Parental origin of de novo cytogenetically balanced reciprocal non-Robertsonian translocations.

De novo cytogenetically balanced reciprocal non-Robertsonian translocations are rare findings in clinical cytogenetics and might be associated with an abnormal phenotype. Knowledge of the parental origin and mechanisms of formation is still limited. By microdissection of the derivative chromosomes and their normal homologs from metaphases followed by microsatellite-mediated marker analysis we identified 7 cases of paternal and 3 cases of maternal origin in a cohort of 10 patients with de novo cytogenetically balanced reciprocal non-Robertsonian translocations. Neither in the maternal nor in the paternal group of our study parental age seems to be increased. Together with the data from the literature our results confirm that the majority of de novo cytogenetically balanced reciprocal translocations are of paternal origin, but the preponderance does not appear to be as distinct as previously thought and the paternal age does not seem to be necessarily a major contributing factor.

Clinical outcome of pretreated B-cell chronic lymphocytic leukemia following alemtuzumab therapy: a retrospective study on various cytogenetic risk categories.

BACKGROUND: Patients with B-cell chronic lymphocytic leukemia (CLL) with 17p deletion respond poorly to chemotherapy. This retrospective study evaluated the benefit of alemtuzumab monotherapy in unselected patients with advanced CLL in the various cytogenetic subgroups. PATIENTS AND METHODS: Data were collected from 105 consecutive, pretreated, cytogenetically defined patients who had received alemtuzumab. Response, progression-free survival (PFS), and overall survival (OS) were assessed. RESULTS: The hierarchic incidence of cytogenetic abnormalities was: 13q deletion (as sole abnormality), 18%; trisomy 12, 13%; 11q deletion, 19%; 17p deletion, 33%; and none of these, 16%. Overall response rate (ORR) was 43% in the total cohort and 49% in the subgroup of 17p-deleted patients (n = 35). From the start of alemtuzumab monotherapy, median PFS in the total cohort and in the subgroup of 17p-deleted patients was 7.0 and 7.1 months, respectively. Median OS in the total cohort and in 17p-deleted patients was 32.8 and 19.1 months, respectively. The poor-risk group of patients with CLL (i.e. fludarabine resistant, 17p deletion; n = 20) showed encouraging ORR, PFS, and OS (35%, 7.0 and 19.2 months, respectively). CONCLUSIONS: Alemtuzumab was effective in treating patients with CLL across the cytogenetic categories evaluated, but there were differences. In patients with CLL with 17p deletion quite favorable ORR, PFS, and OS were achieved.

Parental origin of apparently balanced de novo complex chromosomal rearrangements investigated by microdissection, whole genome amplification, and microsatellite-mediated haplotype analysis.

Complex chromosomal rearrangements (CCRs) are rare findings in clinical cytogenetics. As a result of the high risk of unbalanced segregation, familial cases are even rarer and maternal transmission occurs more frequently than paternal transmission. Analogous to Drosophila and mice, as well as to CCRs involving the Y chromosome or a clinically relevant associated deletion, a preferential origin in spermatogenesis has been assumed but not proven directly and systematically thus far. Here, we investigated three healthy adults, one healthy child, and one child with multiple congenital anomalies and various balanced de novo CCRs. The analyses were performed in each case on 10 copies of a derivative chromosome and their normal homologs by glass-needle microdissection, whole genome amplification (WGA), and microsatellite-mediated haplotype analysis. With respect to the number of chromosomes involved in each case and in all cases together, the number of chromosomal segments in each case and in all cases together, and the number of breakpoints in each case and in all cases together, the conformity for paternal origin of all derivative chromosomes and maternal origin of their normal homologs makes formation in paternal germline more likely than a postzygotic formation with an accidental uniformity. In conclusion, our results confirm the preferential formation of de novo balanced CCRs in the paternal germline.

Whole genome amplification from microdissected chromosomes.

Over the last years various whole genome amplification (WGA) methods have been established for genetic investigations from a limited number of cells or small quantities of DNA but not for molecular analysis of isolated chromosomes, which is important for the direct investigation of haplotypes or molecular rearrangements of derivative chromosomes in clinical cytogenetics and oncology. Here, the results of a pilot study in which the GenomePlex Single Cell Kit linker adapter PCR approach (Sigma-Aldrich, Vienna, Austria) was modified for WGA of glass needle based microdissected chromosomes are presented. Compared with two other WGA strategies (Improved-Primer Extension Preamplification PCR and Multiple Displacement Amplification) the GenomePlex Single Cell Kit shows a higher rate of successfully amplified markers, a lower WGA drop out rate and faster feasibility.

Nail patella syndrome in a cytogenetically balanced t(9;17)(q34.1;q25) carrier.

Nail patella syndrome (NPS) is an autosomal dominant disorder characterized by dysplasia of the nails and patella, decreased mobility of the elbow, iliac horns and in some cases nephropathy. Linkage studies have localized the NPS locus to chromosome 9q34 within a 1-2 cM interval between D9S60 and the adenylate kinase gene (AK1), but the gene has remained elusive. We have identified a balanced t(9;17)(q34.1;q25) associated with NPS. By using FISH with probes from 9q the breakpoint region was narrowed to a 17.0 cM interval between D9S262 and ABL, which includes the NPS critical region. The patient showed the typical clinical features of NPS such as hypoplastic, deep-set nails, a dislocated elbow, iliac horns, and a polygonal patella. This suggests that the translocation has resulted from a break within or near the NPS gene, causing defective expression. The translocation in our patient may aid in the identification of the NPS gene.

Molecular cytogenetic detection of 9q34 breakpoints associated with nail patella syndrome.

The nail patella syndrome (NPS1) is an autosomal dominant disorder characterised by dysplasia of the finger nails and skeletal abnormalities. NPS1 has been mapped to 9q34, to a 1 cM interval between D9S315 and the adenylate kinase gene (AK1). We have mapped the breakpoints within the candidate NPS1 region in two unrelated patients with balanced translocations. One patient [46,XY,t(1;9)(q32.1;q34)] was detected during a systematic survey of old cytogenetic files in Denmark and southern Sweden. The other patient [46,XY,t(9;17)(q34.1;q25)] was reported previously. D9S315 and AK1 were used to isolate YACs, from which endclones were used to isolate PACs. Two overlapping PAC clones span the 9q34 breakpoints in both patients, suggesting that NPS1 is caused by haploinsufficiency due to truncation or otherwise inactivation of a gene at or in the vicinity of the breakpoints.

Characterization of the human gene coding for the swelling-dependent chloride channel ICln at position 11q13.5-14.1 (CLNS1A) and further characterization of the chromosome 6 (CLNS1B) localization.

Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.

Localization of cathepsin B in two human lung cancer cell lines.

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.

Mental and psychomotoric retardation in two brothers with pure partial trisomy 7q32-q34 due to a maternal insertion (14;7).

We present two brothers with mental retardation, seizures disorder, generalized muscular hypertonia, kyphoscoliosis, minor anomalies and a prominent midface. GTG-banded chromosome analysis showed a derivative chromosome 14 without clues toward the origin of the rearrangement. Microdissection of the derivative chromosome 14 and subsequent reverse painting demonstrated partial trisomy 7q32-q34 as the unbalanced product of a maternal insertion (14;7). Thus, we identified two cases with pure trisomy 7q32-q34 that allowed further delineation of this aneusomy syndrome.

Characterisation of a collecting duct carcinoma by cytogenetic analysis and comparative genomic hybridisation.

Collecting duct carcinoma (CDC) is a rare variant of carcinoma of the kidney with aggressive behaviour. CDC arise from the epithelium of the ducts of Bellini in the distal nephron. A CDC in a 53-year old male patient is characterised both by cytogenetic analysis and comparative genomic hybridisation (CGH). The cytogenetic analysis shows a biclonal karyotype 47,XY,+3[2]/40-44,XY,-12[3], -22[4][cp5]/46,XY[42]. Loss of DNA sequences detected by CGH involves chromosome 1, 2, 9, 11 and 18. Gain of DNA sequences affect chromosome 16 and 20. The characterisation of a CDC in this study represents an additional contribution to the poorly explored CDC.

Hypermetaphase and interphase fluorescence in situ hybridisation for monitoring of remission status in Philadelphia chromosome positive chronic myeloid leukaemia.

Interferon (IFN) alone or in combination with cytostatic drugs, can induce major and durable cytogenetic remissions in chronic myelogenous leukaemia (CML) patients. Hypermetaphase (HMF) and interphase (IPF) fluorescence in situ hybridisation (FISH) have been described to be suitable for remission assessment. In the present study we applied HMF and IPF simultaneously to bone marrow (BM) probes from Ph-positive CML patients. As conventional cytogenetics (CC) is still deemed to be the for remission analysis we studied a group of patients analysed with this method as control. A mean of 50 metaphases was available for HMF analysis, whereas only an average of 18.7 metaphases could be analysed by CC. Remission assessment was frequently impossible by CC or HMF due to lack of metaphases, but always possible by applying IPF. Our results show that HMF should replace CC for routinely monitoring the remission status in Ph-positive CML patients and that in case of lack or insufficient number of metaphases in the majority of cases IPF is suitable for remission assessment.

Monitoring of remission status by fluorescence in situ hybridisation in chronic myeloid leukaemia patients treated with interferon-alpha.

Interferon-alpha (IFN-alpha) can be considered as treatment of choice for patients with chronic myeloid leukaemia (CML) in chronic phase. With this treatment major cytogenetic responses can be achieved in 30% to 50% of patients. Regular monitoring of cytogenetic response is essential for the therapeutic management of these patients. As conventional cytogenetics is not always successful, especially under IFN-alpha treatment, molecular cytogenetic methods have been established for the examination of interphase nuclei for the presence of the BCR-ABL fusion gene, the molecular counterpart of the Philadelphia chromosome. To demonstrate the value of these new methods we have analysed interphase nuclei from sequentially cultured bone marrow cells from 14 CML patients who were treated with IFN-alpha and whose bone marrow was investigated regularly during therapy. Dual-colour FISH with a breakpoint spanning BCR-YAC and a flanking cosmid from the ABL region was applied. When compared with conventional cytogenetics the results achieved by FISH were favourable. The most evident advantage of FISH analysis is that in case of failure of conventional cytogenetics a reliable determination of the remission status can be done. Together with other recent studies our results illustrate the advantages and limitations of the interphase FISH method for monitoring CML patients.

Combined study of prostatic carcinoma by classical cytogenetic analysis and comparative genomic hybridization.

Conventional cytogenetic analysis of prostatic carcinoma (PC) is characterized by inefficient growth of tumor cells during in vitro culture, leading to a lack of aberrant karyotypes in many of investigated tumors. In this study we have combined a modified short-term tissue culture method for conventional banding analysis and comparative genomic hybridization (CGH) to examine genetic changes in PC, and to evaluate the effect of the in vitro culture on chromosomal changes by comparing results of the two methods. Cytogenetic analysis was performed on 34 PCs using both, conventional and molecular methods. Tumor tissues were obtained predominantly from untreated primary tumors from 48 patients. For karyotyping all tumor samples were short-term cultured using a feeder layer technique. Additionally DNA from uncultured tumor material from 17 of those patients was isolated and screened for copy number changes using CGH. Conventional banding analysis: clonal aberrations were detected in 65% of the tumor samples. Most of the chromosomal findings were numerical changes, including loss of chromosomes Y (32%), 18, 19 and 21 (each 12%). Less frequent, trisomy of chromosome 7 and monosomy of chromosomes 9, 12 and 22 (each 9%) was found. Additionally an inversion of chromosome 9p and a deletion at chromosome 7q was found in two cases. In 35% no clonal aberrations could be detected. CGH: DNA copy number changes were detected in 65% of the analyzed tumors. Predominantly losses of DNA sequences were found. The most common losses were found at chromosome regions 13q21q33 (29%), 6q11q23 (24%), 16q, and 18 (each 18%), and the most common gains at 19 (18%). In six tumors no copy number changes were found. Both methods showed a similar aneuploidy rate, suggesting that the feeder layer technique is quite a suitable method for in vitro culture of PC cells. However, the two techniques produced substantially differing results for most of the tumor samples, and in some cases the discrepancies are quite striking. Therefore eventual culture effects need to be taken into account when comparing results from conventional cytogenetics and CGH. Some contrary findings from the two methods are discussed.

Unfavourable prognosis of patients with trisomy 18q21 detected by fluorescence in situ hybridisation in t(11;18) negative, surgically resected, gastrointestinal B cell lymphomas.

BACKGROUND: The most frequent cytogenetic alteration in gastrointestinal (GI) B cell lymphoma (BCL) is t(11;18)(q21;q21). GI B cell non-Hodgkin lymphomas lacking this translocation vary in their biology and clinical outcome. The t(11;18) negative subgroup shows increased numerical changes of chromosome 18, although its clinical relevance remains unknown. METHODS: Thirty surgically resected primary GI BCLs were examined-11 low grade marginal zone mucosa associated lymphoid tissue (MALT) lymphomas, four marginal zone lymphomas with diffuse large BCL (DLBCL), and 15 de novo DLBCLs. Chromosome 18 aberrations were examined using interphase fluorescence in situ hybridisation. Trisomy 18 was studied applying a centromere 18 probe and a dual colour probe for the MALT1 gene at 18q21. RESULTS: Using the MALT1 probe, only one marginal zone MALT lymphoma had a break apart pattern, indicating t(11;18) or variants. In the GI BCLs lacking MALT1 breaks, trisomy 18q21 was seen in seven patients (four with complete trisomy 18 and three with partial trisomy of 18q21). Trisomy 18q21 was found in two of 10 low grade MALT lymphomas and five of 19 GI BCLs with large cell component. Six of 17 patients with trisomy 18q21 presented with >/= stage II and one of 12 with stage I disease. Trisomy 18q21 was associated with significantly shorter disease specific survival in the whole group and GI BCLs with large cell component, but not in the low grade group. CONCLUSIONS: Trisomy 18q21, including MALT1, may be associated with advanced tumour stage and may be a predictor of poor outcome in surgically resected primary GI BCLs.

Karyotypic characterization of established cell lines derived from a squamous cell carcinoma and an adenocarcinoma of human lung cancers.

Two non-small cell carcinoma cell lines from the major histopathologic groups of human lung cancers have been karyotyped: HS-24 was established from a squamous cell carcinoma, and SB-3 was obtained from a metastasis of a poorly differentiated adenocarcinoma. Endoreduplication is characteristic for both cell lines. Subsequent loss of chromosomes led finally to hypotetraploid karyotypes with modal chromosome numbers of 66-68 and 70-72 for HS-24 and SB-3, respectively. The structural analysis was performed by G- and C-banding. Stable overrepresentation of chromosomes 7, 8, 12, and 16 was found. Both cell lines developed a characteristic set of disomic and stable markers. Chromosomes involved in markers were 1, 2, 5, 6, 10, 11, 16, and 17. Consistent numerical and structural normality for chromosomes 4, 18, and 21 was observed.

Cytogenetic characterization of 22 human renal cell tumors in relation to a histopathological classification.

In this study, cytogenetic and fluorescence in situ hybridization analyses were performed on 22 sporadic, unilateral primary renal cell tumors. The tumors were classified according to cell types, growth patterns, and grades of malignancy. A feeder layer technique was used for the cell culture of 13 clear-cell carcinomas, 4 chromophilic carcinomas, 3 chromophobe carcinomas, 1 oncocytoma, and 1 spindle-shaped pleomorphic carcinoma. Eighty-six percent (19/22) of renal tumors showed clonal abnormalities. The most frequent finding in the 15 male patients was loss of chromosome Y (9/15). In 3/15, it was the only observed aberration. The second most visible aberration was regional loss or entire loss of chromosome 9, which was detected in 36% (8/22) of the cases. Four cases showed loss of chromosome 9 and 4 cases a deletion of the short arm with breakpoints on 9p11 and 9p21. Loss of 3p material was observed in 32% (7/22) of the cases but only in 2/13 patients with clear-cell carcinoma. Gain of chromosome 12 or 12p was observed in 27% (6/22). In 23% (5/22) of the patients, gain of whole or partial chromosomes 2, 5, and 7 was found. Less-frequent findings were loss of chromosomes 8, 14, and 21; gain of chromosome 16; and structural abnormalities of chromosome 1 (each 18%; 4/22). Only some of the karyotypes described as typical for the various renal tumor types were confirmed. In contrast with previous reports, chromosome 3 and 9 aberrations did not allow differentiation between tumor types in our study.

Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors.

The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.

Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer.

Recently we showed an integral epidermal growth factor receptor (EGFR)-E2F3a signaling path, in which E2F3a was found to be essential in EGFR-mediated proliferation in ovarian cancer cells. The present work evaluates the clinical relevance of this novel axis and of E2F3a itself in a large set of 130 ovarian cancer specimens. For this purpose E2F3a and its counterpart, E2F3b, were measured by RT-PCR and activated EGFR was assessed by immunohistochemistry. When compared with healthy control tissue, both E2F3 isoforms were overexpressed in the cancers, but only E2F3a expression correlated with tumor stage (ρ=0.349, P=0.0001) and residual disease (ρ=0.254, P=0.004). Univariate survival analyses showed E2F3a and activated EGFR to be associated with poor PFS and OS. Furthermore, a strong, positive correlation between activated EGFR and E2F3a expression was shown (P=0.0001). We further identified two EGFR-independent mechanisms that regulate E2F3a expression, namely one, acting by promoter methylation of miR-34a, which by its physical interaction with E2F3a transcripts causes their degradation, and the second based on 6p22 gene locus amplification. MiRIDIAN-based knockdown and induction of miR-34a evidenced a direct regulatory link between miR-34a and E2F3a, and the tumor-suppressive character of miR-34a was documented by its association with improved survival. Although, 6p22 gene locus amplification was detected in a significant number of ovarian cancer specimens, 6p22 ploidy was not relevant in predicting survival. In Cox regression analysis, E2F3a, but not activated EGFR or miR-34a expression, retained independent prognostic significance (PFS: hazards ratio 3.785 (1.326-9.840), P=0.013; OS: hazards ratio 4.651 (1.189-15.572), P=0.013). These clinical findings highlight the relevance of E2F3a in the biology of ovarian cancer. Moreover, identification of EGFR-independent mechanisms in E2F3a control can be helpful in explaining the non-responsiveness of therapeutic EGFR targeting in ovarian cancer.