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Ovarian cancer has the highest mortality rate among gynecological tumors. This is based on late diagnosis and the lack of early symptoms. To improve early detection, it is essential to find reliable biomarkers. The metalloprotease ADAM17 could be a potential marker, as it is highly expressed in many solid tumors, including ovarian and breast cancer. The aim of this work is to evaluate the relevance of ADAM17 as a potential diagnostic blood-based biomarker in ovarian cancer. Ovarian cancer cell lines IGROV-1 and A2780, as well as primary patient-derived tumor cells obtained from tumor tissue and ascitic fluid, were cultured to analyze ADAM17 abundance in the culture supernatant. In a translational approach, a cohort of 117 well-characterized ovarian cancer patients was assembled and ADAM17 levels in serum and corresponding ascitic fluid were determined at primary diagnosis. ADAM17 was quantified by enzyme-linked immunosorbent assay (ELISA). In the present study, ADAM17 was detected in the culture supernatant of ovarian cancer cell lines and primary cells. In addition, ADAM17 was found in serum and ascites of ovarian cancer patients. ADAM17 level was significantly increased in ovarian cancer patients compared to an age-matched control group (p &lt, 0.0001). Importantly early FIGO I/II stages, which would not have been detected by CA-125, were associated with higher ADAM17 concentrations (p = 0.007). This is the first study proposing ADAM17 as a serum tumor marker in the setting of a gynecological tumor disease. Usage of ADAM17 in combination with CA-125 and other markers could help detect early stages of ovarian cancer.

Epithelial ovarian cancer displays the highest mortality of all gynecological tumors. A relapse of the disease even after successful surgical treatment is a significant problem. Resistance against the current platinum‐based chemotherapeutic standard regime requires a detailed ex vivo immune profiling of tumor‐infiltrating cells and the development of new therapeutic strategies. In this study, we phenotypically and functionally characterize tumor cells and autologous tumor‐derived αβ and γδ T lymphocyte subsets. Tumor‐infiltrating (TIL) and tumor‐ascites lymphocytes (TAL) were ex vivo isolated out of tumor tissue and ascites, respectively, from high‐grade ovarian carcinoma patients (FIGO‐stage IIIa‐IV). We observed an increased γδ T cell percentage in ascites compared to tumor‐tissue and blood of these patients, whereas CD8+ αβ T cells were increased within TAL and TIL. The number of Vδ1 and non‐Vδ1/Vδ2‐expressing γδ T cells was increased in the ascites and in the tumor tissue compared to the blood of the same donors. Commonly in PBL, the Vγ9 chain of the γδ T cell receptor is usually associated exclusively with the Vδ2 chain. Interestingly, we detected Vδ1 and non‐Vδ1/Vδ2 T cells co‐expressing Vγ9, which is so far not described for TAL and TIL. Importantly, our data demonstrated an expression of human epidermal growth factor receptor (HER)‐2 on high‐grade ovarian tumors, which can serve as an efficient tumor antigen to target CD3 TIL or selectively Vγ9‐expressing γδ T cells by bispecific antibodies (bsAbs) to ovarian cancer cells. Our bsAbs efficiently enhance cytotoxicity of TIL and TAL against autologous HER‐2‐expressing ovarian cells., Tumor‐infiltrating γδ T cells are attractive effector cells for targeting with bispecific antibodies in the treatment of ovarian cancer cells.

Introduction Mammography is the gold standard for early breast cancer detection, but shows important limitations. Blood-based approaches on basis of cell-free DNA (cfDNA) provide minimally invasive screening tools to characterize epigenetic alterations of tumor suppressor genes and could serve as a liquid biopsy, complementing mammography. Methods Potential biomarkers were identified from The Cancer Genome Atlas (TCGA), using HumanMethylation450-BeadChip data. Promoter methylation status was evaluated quantitatively by pyrosequencing in a serum test cohort (n = 103), a serum validation cohort (n = 368) and a plasma cohort (n = 125). Results SPAG6, NKX2-6 and PER1 were identified as novel biomarker candidates. ITIH5 was included on basis of our previous work. In the serum test cohort, a panel of SPAG6 and ITIH5 showed 63% sensitivity for DCIS and 51% sensitivity for early invasive tumor (pT1, pN0) detection at 80% specificity. The serum validation cohort revealed 50% sensitivity for DCIS detection on basis of NKX2-6 and ITIH5. Furthermore, an inverse correlation between methylation frequency and cfDNA concentration was uncovered. Therefore, markers were tested in a plasma cohort, achieving a 64% sensitivity for breast cancer detection using SPAG6, PER1 and ITIH5. Conclusions Although liquid biopsy remains challenging, a combination of SPAG6, NKX2-6, ITIH5 and PER1 (SNiPER) provides a promising tool for blood-based breast cancer detection.

Simple Summary Ovarian cancer (OvCa) treatment is still a challenge, mainly due to acquired resistance mechanisms during the course of chemotherapy. Here, we show the enhanced cytotoxicity of the combined treatment with the ADAM17 inhibitor GW280264X and cisplatin in comparison with cisplatin monotherapy. This effect was visible in five of five ovarian cancer cell lines grown as a monolayer and two of three tested cell lines in three-dimensional tumor spheroids. Tumor spheroids derived from primary tumor and ascites cells were sensitized to cisplatin treatment by GW280264X. In summary, the combination of ADAM17 inhibition with conventional chemotherapy seems to be a promising strategy to overcome chemotherapy resistance in OvCa. Abstract Chemotherapy resistance is a major challenge in ovarian cancer (OvCa). Thus, novel treatment combinations are highly warranted. However, many promising drug candidates tested in two-dimensional (2D) cell culture have not proved successful in the clinic. For this reason, we analyzed our drug combination not only in monolayers but also in three-dimensional (3D) tumor spheroids. One potential therapeutic target for OvCa is A disintegrin and metalloprotease 17 (ADAM17). ADAM17 can be activated by chemotherapeutics, which leads to enhanced tumor growth due to concomitant substrate cleavage. Therefore, blocking ADAM17 during chemotherapy may overcome resistance. Here, we tested the effect of the ADAM17 inhibitor GW280264X in combination with cisplatin on ovarian cancer cells in 2D and 3D. In 2D, the effect on five cell lines was analyzed with two readouts. Three of these cell lines formed dense aggregates or spheroids (HEY, SKOV-3, and OVCAR-8) in 3D and the treatment effect was analyzed with a multicontent readout (cytotoxicity, viability, and caspase3/7 activation). We tested the combined therapy on tumor spheroids derived from primary patient cells. In 2D, we found a significant reduction in the half minimal (50%) inhibitory concentration (IC50) value of the combined treatment (GW280264X plus cisplatin) in comparison with cisplatin monotherapy in all five cell lines with both 2D readout assays (viability and caspase activation). In contrast, the combined treatment only showed an IC50 reduction in HEY and OVCAR-8 3D tumor spheroid models using caspase3/7 activity or CelltoxTM Green as the readout. Finally, we found an improved effect of GW280264X with cisplatin in tumor spheroids derived from patient samples. In summary, we demonstrate that ADAM17 inhibition is a promising treatment strategy in ovarian cancer.

Purpose: Seventy percent of all ovarian cancer (OvCa) diseases are diagnosed at an advanced stage due to poor screening methods. In general, the therapy consists of cyto-reductive surgery followed by first line platinum based chemo therapy, which frequently leads to a long-term response. A resistance against platinum therapy was detected in some cases by refractory recurrence within six months after the last donation. Identifying a marker predicting platinum effectiveness could be of great benefit for affected patients.Patients and methods: In this study we propose cyto-genetic procedures identifying markers of platinum resistance. Fresh tumor material was obtained from nine OvCa patients. After the last platinum-based chemotherapy, five patients had refractory recurrence within six month. DNA was isolated from tumor tissues for array-based comparative genomic hybridization (aCGH) analysis. Ten cultured metaphases per case were analyzed by spectral karyotyping (SKY) to detect repeated chromosome rearrangements. The obtained genomic data were correlated to the patients’ clinical follow-up data.Results: Eight chromosome aberrations correlated with recurrence later than six months were detected in tumors by SKY. Further seven chromosome aberrations correlated with a refractory recurrence. Non refractory tumors more frequently showed losses in 19q13.31-q13.42 by aCGH. Tumors with refractory recurrence more frequently showed gains in 17q21.32-17q24.3 and losses in 14q11.2, 9p22-p21.1, and 1p22.3-p22.1. OvCa showed separating cyto-genetic changes differentiated in platinum-refractory and non-refractory cases.Conclusion: Our results suggest that aCGH is the most suitable strategy for this, due to a significantly higher resolution. The loss in 19q13 correlated with better prognosis in OvCa

// Nina Hedemann 1 , Christoph Rogmans 1 , Susanne Sebens 2 , Daniela Wesch 3 , Manuel Reichert 4 , Dirk Schmidt-Arras 4 , Hans-Heinrich Oberg 3 , Ulrich Pecks 1 , Marion van Mackelenbergh 1 , Jorg Weimer 1 , Norbert Arnold 1 , Nicolai Maass 1 and Dirk O. Bauerschlag 1 1 Department of Gynecology and Obstetrics, Christian-Albrechts-University Kiel and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany 2 Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany 3 Institute of Immunology, Christian-Albrechts-University and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany 4 Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany Correspondence to: Nina Hedemann, email: Nina.Hedemann@uksh.de Dirk O. Bauerschlag, email: Dirk.Bauerschlag@uksh.de Keywords: ovarian cancer; chemo resistance; ADAM17; amphiregulin; tumor therapy Received: April 04, 2017 Accepted: February 28, 2018 Published: March 23, 2018 ABSTRACT Chemotherapeutic resistance evolves in about 70 % of ovarian cancer patients and is a major cause of death in this tumor entity. Novel approaches to overcome these therapeutic limitations are therefore highly warranted. A disintegrin and metalloprotease 17 (ADAM17) is highly expressed in ovarian cancer and required for releasing epidermal growth factor receptor (EGFR) ligands like amphiregulin (AREG). This factor has recently been detected in ascites of advanced stage ovarian cancer patients. However, it is not well understood, whether and how ADAM17 might contribute to chemo resistance of ovarian cancer. In this study, we identified ADAM17 as an essential upstream regulator of AREG release under chemotherapeutic treatment in ovarian cancer cell lines and patient derived cells. In the majority of ovarian cancer cells cisplatin treatment resulted in enhanced ADAM17 activity, as shown by an increased shedding of AREG. Moreover, both mRNA and the protein content of AREG were dose-dependently increased by cisplatin exposure. Consequently, cisplatin strongly induced phosphorylation of ADAM17-downstream mediators, the EGFR and extracellular signal-regulated kinases (ERK). Phorbol 12-myristate 13-acetate (PMA), similarly to cisplatin, mediated AREG shedding and membrane fading of surface ADAM17. Inhibition of ADAM17 with either GW280264X or the anti-ADAM17 antibody D1 (A12) as well as silencing of ADAM17 by siRNA selectively reduced AREG release. Thus, ADAM17 inhibition sensitized cancer cells to cisplatin-induced apoptosis, and significantly reduced cell viability. Based on these findings, we propose that targeting of ADAM17 in parallel to chemotherapeutic treatment suppresses survival pathways and potentially diminish evolving secondary chemo resistance mechanisms.

Endometrial cancer (EC) therapy is characterized by the heterogeneity of EC subtypes resulting in unclear clinical behavior as well as in unsatisfactory treatment options. The available biomarkers, such as cellular tumor antigen p53 (TP53), phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN), and phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) genes alone might not be sufficient, and thus, new predictive and prognostic biomarkers are urgently required. The biomolecule class of microRNA represents a group of endogenously expressed regulatory factors primarily involved in control of pivotal cancer-related mechanisms including cell cycle, proliferation, apoptosis, and metastasis. Here, we review the current state of science regarding microRNA functionality in EC progression.

Mammography is the gold standard for early breast cancer detection, but shows important limitations. Blood-based approaches on basis of cell-free DNA (cfDNA) provide minimally invasive screening tools to characterize epigenetic alterations of tumor suppressor genes and could serve as a liquid biopsy, complementing mammography.Potential biomarkers were identified from The Cancer Genome Atlas (TCGA), using HumanMethylation450-BeadChip data. Promoter methylation status was evaluated quantitatively by pyrosequencing in a serum test cohort (Although liquid biopsy remains challenging, a combination of

The significance of human biorepositories for modern medical research, particularly for comprehensive population-based genetic analyses, is constantly growing. While large and centralized institutions are usually considered best suited to meet the increasing demand for high-quality “biobanks,” most medical research institutions still host rather heterogeneous and fragmented biobanking activities, undertaken by clinical departments with oftentimes rather different scientific scope. Undoubtedly, most clinicians and medical researchers would appreciate infrastructural support in terms of the storage and handling of their biosamples, but they are also likely to expect access to their samples avoiding extensive formal requirements. We report on the establishment of the PopGen 2.0 Network (P2N), an overarching alliance of initially seven biobanks from Northern Germany which adopted a joint but lean governance structure and use-and-access policy for their samples and data. In addition, the members of P2N have pursued an intense collaboration on ethical, legal and social issues and maintain a common IT infrastructure. The implementation of P2N has substantially improved the prospects of biobank-based research at the participating institutions. The network may thus serve as a role model for similar initiatives geared at linking pre-existing biorepositories for the benefit of research quality, efficiency, and transparency.

Aberrations of chromosome arm 19p in ovarian cancer were first described decades ago and have been confirmed in recent publications, which have focused on chromosome 11 as a translocation partner. Recently, genetic analysis of the ovarian cancer cell line SKOV3 revealed a rearrangement described as der(19)t(11;19)(q13.2;p13.2), which lead to a fusion protein containing parts of HOOK2 and frame shifted ACTN3 that had unknown functionality. To evaluate the frequency of these breakpoints, we used fluorescence in situ hybridization (FISH) probes flanking these genes for interphase analysis of ovarian cancer cells. We analyzed 49 primary cell cultures of ovarian cancers using FISH probes next to these breakpoints on chromosomes 11 and 19 defined in SKOV3. Co-localizations of the signals in interphase nuclei were considered to be positive fusions when the frequency was over the experimentally calculated cutoff of 24.3% (mean average value for normal ovary cells plus three times the standard deviation). Fusions between 11q13.2 and 19p13.2 were confirmed in 22 (45%) primary cell cultures of ovarian cancers. However, by PCR, the fusion originally described in SKOV3 was not detected in any of the primary cell cultures. Our results confirm other reports and show that these regions are very frequently involved in chromosomal rearrangements in ovarian cancer. Furthermore, they reveal a significant correlation (P = 0.023) of co-localized signals of 11q13.2 and 19p13.2 with low and intermediate grades in ovarian cancer.

For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4–13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern “1, 2, 3 and fine” in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.

Preimplantation genetic screening wants to improve artificial reproductive technologies, primarily by raising the rates of pregnancy, implantation and birth. We investigated if embryos derived from oocytes detected euploid for five chromosomes implant better than those which were biopsied but where the genetic detection failed. They were nevertheless transferred, thus serving as a sham control. From 2004 to 2008 we performed 104 cycles of PGS with laser biopsy of the first polar body and FISH with five chromosomes. It was offered to all patients with eight or more oocytes, free of charge. The average female age was 36 years. If no euploid oocytes were available, not detected oocytes were transferred. In 104 cycles 99 embryo transfers (95 %) were performed, resulting in 28 pregnancies (27 %), 20 births (71 %) and 8 miscarriages (29 %). The implantation rate in the euploid group was 19 vs. 13 % in the not detected group (n.s.). This trend was the same independent of age and embryo morphology. The pregnancy rate does not differ significantly from the national average. The trend in better implantation rates of euploid oocytes justifies a continuation of studies in this matter.

The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum. We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells. We could show that the combination of both medicines in the preclinical experiment produces a working advantage.

Zielsetzung: Das Ovarialkarzinom besitzt die hochste Letalitat unter den gynakologischen Tumoren. Ursachlich hierfur sind insbesondere die spate Diagnose sowie das ausgepragte Auftreten von Chemotherapeutika-Resistenzen. Um die Fruherkennung sowie Therapiepradiktion zu verbessern, ist es daher unabdingbar zuverlassige Marker zu evaluieren. Ein aussichtsreicher Marker konnte die Metalloprotease ADAM17 darstellen. Diese wird in vielen soliden Tumoren, unter anderem dem Ovarialkarzinom und dem Mammakarzinom erhoht exprimiert. Durch Freisetzung zahlreicher Wachstumsfaktoren und der engen Verknupfung mit dem EGFR-Signaling beeinflusst ADAM17 die Tumorinitiation sowie Metastasierungsprozesse. Ziel dieser Arbeit ist es, ADAM17 in Seren von Ovarialkarzinompatientinnen zu detektieren und mit klinischen Parametern zu korrelieren. Material und Methoden: In Seren von 60 Ovarialkarzinomfallen mit je durchschnittlich 4 Abnahmepunkten im Krankheitsverlauf soll mittels spezifischem ELISA, der ADAM17-Gehalt ermittelt werden. Verglichen wird die ADAM17-Expression mit Seren von gesunden Probanden. Gepruft wird, ob eine Korrelation der bereits erhobenen klinischen Parameter, wie Tumorstadium, Tumorrest nach Initial-OP und Verlauf der Erkrankung unter besonderer Berucksichtigung der moglichen Resistenz gegenuber der Chemotherapie besteht. Ergebnisse: ADAM17 konnte mittels ELISA in Zelllysaten von Krebszellen detektiert werden. Initiale Experimente zur Detektion von ADAM17 in Patientenserum zeigten eine durchschnittlich dreifach erhohte Expression von ADAM17 im Vergleich zu Kontrollseren gesunder Spender. In einem Testkollektiv an Seren von Ovarialkarzinomen wurde eine differentielle Expression von ADAM17 festgestellt. Eine Korrelation der Ergebnisse mit den klinischen Parametern ist in Arbeit. Zusammenfassung: In dieser Studie wird die Metalloprotease ADAM17, die mit ursachlich fur die Freisetzung tumorformierender Faktoren ist, mittels ELISA in Seren von Ovarialkarzinompatientinnen nachgewiesen und mit klinischen Daten korreliert.

The dysregulation of cell cycle kinases plays a crucial role in carcinogenesis and the expression of various kinases has been attributed to aggressive tumor growth and an unfavourable prognosis in oncological patients. We, therefore, aimed to evaluate the expression of Ki67 among five additional cell cycle kinases in a collective of mammary and ovarian tumor specimens and to find a correlation with clinicopathological parameters. 76 mammary and 93 ovarian benign and malignant tumor samples were immunohistochemically stained and evaluated for the expression of Aurora A and B, Repp86, CDK1 and 2 (only breast specimens) and Ki67. The expression patterns of these cell cycle kinases were matched with retrospectively collected clinicopathological parameters. All examined cell cycle kinases accurately discriminated benign from malignant breast and ovarian tissues. In breast cancer, Aurora A and B-, Repp86-, CDK2- and Ki67-expression was inversely associated with ER expression. No correlation with the HER2-status was found in our collective. Importantly, we found a significant correlation between the expression of Aurora A and CDK1 and axillary lymph node metastasis in breast cancer. Furthermore, a shortened disease free survival (DFS) upon expression of Aurora B and CDK2 was shown in breast cancer patients. None of the cell cycle kinases was associated with predictive or prognostic factors in epithelial ovarian cancer. The prognostic value of the expression of Ki67 is overtrumped by alternative cell cycle kinases when it comes to prediction of axillary tumor spread and a shortened DFS, which might allow a further risk stratification in breast cancer patients.

Alterations of chromosome 19 are among the most frequent cytogenetic changes in ovarian carcinomas. They usually occur as added extra material of unknown origin to 19p or, less frequently, 19q but sometimes as homogeneously staining regions. The precise nature of these markers, i.e., exactly which regions of chromosome 19 are involved and from which chromosome(s) the additional material comes, could only rarely be established. We have investigated by high resolution array-CGH a series of 29 chromosome 19 markers after previous microdissection of ovarian carcinoma metaphases followed by FISH to determine where in chromosome 19 the rearrangements took place as well as from which partner chromosomes the additional material stems, obtaining informative results on 23 markers from 18 carcinomas. Along the entire chromosome 19, a total of nine regions were found gained in 10 or more carcinomas (from 10 to 16) whereas 15 regions were gained in 6 to 10 markers. The most commonly gained region (16 markers) was observed in 19p13 between 20.80 Mbp and 20.85 Mbp from 19pter. According to the human genome 18 (hg18) NCBI 36, a total of 43 genes reside in the most commonly gained regions. Most of them (n = 31) code for zinc finger proteins. None of these genes is known to be involved in human neoplasia (the only exception is the ZNF91, which is found highly expressed in seminomas) but their frequent gain in the examined tumors makes all of them candidates for a pathogenetic role in ovarian carcinogenesis.

Alterations of chromosome bands 19p13 and 19q13 in the form of added extra material of unknown origin are among the most frequent cytogenetic changes in ovarian carcinomas. To investigate the chromosomal composition of the 19p+ and/or 19q+ markers, we selected for examination 26 ovarian carcinomas which by G-banding had one to four 19p+ and/or 19q+, in total 37 markers. These cases were then subjected to chromosomal microdissection with subsequent reverse painting, which gave informative results on 29 markers. The breakpoints on chromosome 19 were located in both the short (p; n = 15) and the long (q; n = 10) arms, as well as in the centromeric (n = 2) and pericentromeric (n = 6) region. The analysis showed that many chromosomes added material to chromosome 19, but the chromosome arms 11q, 21q, and 22q were particularly common donors. Homogeneously staining regions (hsr) were seen in only three markers, in all of them consisting of 19p material. Eighteen markers were derived from an unbalanced translocation involving chromosome 19. In five markers, chromosome 19 was rearranged with two chromosomes. The most complex marker showed chromosome 19 rearranged with three other chromosomes, i.e., X, 13, and 16. In five markers, all of the additional material stemmed from chromosome 19 itself. This is the first large chromosome microdissection/FISH study of chromosome 19 markers in ovarian carcinomas. A detailed map of the rearrangements should provide clues to the positions of oncogenes and potential fusion genes important in ovarian carcinogenesis.

Cell authentication is a necessary procedure to avoid scientific data from cell culture experiments with cross-contamination or false classification. A genetic fingerprint pattern of a specimen by short tandem repeats (STR) is self-evident. Due to high amount of chromosomal rearrangements, known in epithelia ovary cancer cells and the instable STR pattern described in other tumour entities like leukaemia, this study explores the suitability of STR profiling for primary cultured epithelial ovary cancer cells. STR profiles of epithelial ovary cancers of 16 patients were compared with corresponding blood and corresponding primary cell cultures. The primary cell cultures of epithelial ovary tumours were passaged up to 28 times. In between, cultures were cryo conserved and recultured again, two to five times per patient. In two cases, the STR pattern of tumour lost alleles (1/16 and 3/16) in comparison of corresponding STR-pattern from blood. In comparison to blood, cell culture of a third case, lost four alleles (4/16) accompanied with morphologic changes after 14th passage. It is equal after cryo conservation of the seventh passage from the same patient. The only changes in STR profiles we recognized are losses of alleles. Remaining STR markers allow authentication. Very likely, the allelic drop-outs beyond passage 14 assume complex genetic losses of heterozygosis resulting in changed growth behaviour of cells. All other STR-profiles of remaining 15 patients analysed in this study are stable over all passages and freeze–thaw processes. Thus, ovary cancer cell cultures in research should be authenticated by STR-profile in general.

We report on a family in which a daughter is described with mental retardation, as well as malformations of the heart, and of the brain (Dandy-Walker variant). The patient’s phenotype suggests a chromosomal rearrangement. However, her karyotype was unremarkable by conventional cytogenetic analysis. In order to detect chromosome rearrangements overseen by this method, the subtelomere regions of suspicious chromosomes were verified by fluorescence in situ hybridization (FISH). A rearranged derivative chromosome 6 was identified. Further examinations by FISH-microdissection (FISH-MD) revealed a maternal complex balanced translocation. The patient inherited the derivative chromosome 6 from her mother and therefore carries a partial monosomy 6q26→qter and a partial trisomy 11q23.3→qter.

A boy with signs of Klinefelter syndrome, mild facial dysmorphic features, and severely retarded speech development displayed a female karyotype with mosaicism for two marker chromosomes 48,XX,+mar1,+mar2[68]/47,XX,+mar1[19]/47,XX,+mar2[6]/46,XX[8]. Using chromosomal microdissection, locus-specific fluorescence in situ hybridization (FISH), and PCR with several Y-chromosome markers, the larger supernumerary marker chromosome (SMC) was characterized as a ring Y-chromosome. Detection of the SRY-region explained the male phenotype. The smaller second marker chromosome contained the pericentromeric region of chromosome 8. We suggest that the co-occurrence of a partial Y-chromosome and partial trisomy 8 explain the severe speech delay and the facial dysmorphic features.

Several investigations have supposed that tumor suppressor genes might be located on human chromosome 8. We used microcell-mediated transfer of chromosome 8 into MDA-MB-231 breast cancer cells and generated independent hybrids with strongly reduced tumorigenic potential. Loss of the transferred chromosome results in reappearance of the malignant phenotype. Expression analysis identified a set of 109 genes (CT8-ps) differentially expressed in microcell hybrids as compared to the tumorigenic MDA-MB-231 and rerevertant cells. Of these, 44.9% are differentially expressed in human breast tumors. The expression pattern of CT8-ps was associated with prognostic factors such as tumor size and grading as well as loss of heterozygosity at the short arm of chromosome 8. We identified CT8-ps networks suggesting that these genes act cooperatively to cause reversion of tumorigenicity in MDA-MB-231 cells. Our findings provide a conceptual basis and experimental system to identify and evaluate genes and gene networks involved in the development and/or progression of breast cancer.

Um Verstandnis fur jene Vorgange zu erlangen, die eine normale, epitheliale Korperzelle, uber das Stadium verschiedener adenomatoser Formen, zur Karzinomzelle entarten lasst, ist es notwendig, das Augenmerk auf veranderte Genstrukturen in solchen Zellen zu richten. Hinweise auf die in diesem Entartungsprozess involvierten Gene lassen sich durch verschiedene zytogenetische Analysetechniken ermitteln. Diese Techniken unterscheiden sich in ihrer Leistungsfahigkeit und mussen je nach Fragestellung einzeln oder in Kombination eingesetzt werden. Dieser Ubersichtsartikel soll dem onkologisch interessierten Kliniker einen Uberblick in die Moglichkeiten der molekularen Zytogenetik verschaffen. An understanding of the processes by which a normal epithelial cell is transformed via adenomatosis to a cancer cell requires examination of altered gene structures in these cells. Several cytogenetic techniques provide insights into affected genes. Cytogenetic techniques differ in their capability and can be used alone or in combination. This article reviews for clinicians the possibilities afforded by cytogenetic techniques.

Comparative genomic hybridization (CGH) is a valuable technique for cytogenetic analysis of solid tumors. To evaluate the reliability of CGH, we examined DNA of 10 ovarian carcinomas after CGH analysis with single- and double-locus fluorescence in situ hybridization (FISH). The FISH experiments, involving 5 chromosomes (chromosomes 3, 6, 8, 12, and 18) with different FISH probes, confirmed the CGH results in 66.2% of cases (92 of 139 investigated loci). In 4 patients, inconsistent results (41 loci) were related to polyploidy, because CGH cannot detect polyploid karyotypes. The remaining 6 discordant loci can be referred to limitations in both techniques. Re-evaluation of FISH and CGH results by one other is therefore recommended to overcome these technical artifacts. Nevertheless, CGH is of potential value in characterizing chromosomal alterations and might help in generating tumor-specific sets of FISH probes to obtain genetic information of prognostic value within a few days.

We report on a male patient and members of his family with additional material in chromosome 3. This derivative chromosome 3 was transmitted from his mother who had a complex rearrangement between chromosomes 2, 3, and 7. It was possible to delineate her chromosomal rearrangement by microdissection and reverse painting and to exclude these aberrations from being responsible for neonatal deaths and several abortions in this family. Two members of this family suffer from ectrodactyly or split hand/foot malformations (SHFM) of the feet which possibly correlates with the derivative chromosome 7 containing a breakpoint in the SHFM1 critical region involving several homeobox genes. Keywords: microdissection; CGH; SHFM; ectrodactyly

Microdissection in combination with reverse painting fluorescence in-situ hybridization (FISH) is a very effective method to identify breakpoints and rearrangements of derived chromosomes and reveal the chromosomal origin of marker chromosomes. We describe an innovation that allows a convenient, fast and safe isolation of microdissected fragments as currently available protocols. The microdissected chromosomes are harvested in a collection drop located in a movable micropipette adjusted to a second micromanipulator under microscopic observation. We used this technique to analyze several cytogenetic aberrations. In order to evaluate the efficiency of our microdissection procedure, we compared the results obtained with microdissection probes made from only one fragment with those obtained with more than six microdissected fragments. In all cases, the single- fragment microdissections were sufficient to provide probes.

Two strains of the potato pathogen Gibberella pulicaris were analysed for the ability to metabolize the phytoalexin rishitin and for virulence on potato tubers. While strain R-6380 was always virulent and able to metabolize the phytoalexin, strain R-7843 showed significant variation in both traits. Analysis of eight sectors of a radially grown culture of this strain revealed that six had a virulent, rishitin-metabolizing phenotype, one had an avirulent, rishitin-nonmetabolizing phenotype, and one had an intermediate phenotype for both traits. Similar results were found for single spores isolated from one of two spore suspensions from different agar cultures. In all cases high virulence was associated with fast metabolism of rishitin; weakly virulent strains only slowly degraded the phytoalexin. Restriction-fragment-length-polymorphism (RFLP) analysis of sectors with contrasting phenotypes and measurement of the nuclear DNA content from these sectors indicate that the sectors were identical to the original strain (R-7843) with respect to these traits. It is therefore concluded that the variations observed in virulence and in the ability to metabolize rishitin are the result of spontaneous genetic changes in strain R-7843, The concurrent changes in tolerance, metabolism and virulence are further indicators of a relationship among these traits and lend support to the hypothesis that phytoalexin metabolism is important in pathogenicity. Zusammenfassung Zwei Stamme des Kartoffelpathogens Gibberella pulicaris wurden auf ihre Fahigkeit zum Abbau des Phytoalexins Rishitin und auf Vinilenz gegenuber Kartoffelknollen untersucht, Wahrend der Stamm R-6380 in alien Experimenten viruletit und in der Lage war, das Phytoalexin abzubauen, zeigte der Stamm R-7843 eine deutliche Variabilitat dieser beiden Merkmale, Die Analyse von acht Sektoren einer radial gewachsenen Kultur dieses Stammes ergab, das sechs einen virulenten, Rishitin metabolisierenden, einer einen avirulenten und Rishitin nicht-abbauenden und der achte Sektor einen intermediaren Phanotyp beider Merkmale aufwiesen, Ahnliche Ergebnisse resultierten aus der Analyse einer von zwei Sporensuspensionen verschiedener Agarkulturen. In alien Fallen war hohe Virulenz mit schnellem Abbau von Rishitin korreliert und schw ache Virulenz mit langsamem Abbau des Phytoalexins. RFLP Analysen und die Messung des nuklearen DNA-Gehaltes von Sektoren mit entgegengesetztem Phanotyp zeigten, das sich diese Sektoren in den untersuchten Merkmalen nicht von dem Ausgangsstamm (R-7843) unterschieden, Daraus ergibt sich, das die beobachtete Variabilitat der Virulenz und die Fahigkeit zum Abbau von Rishitin das Resultat spontaner genetischer Veranderungen des Stammes R-7843 sind. Die gemeinsame Veranderung von Toleranz, Rishitin- Metabolismus und Virulenz ist ein weiteres Indiz fur eine kausale Beziehung zwischen diesen Merkmalen und unterstutzt die Hypothese, das der Abbau von Phytoalexinen fur Pathogenitat von Bedeutung ist.

Aberrations of chromosome arm 19p in ovarian cancer were first described decades ago and have been confirmed in recent publications, which have focused on chromosome 11 as a translocation partner. Recently, genetic analysis of the ovarian cancer cell line SKOV3 revealed a rearrangement described as der(19)t(11;19)(q13.2;p13.2), which lead to a fusion protein containing parts of HOOK2 and frame shifted ACTN3 that had unknown functionality. To evaluate the frequency of these breakpoints, we used fluorescence in situ hybridization (FISH) probes flanking these genes for interphase analysis of ovarian cancer cells. We analyzed 49 primary cell cultures of ovarian cancers using FISH probes next to these breakpoints on chromosomes 11 and 19 defined in SKOV3. Co-localizations of the signals in interphase nuclei were considered to be positive fusions when the frequency was over the experimentally calculated cutoff of 24.3% (mean average value for normal ovary cells plus three times the standard deviation). Fusions between 11q13.2 and 19p13.2 were confirmed in 22 (45%) primary cell cultures of ovarian cancers. However, by PCR, the fusion originally described in SKOV3 was not detected in any of the primary cell cultures. Our results confirm other reports and show that these regions are very frequently involved in chromosomal rearrangements in ovarian cancer. Furthermore, they reveal a significant correlation (P = 0.023) of co-localized signals of 11q13.2 and 19p13.2 with low and intermediate grades in ovarian cancer.

Background Genotype-phenotype correlations for chromosomal imbalances are often limited by overlapping effects of partial trisomy and monosomy resulting from unbalanced translocations and by poor resolution of banding analysis for breakpoint designation. Here we report the clinical features of isolated partial trisomy 7q21.2 to 7q31.31 without overlapping phenotypic effects of partial monosomy in an 8 years old girl. The breakpoints of the unbalanced rearranged chromosome 7 could be defined precisely by array-CGH and a further imbalance could be excluded. The breakpoints of the balanced rearranged chromosomes 9 and 10 were identified by microdissection of fluorescence labelled derivative chromosomes 9 and 10. Results The proband's mother showed a complex balanced translocation t(9;10)(p13;q23) with insertion of 7q21.2-31.31 at the translocation breakpoint at 9p13. The daughter inherited the rearranged chromosomes 9 and 10 but the normal chromosome 7 from her mother, resulting in partial trisomy 7q21.2 to 7q31.31. The phenotype of the patient consisted of marked developmental retardation, facial dysmorphism, short stature, strabism, and hyperextensible metacarpophalangeal joints. Discussion For better understanding of genotype-phenotype correlation a new classification of 7q duplications which will be based on findings of molecular karyotyping is needed. Therefore, the description of well-defined patients is valuable. This case shows that FISH-microdissection is of great benefit for precise breakpoint designation in balanced rearrangements.

The mouse strain CBA/CaH-T(14;15)6Ca/J carries a homozygous balanced reciprocal translocation between mouse chromosomes 14 and 15, but the break points of this translocation have not previously been examined in detail. Using fluorescent in situ hybridization, we assigned the break point in 14qE3 to a 200-kb region devoid of any known gene. We similarly defined the break point in 15qA1 to a 27-kb region containing involving ADAMTS12. The chromosomal break likely is between exons 2 and 3 of ADAMTS12. This gene encodes a disintegrin and metalloproteinase with thrombospondin motifs, and this product plays crucial roles in both vascularization and cancer progression and has been implicated in the development of arthritis. The CBA/CaH-T(14;15)6Ca/J mouse strain likely is a suitable model for further examination of the influences of defective ADAMTS12 in various pathologic processes.

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor suppressor gene(s) involved in breast cancer. We have shown previously that microcell-mediated transfer of human chromosome 8 into the breast cancer cell line MDA-MB-231 results in reversion of tumorigenicity of these cells and is accompanied by expression changes of a clinically relevant set of genes. In the present study we demonstrate that the transfer of human chromosome 8 into another breast cancer cell line, CAL51, results in hybrids characterized by suppression of tumorigenicity in vitro and in vivo as compared with the parental cells. Loss of the transferred chromosome 8 results in reappearance of the CAL51 phenotype. Oligonucleotide microarray analysis identified 78 probe sets differentially expressed in the hybrids as compared with CAL51 and the rerevertant cells. The majority of these genes is involved in signal transduction, developmental processes, angiogenesis, cadherin signaling, Wnt signaling or inflammation. It is of particular interest that the 78-gene signature is also reflected in a panel of breast tumors, lymph node and distant metastases, and is correlated with several prognostic markers including tumor size, grading, metastatic behavior and estrogen receptor status. As opposed to the corresponding non-tumorigenic phenotypes demonstrated for the MDA-MB-231-derived and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differ from each other. However, it was of special interest that the majority of genes of both gene sets could be integrated into a similar spectrum of biological processes and pathways. Our findings provide an experimental system to identify and evaluate genes but, more importantly, signatures of biological processes and pathways involved in the development and/or progression of breast cancer.

The technique of chromosome microdissection was initially developed for generating DNA libraries from specific parts of polytene chromosomes of Drosophila (Scalenghe 1981) in order to isolate certain genes known to be localized in those regions. First improvements of this original technique such as the use of GTG-banded chromosomes and the invention of the first sequence independent DNA amplification system (Ludecke 1989, Senger 1990, Ludecke 1990) allowed the generation of microdissection libraries from precisely defined regions of human chromosomes and a stepwise reduction of the number of chromosomes that had to be dissected for one library. By Trautmann and co-worker (1991) it was shown for the first time that microdissection libraries can be used as painting probes for in situ hybridization.

We examined 88 nonpapillary renal cell carcinomas for allelic loss at chromosome arm 14q and correlated the results to size, grade, and stage of these tumors. Fourteen highly polymorphic microsatellite markers on the long arm of chromosome 14 were used for deletion mapping. Loss of heterozygosity (LOH) at the smallest overlapping segment of 14q24.2-qter was seen in 42 of 88 tumors. There was no significant correlation between frequency of 14q LOH and size of tumors (P = 0.11). LOH was frequently seen in grade 2 and 3 tumors (55% and 73%, respectively) and in stage III and IV tumors (53% and 80%, respectively). We found a significant correlation between chromosome arm 14q LOH and nuclear grade (P < 0.001) and stage (P < 0.001) of tumors. These observations indicate the presence of a tumor-suppressor gene at chromosome segment 14q24.2-qter and demonstrate the usefulness of microsatellite analysis for assessing the possible clinical outcome of nonpapillary renal cell carcinomas. Genes Chromosom. Cancer 19:29–35, 1997. © 1997 Wiley-Liss, Inc.

Background Complex small supernumerary marker chromosomes (sSMC) constitute one of the smallest subgroups of sSMC in general. Complex sSMC consist of chromosomal material derived from more than one chromosome; the best known representative of this group is the derivative chromosome 22 {der(22)t(11;22)} or Emanuel syndrome. In 2008 we speculated that complex sSMC could be part of an underestimated entity. Results Here, the overall yet reported 412 complex sSMC are summarized. They constitute 8.4% of all yet in detail characterized sSMC cases. The majority of the complex sSMC is contributed by patients suffering from Emanuel syndrome (82%). Besides there are a der(22)t(8;22)(q24.1;q11.1) and a der(13)t(13;18)(q11;p11.21) or der(21)t(18;21)(p11.21;q11.1) = der(13 or 21)t(13 or 21;18) syndrome. The latter two represent another 2.6% and 2.2% of the complex sSMC-cases, respectively. The large majority of complex sSMC has a centric minute shape and derives from an acrocentric chromosome. Nonetheless, complex sSMC can involve material from each chromosomal origin. Most complex sSMC are inherited form a balanced translocation in one parent and are non-mosaic. Interestingly, there are hot spots for the chromosomal breakpoints involved. Conclusions Complex sSMC need to be considered in diagnostics, especially in non-mosaic, centric minute shaped sSMC. As yet three complex-sSMC-associated syndromes are identified. As recurrent breakpoints in the complex sSMC were characterized, it is to be expected that more syndromes are identified in this subgroup of sSMC. Overall, complex sSMC emphasize once more the importance of detailed cytogenetic analyses, especially in patients with idiopathic mental retardation.