Paratesticular desmoplastic small round cell tumour: an unusual tumour with an unusual fusion; cytogenetic and molecular genetic analysis combining RT-PCR and COBRA-FISH

Desmoplastic small round cell tumour is a rare malignant tumour with a male to female ratio of 4:1. It manifests mostly at serosal sites. Here we present a case of a 28-year-old male patient, who presented with a fast growing paratesticular mass. On biopsy nests and cords of small round cells, without a clear morphological lineage of differentiation were seen. Occasionally desmoplatic small round cell tumour shows different lines of differentiation. An unequivocal histological diagnosis might be difficult in such cases. Here we demonstrate by a combination of methods the characteristic immunohistochemical profile and - albeit unusual - molecular background and discuss the eventual link with Ewing sarcoma

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms

A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed

Molekuláris citogenetikai vizsgálatok Baranya és Tolna megye plazmasejtes myelomában szenvedő betegein = Molecular cytogenetic analyses of patients with plasma cell myeloma in Tolna and Baranya counties in Hungary

Bevezetés: A plazmasejtes myeloma változatos klinikai lefolyással járó hematológiai malignitás, melyhez heterogén genetikai háttér társul. A betegség patogeneziséhez és progressziójához asszociáltan gyakran jelennek meg visszatérő kromoszomális és szubkromoszomális eltérések, melyek diagnóziskor való kimutatása segíti a betegek genetikai karakterizálását, klasszifikációját és prognosztikai besorolását. Célkitűzés: Tanulmányunkban átfogóan értékeltük a Pécsi Klinikai Központ és a Tolna Megyei Balassa János Kórház plazmasejtes myelomában szenvedő betegein 2005 és 2018 között általunk elvégzett molekuláris citogenetikai vizsgálatok eredményeit. Módszer: Az említett periódusban 231 beteg csontvelői és perifériás vérmintájában szűrtünk visszatérő genetikai aberrációkat fluoreszcens in situ hibridizációval. A módszerrel az immunglobulin-nehézlánc-gént érintő kromoszómatranszlokációkat, az 1p és 17p kromoszómakarokat érintő vesztéseket, az 1q kromoszómakart érintő többletet, valamint a 13-as kromoszómát érintő kiegyensúlyozatlan aberrációkat vizsgáltuk. Negyvenkét beteg mintáján multiplex ligatiofüggő szondaamplifikációval vizsgáltuk az 1p, 1q, 5q, 12p, 13q, 16q és 17p kromoszómakarok jellemző vesztéseit és többleteit. A vizsgált időszakban 116 csontvelői mintán kariotipizálásra is sor került. Eredmények: Összesen 233 genetikai eltérést azonosítottunk célzottan, az aberrációk gyakorisága megfelelt a korábbi nemzetközi tanulmányok által látottaknak. Azonos kromoszómakarokat fluoreszcens in situ hibridizációval, valamint multiplex ligatiofüggő szondaamplifikációval vizsgálva az eredmények 96,2%-os egyezést mutattak. Az utóbbi technikával a fluoreszcens in situ hibridizációval detektált abnormalitásokon túl további 21 kiegyensúlyozatlan genetikai aberrációt azonosítottunk 16/42 betegben (38%). Következtetés: Eredményeink alapján az általunk használt két molekuláris citogenetikai módszer együttes alkalmazása jelentősen segítheti a jövőben a plazmasejtes myelomában szenvedő hazai betegek átfogóbb genetikai karakterizálását. Introduction: Plasma cell myeloma is a hematological malignancy with heterogeneous genomic landscape and diverse clinical course. Recurrent chromosomal and subchromosomal aberrations commonly occur in this entity and are associated with the pathogenesis and progression of the disease. The identification of these alterations aids genetic characterization, classification and prognostication of patients. Aim: Molecular cytogenetic investigations of plasma cell myeloma patients treated at the University of Pécs Clinical Center and János Balassa County Hospital of Tolna County, Szekszárd, between 2005 and 2018 were evaluated in our study. Method: 231 patients were screened for genetic aberrations using fluorescence in situ hybridization. Translocations involving the immunoglobulin heavy chain gene, losses of 1p and 17p chromosome arms, gains of 1q chromosome arm and unbalanced aberrations of chromosome 13 were investigated. Losses and gains of 1p, 1q, 5q, 12p, 13q, 16q and 17p chromosome arms were analyzed using multiplex ligation-dependent probe implification in 42 patients. During the investigated period, 116 bone marrow karyotyping was also performed. Results: In total, 233 genetic aberrations were identified using our targeted approaches; the frequency of specific aberrations correlated with data of the recent literature. Concordance of results gained by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification was 96.2% by analyzing the same chromosome arms. The latter technique revealed 21 additional genetic aberrations in 16/42 patient samples (38%) as compared to fluorescence in situ hybridization. Conclusions: Our results suggest that the combined application of the two molecular cytogenetic methods may facilitate a more detailed characterization of genetic aberrations of plasma cell myeloma patients in Hungary

Opening the archives for state of the art tumour genetic research: sample processing for array-CGH using decalcified, formalin-fixed, paraffin-embedded tissue-derived DNA samples

<p>Abstract</p> <p>Background</p> <p>Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.</p> <p>Findings</p> <p>The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements.</p> <p>Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.</p> <p>Conclusions</p> <p>We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.</p

A short-term in vivo model for giant cell tumor of bone

<p>Abstract</p> <p>Background</p> <p>Because of the lack of suitable <it>in vivo </it>models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus <it>in vitro </it>testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.</p> <p>Methods</p> <p>Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using <it>in vivo </it>biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>The suspension of all 10 patients formed solid tumors when grafted on the CAM. <it>In vivo </it>microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.</p> <p>Conclusions</p> <p>A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first <it>in vivo </it>model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.</p

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells