancer biomarkers and related drugs, analysis of relevant genetic alterations for individual MDS patients might become part of routine evaluation. Therefore and in order to cover the entire bone marrow microenvironment involved in the pathogenesis of MDS, SNP array analysis and targeted next generation sequencing (tNGS) for the mostly therapy relevant 46 onco- and tumor-suppressor genes were performed on bone marrow biopsies from 29 MDS patients. In addition to the detection of mutations known to be associated with MDS in NRAS, KRAS, MPL, NPM1, IDH1, PTPN11, APC and MET, single nucleotide variants so far unrelated to MDS in STK11 (n=1), KDR (n=3), ATM (n=1) and JAK3 (n=2) were identified. Moreover, a recurrent microdeletion was detected in Xq26.3 (n=2), causing loss of PHF6 expression, a potential tumor suppressor gene, and the miR-424, which is involved in the development of acute myeloid leukemia. Finally, combined genetic aberrations affecting the VEGF/VEGFR pathway were found in the majority of cases demonstrating the diversity of mutations affecting different nodes of a particular signaling network as an intrinsic feature in MDS patients. We conclude that combined SNP array analyses and tNGS can identify established and novel therapy relevant genomic aberrations in MDS patients and track them in a clinical setting for individual therapy selection.

Phosphoinositid-3-Kinase- (PI3-K) Expression

ZusammenfassungPhospoinositid-3-Kinase (PI3-K) ist ein heterodimeres Enzym und in die Regulation von Zellzyklus, Apoptose, Zelladhäsion und Zellmotilität eingebunden. Es wird als Proto-Onkogen in humanen Karzinomen diskutiert. In der vorliegenden Arbeit wurde die PI3-K-Expression in normalem Plattenepithel der Mundhöhle, Dysplasien, Carcinomata in situ, invasiven Karzinomen und Lymphknotenmetastasen immunhistologisch untersucht. Die stärkste Immunreaktivität für die regulatorische p85α- und die katalytische p110α-Untereinheit wurde in invasiven Tumoren und Metastasen gefunden. Carcinomata in situ zeigten eine herdförmige Reaktion, Dysplasien und normales Epithel reagierten überwiegend negativ. Zusätzlich hemmte der PI3-K-Inhibitor LY294002 die Proliferations- und Invasionsfähigkeit der humanen HNSCC-Zelllinie CAL-27 und induzierte die Apoptose in vitro.Unsere Ergebnisse deuten darauf hin, dass PI3-K ein Malignitäts- und Invasionsmarker beim Plattenepithelkarzinom des oberen Aerodigestivtraktes (HNSCC) ist. Wir schlagen vor, PI3-K als Proto-Onkogen in das vorläufige Mehrschrittkanzerogenesemodell des HNSCC aufzunehmen. PI3-K ist darüber hinaus ein potenzielles Ziel pharmakologischer Intervention.AbstractPhosphoinositide 3-kinase (PI3-K) is a heterodimeric enzyme involved in the regulation of mitogenesis, apoptosis, cell adhesion, and motility. PI3-K was suggested as a protooncogene in human cancer. To determine the expression of PI3-K during cancerogenesis and tumor invasion of HNSCC, we investigated normal and dysplastic epithelium of the oral cavity, squamous cell carcinoma and lymph node metastasis by immunohistochemistry. The strongest immunoreactivity for p85α and p110α was found in invasive tumors and their metastases. Carcinomas in situ showed a focal positivity. Dysplasias and normal epithelium reacted predominantly negatively. The PI3-K inhibitor LY294002 inhibited proliferation and invasion of the HNSCC cell line CAL-27 and induced apoptosis in vitro.Our data suggest PI3-K as a marker of malignancy and tumor invasion. We suggest including PI3-K in the multistep carcinogenesis model of HNSCC. In addition, PI3-K is a potential target for pharmacological intervention.

Characterization of the Karyotype in Patients with Multiple Myeloma by the Combination of Karyotype Analysis, FISH and CGH

Abstract Cytogenetic and molecular cytogenetic investigations from bone marrow samples were performed in 83 patients with multiple myeloma. Karyotype analyses were made after cultivation with and without growth factors. The polyploidy level ranged from 3n to 6n. For each patient the composite karyotype was delineated. The number of abnormalities per aberrant cell was in a range from 1 to 17 (x =3,7). The comparison of CGH and FISH results showed an accordance of 92%. In total, 57% of cells showed chromosomal aberrations.

Mosaic Trisomy 1q Due to a de novo Translocation in a Foetus with Early Developmental Abnormalities (Karyotype 46,XY,der(14),t(1;14)(p11;p11.2)/46,XY) Delineation of Parent and Cell Stage of Origin

Abstract Pure trisomies of the whole long arm of chromosome 1 are extremely rare and have been reported only once in association with mosaicism. We report on a malformed foetus with mosaic trisomy 1p11 to 1qter whose clinical features were partially in accordance with those of previously described trisomy 1q patients. An additional long arm of chromosome 1 was translocated onto 14p11.2 (karyotype: mos46,XY, der(14)t(1;14)(p11;p11.2)/ 46,XY). Mosaic formation of the partial trisomy 1 was investigated in seven different somatic tissues of first and second trimester pregnancy. The distribution of the pathologic cells was unequal, ranging from 4 to 93%. The duplicated region was paternal in origin. We were able to delineate two possible complex formation mechanisms involving paternal meiosis and postzygotic mitoses.