Compelling arguments, documentation, and published data have always been the basis of scholarly examination, discussion, and reasoning. Rhetorical catch phrases, biased ideologies, and anecdotal reports, when used to prove erroneous and often unrelated points, are creating condemnation and polarization instead of awareness on the important issues of research ethics boards. The statement that “they are in a client–provider business relationship” as expressed in the article by Lemmens and Elliott [1] implies a direct financial relationship between sponsors and those reviewing their research protocols. This is incorrect. Since we cannot speak on behalf of other for-profit Institutional Review Boards (IRBs), we will describe how our Canadian for-profit IRB functions. The owners of the company and those involved in the running of the business are not IRB members and have no voting privileges. IRB members are external consultants who are paid by the company providing the review services for the time spent in reviewing a protocol and providing an expert ruling regarding the ethics and science of a particular study. IRB members have no business interests in the company (e.g., partnership or profit-sharing) and no vested interest in the approval or non-approval of a specific study. It is worth noting that members of academic or governmental not-for-profit IRBs are often paid for the ethical review services they provide, in recognition of the time and skill required to carry out their duties. Respected, well-organized, and accredited IRBs (for-profit and not-for-profit) are not an enterprise of “five people”. This is merely the minimal regulatory requirement, which we consistently exceed in our meetings. IRBs are complex bodies of highly qualified professional experts from various disciplines, such as physicians, pharmacists, scientists, lawyers, philosophers, ethicists, and lay persons. They are multicultural, balanced in gender, diverse in social background, and dedicated to the protection of research subjects (which remains the main mandate for all IRBs). Often, for-profit IRB members are serving or have served as IRB members on academic or governmental not-for-profit IRBs. Is it expected that the work of these individuals is ethical when they are volunteers, and unethical when they are remunerated for their expertise? Rather than fixate on the concept that members of IRBs are paid, we should concentrate on the real challenges regarding IRBs (for-profit and not-for-profit) and try to propose some meaningful insight. Ethics in biomedical research is a serious and complex affair with huge societal implications. Expert advisors that are paid or not paid are not the problem that IRBs face today. It is rather the rapidly growing complexity of biomedical research that confronts IRBs, together with the lack of a timely transfer of knowledge, often due to insufficient resources, the lack of continuing education, and the lack of sufficient and appropriate member expertise. IRBs are expected to function as multifaceted oversight bodies and assume many duties, including ethics consultation, education, peer reviewing, adverse events reporting, publication policies, and the review of financial conflicts of interest. The attention should be focused on whether IRBs are performing to the highest ethical standards and conforming to existing regulations rather than if their members are remunerated or not. Singling out the issue of payment distracts from sincere discussion on how to improve and ensure high-quality reviews of biomedical research and maintain the protection of the human subjects involved.

Rearrangements of the neuregulin (NRG1) gene have been implicated in breast carcinoma oncogenesis. To determine the frequency and clinical significance of NRG1 aberrations in clinical breast tumors, a breast cancer tissue microarray was screened for NRG1 aberrations by fluorescent in situ hybridization (FISH) using a two-color split-apart probe combination flanking the NRG1 gene. Rearrangements of NRG1 were identified in 17/382 cases by FISH, and bacterial artificial chromosome array comparative genomic hybridization was applied to five of these cases to further map the chromosome 8p abnormalities. In all five cases, there was a novel amplicon centromeric to NRG1 with a minimum common region of amplification encompassing two genes, SPFH2 and FLJ14299. Subsequent FISH analysis for the novel amplicon revealed that it was present in 63/262 cases. Abnormalities of NRG1 did not correlate with patient outcome, but the novel amplicon was associated with poor prognosis in univariate analysis, and in multivariate analysis was of prognostic significance independent of nodal status, tumor grade, estrogen receptor status, and human epidermal growth factor receptor (HER)2 overexpression. Of the two genes in the novel amplicon, expression of SPFH2 correlated most significantly with amplification. This amplicon may emerge as a result of breakpoints and chromosomal rearrangements within the NRG1 locus.

A hybrid cell line, IOSE-Ov29, was created through fusion of cells from the human ovarian adenocarcinoma line OVCAR3 and the non-tumorigenic SV40 Tag-transfected human ovarian surface epithelial line IOSE-29. OVCAR3 cells exhibit a differentiated epithelial phenotype, whereas line IOSE-29 expresses mesenchymal characteristics that were acquired in culture by epithelio-mesenchymal transition. Microsatellite analysis, comparative genomic hybridization (CGH), and MFISH showed the genotype of the IOSE-Ov29 cells to contain components of both parent cell lines, but to be predominantly OVCAR3 derived. IOSE-Ov29 resembled OVCAR3 and differed from IOSE-29 as shown by its unlimited life span, tumorigenicity, epithelial morphology, keratin, occludin, E-cadherin and CA125 expression, increased expression of kinases of the PI3K pathway, and loss of cGMP-dependent protein kinase expression. IOSE-29-derived properties included SV40 Tag expression, growth inhibition by activin, collagen type III secretion, increased adhesion and spreading on tissue culture plastic, and increased growth rate. Proliferation of all three lines was stimulated by FSH and ATP and inhibited by GnRH I and GnRH II. Interestingly, IOSE-Ov29 was more anchorage independent than either parent line and was the only line that invaded Matrigel in Boyden chambers and formed invasive branches in collagen gels. The results indicate that IOSE-Ov29 is an IOSE-29/OVCAR3 hybrid, which differs from both parent lines genetically and phenotypically. Unexpectedly, fusion with the non-tumorigenic IOSE-29 cells enhanced malignancy-associated characteristics of OVCAR3, presumably as a result of the expression of IOSE-29-derived mesenchymal properties that are usually acquired by carcinoma cells through epithelio-mesenchymal transition during metastatic progression.

Regional deletions of 6q are frequent karyotypic alterations in malignant lymphoma and are associated with an adverse clinical outcome. One such region of recurrent deletion is 6q16−q21; however, the specific genes affected have not been identified. Our objective in this study was to identify cases with deletion of 6q16−q21 in follicular lymphoma and to define a minimal region of deletion. A physical map of 6q16.2−q21 was constructed using map information from both sequence‐based and bacterial artificial chromosome (BAC) fingerprint–based maps. Forty‐three BAC clones spanning a 6‐Mb region of 6q16.2–q21 were identified and obtained from the RP‐11 library. Selected BACs were fluorescence‐labeled and hybridized to a series of 34 follicular lymphomas with a regional 6q deletion detected by G banding. Twenty‐four cases with deletion of the 6q16.3 region were detected. A minimal deletion of 2.3 Mb was defined. Our study has identified a limited region of deletion of 6q16.3 that may implicate four known genes in follicular lymphoma and possibly in other cancers. A BAC contig spanning a 6‐Mb region has been anchored to the 6q16.2–q21 region. This map represents a useful resource for gene identification in this region, not only in lymphoma but also in other neoplasms with 6q alterations. © 2004 Wiley‐Liss, Inc.

Epstein–Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein–Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein–Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.

Intravascular lymphoma (IVL) is a rare neoplastic disease characterized by the presence of large malignant lymphoid cells in small vessels. It is often diagnosed at autopsy. Clinical manifestations are typically neurologic and dermatologic. Karyotypic abnormalities have been described in a small number of cases and have revealed complex alterations in the majority of cases. We have identified three cases of IVL with varied clinicopathological findings. Karyotypic analysis was undertaken by standard G-banding and supplemented by multi-colored karyotyping (M-FISH) to decipher the chromosomal content of marker chromosomes and undefined additions. M-FISH clarified the chromosomal abnormalities in two cases and unveiled cryptic translocations der(10)t(10;22), der(17)t(17;22), and balanced t(11;14). Comparison with previously published karyotypes revealed prominent involvement of chromosomes 1, 3, 6, 11, 14, and 18, similar to the pattern of clonal evolution in other B-cell lymphomas. The most frequent alterations seen were −6 or 6q− and +18 or dup(18q), with a minimally deleted region located at 6q21-q23 and a commonly amplified region located at 18q13-q23, respectively. Few differences between the classical and Asian variant of this disease were apparent at the karyotypic level. Cytogenetic analysis of additional cases supplemented by multicolor karyotyping may help identify the full spectrum of genetic alterations associated with IVL and assist in the delineation of the critical mutations associated with initiation and progression of this disease.

Summary. In order fully to identify secondary chromosomal alterations, such as duplications, additions and marker chromosomes that remained unresolved by G banding, 60 cases of t(14;18)‐positive follicular lymphoma (FL) were analysed by multicolour karyotyping techniques [multicolour fluorescence in situ hybridization (MFISH)/multicolour banding for chromosome 1 (MBAND1)]. A total of 165 additional structural chromosomal aberrations were delineated. An increased frequency of chromosomal gains involving X, 1q, 2, 3q27‐q29, 5, 6p11‐p21, 7, 8, 11, 12, 14q32, 17q, 18 and 21 and deletions of 1p36, 3q28‐q29, 6q, 10q22‐q24 and 17p11‐p13 was revealed by the MFISH/MBAND1 analysis. Balanced translocations other than t(14;18) were uncommon, whereas unbalanced translocations were numerous. Deletion of 1p36 and duplication of 1p33‐p35, 1p12‐p21 and 1q21‐q41 were regularly involved in chromosome 1 alterations, seen in 53% of the cases. A strong correlation was demonstrated between gains of individual chromosomal bands and increased gene expression, including 1q22/MNDA, 6p21/CDKN1A, 12q13‐q14/SAS, 17q23/ZNF161, 18q21/BCL2 and Xq13/IL2RG. Unfavourable overall survival was associated with del(1)(p36) and dup(18q). These data support the notion that translocation events are primarily responsible for FL disease initiation, whereas the unbalanced chromosomal gains and losses that mirror the gene expression patterns characterize clonal evolution and disease progression, and thus provide further insights into the biology of FL.

Aberrations of chromosomal bands 1p36 and 1q11-q23 are among the most common chromosomal alterations in non-Hodgkin lymphoma (NHL). In this study, 16 cases of NHL showing recurrent unbalanced translocation t(1;1)(p36;q11-23) by G-band analysis were selected for further analysis. To delineate the exact breakpoints, multicolor band analysis for chromosome 1 (M-BAND1), and locus-specific fluorescence in situ hybridization (LS-FISH) using human genome designated BAC clones were performed. In all but one dicentric case, the breakpoint was determined to involve chromosomal bands 1p36.3 and 1q21.1-2. LS-FISH analysis for the TP73, MEL1, SKI, and CASP9 loci at 1p36, and the loci IRTA1, IRTA2, BCL9, AF1Q, JTB, and MUC1 at 1q21, verified the MBAND1 results and further delineated the breakpoints. In band 1p36, two hybridization patterns were observed, one involving deletions of MEL1, TP73, and SKI, but not CASP9, and the second involving a breakpoint telomeric to TP73. In region 1q21, four hybridization patterns were observed, the first involving duplication/translocation of all five genes; the second involving duplication/translocation of BCL9, AF1Q, JTB, and MUC1; the third involving duplication/translocation of AF1Q, JTB, and MUC1; and the fourth with a breakpoint telomeric to MUC1. Using an alpha-satellite probe for chromosome 1 (D1Z5), centromeric involvement in the unbalanced translocation t(1;1)(p36.3;q21.1-2) was excluded in all but the one dicentric case, that is, dic(1;1)(p36.3;q10). In conclusion, deletion of 1p36 and duplication of 1q21 through formation of an unbalanced translocation t(1;1)(p36.3;q21.1-2) is a non-random event in NHL, suggesting a deletion-duplication mechanism involved in lymphoma progression and justifying further systematic research.

Expression of ALK protein by lymphoid cells and the description of variant anaplastic lymphoma kinase (ALK) translocations have typically been restricted to cases of T-cell and null anaplastic large-cell lymphoma (ALCL). All such cases result from a novel fusion created by the ALK gene on chromosome 2p23 and NPM on 5q35 or other variant translocation partners. A rare variant of diffuse large B-cell lymphoma (DLBCL), originally described in 1997, was thought to overexpress full-length ALK in contrast to a chimeric protein characteristic of ALCL. However, full-length ALK protein lacks tyrosine kinase activity and thus the mechanism of oncogenesis has remained elusive. We describe 6 cases of ALK+ DLBCL characterized by a simple or complex t(2;17)(p23;q23) involving the clathrin gene (CLTC) at chromosome band 17q23 and the ALK gene at chromosome band 2p23. All cases were studied using fluorescence in situ hybridization (FISH), complemented in one case with standard cytogenetic analysis, multicolor karyotyping (M-FISH), and reverse transcriptase-polymerase chain reaction. These results clearly demonstrate that most cases of ALK+ DLBCL share the same mechanism of deregulated ALK expression. Moreover, these results demonstrate the presence of CLTC-ALK fusions in these tumors and extend the list of diseases associated with this genetic abnormality to include classical T-cell or null ALCL, ALK+ DLBCL, and inflammatory myofibroblastic tumors.

A 54-year-old male presented with a spontaneous peroneal nerve palsy and a diagnosis of monophasic synovial sarcoma (SS) was rendered by histologic examination. Cytogenetic analysis revealed a complex abnormal karyotype without evidence of the typical t(X;18)(p11;q11) associated with SS. Subsequent reverse transcriptase polymerase chain reaction analysis showed the presence of an SYT/SSX2 fusion transcript, confirming the presence of a cyptic t(X;18). In light of −X, −18 and marker chromosomes evident in the G-band karyotype, it was suspected that a cryptic chromosomal rearrangement involving the marker chromosomes would harbor an X;18 fusion. Multi-colored karytotyping (M-FISH) revealed a previously unrecognized t(X;18) and t(5;19) in the marker chromosomes as well as unrecognized ins(6;18) and t(16;20). The addition of M-FISH analysis in this case led to the identification of complex inter-chromosomal rearrangements, thus providing an accurate karyotype.

Follicular lymphoma (FL) is characterized by t(14;18)(q32;q21), which is the initial genetic perturbation in this disease. Additional genetic mutations are required to generate a fully malignant phenotype. Secondary chromosomal alterations seen in FL include prominent involvement of chromosome 1 in the form of balanced or unbalanced translocations, insertions, deletions, and duplications involving both the p and q arms. We investigated a diagnostically well defined set of 55 t(14;18)‐positive FL cases with complex karyotypes by means of multicolor karyotyping. Sixteen cases showed involvement of chromosome 1 and were analyzed in further detail by a novel multicolor banding technique for this chromosome. We defined three groups showing varying complexity of chromosome 1 alterations. The first group revealed simple translocations, such as t(1;2), t(1;6), t(1;8), and t(1;17), involving breakpoints on either the p or the q arm of chromosome 1. The second group showed more complex rearrangements with translocations, insertions, regional duplications, and involvement of more than one partner chromosome with either the p or the q arm of chromosome 1. The third group was defined by highly complex rearrangements involving translocations, regional duplications, amplifications, and intrachromosomal band relocations affecting the entire chromosome 1. All three groups shared interchromosomal rearrangements of chromosome 1 with chromosome 8, often involving the MYC protooncogene site, amplification involving region 1q21–q31, and deletion involving region 1p36. Thus, the use of sophisticated multicolor molecular cytogenetic assays in the investigation of malignant lymphoma allows precise characterization of chromosomal alterations and will provide a better understanding of their biology. © 2002 Wiley‐Liss, Inc.

An 80-year-old male presented with a lobulated mass in the lower abdominal wall. A diagnosis of an intermediate grade myofibroblastic spindle cell sarcoma was made. Cytogenetic analysis demonstrated a complex karyotype with a der(6), a small marker and five, different in size, ring chromosomes. Fluorescence in situ hybridization (FISH), multiplex FISH, and multicolor banding analysis was used to further delineate this complex karyotype. The der(6) was shown to be a der(18)t(6;18;9;12;18), the marker chromosome was identified as del(17), and the ring chromosomes as r(9) and r(12;18)×4. Amplification of 18 and coamplification of 12p and 12q was detected in the ring and marker chromosomes. No intercellular heterogeneity was observed although a few micronuclei containing chromosome 18 and anaphase bridges, containing chromosome 12 material, the result of bridge–fusion–bridge (BFB) cycles, were observed. Our findings combined with results from others indicate that amplification of chromosomes 12 and 18 as well as BFB phenomena characterize this type of sarcoma.

A spontaneously EBV transformed follicular lymphoma (FL) cell line, Tat-1, was established from the lymph node biopsy specimen of a patient with B cell FL, grade 1 in transformation to high grade disease. Tat-1 cells expressed lymphoid markers and developed tumor masses in immunodeficient mice. Bcl-2, Bcl-X(L), Bax and p53 protein expression was revealed by Western blotting. Flow cytometric analysis confirmed P-gp expression. Cytogenetically, the Tat-1 cell line showed identical chromosomal alterations to that of the initial biopsy specimen, among which the most notable were the t(14;18) typical of FL and additional abnormalities involving chromosomes 1, 8 and 13. Multicolor FISH analysis delineated all abnormalities, including a t(1p;8q), a der(8)(8q24::14q32::18q21) and a der(13)(13q32::8q24::14q32::18q21). Further FISH investigations using a locus-specific probe cocktail containing c-myc, IgH and bcl-2 revealed fusion of these three loci on the derivatives 8 and 13, in addition to the derivative 14 IgH/bcl-2 fusion and an extra copy of c-myc on derivative chromosome 1. These results demonstrate an additional example of the deregulation of bcl-2 and c-myc expression through recombination with a single IgH enhancer region. The unusual molecular features of the Tat-1 cell line render it a unique tool for studies focused on cytogenetic alterations, expression of multidrug resistance phenotype and expression of anti-apoptotic proteins in FL.

We report a novel cytogenetic finding in a favorable histology Wilms tumor occurring in a 4-month-old boy. Karyotypic analysis demonstrated a t(6;15)(q21;q21) in all tumor cells examined. This was confirmed using fluorescence in situ hybridization analysis. Molecular analysis of this rearrangement may provide clues to understanding the pathobiology of Wilms tumor.

OBJECTIVE:To demonstrate the effectiveness of comparative genomic hybridization (CGH) for analysis of reproductive pathology specimens in clinical cytogenetics laboratories.DESIGN:A total of 856 CGH analyses were performed on various placental and fetal tissues derived from 368 specimens of spontaneous abortions and on placentas from 219 pregnancies with live-born infants. The live-born infants were clinically evaluated as normally developed, with either a normal birth weight or with intrauterine growth restriction; some live-born infants had an abnormal prenatal triple screen with normal cytogenetic results on amniotic fluid cell cultures.RESULTS:Comparative genomic hybridization analysis was successfully performed on 856 samples from spontaneously aborted specimens and term placentas. Failure of analysis occurred in 1.6% of samples and was due to an insufficient amount of tissue for DNA extraction. Comparative genomic hybridization identified aneuploidy in 53% of spontaneous abortion samples and 3.1% of term placentas.CONCLUSIONS:Comparative genomic hybridization analysis is a useful clinical tool for detection of aneuploidy in placental and fetal tissues. It provides a genome-wide screen while eliminating tissue culture failures, culture artifacts, and maternal cell contamination. We present practical guidelines for interpreting CGH profiles derived from human reproductive specimens.

In the practice of clinical genetics chromosomal aneuploidy in both mosaic and nonmosaic forms has long been recognized as a cause of abnormal prenatal and postnatal development. Traditionally, cytogenetic analysis of cultured lymphocytes has been used as a standard test for detection of constitutional aneuploidies. As lymphocytes represent only one lineage, chromosomal mosaicism expressed in other tissues often remains undetected. The purpose of this study was to assess the utilization of molecular cytogenetic analysis for detection of chromosomal aneuploidy in placental tissues. Using placentas from 100 pregnancies with viable nonmalformed livebirths, both trophoblast and chorionic stroma were analyzed using comparative genomic hybridization (CGH). In all cases with an indication of chromosomal imbalance by CGH, fluorescence in situ hybridization (FISH) analysis was performed to confirm the presence of aneuploidy. To differentiate between constitutional aneuploidy and confined placental mosaicism (CPM), amniotic membrane was analyzed by CGH and FISH techniques. Our results demonstrated five placentas with CPM for chromosomes 2, 4, 12, 13, and 18, respectively, and two constitutional nonmosaic aneuploidies (47,XXX and 47,XXY). Molecular cytogenetic studies of human placental tissues enables easy analysis of both embryonic (amnion) and extraembryonic (chorion) cell lineages. Detection at birth of chromosomal defects affecting intrauterine placental and fetal development is important because these chromosomal defects may continue to have an influence on postnatal development.