Diffuse Large B-Cell Lymphoma with t(1;22)(q21;q11.2) and t(6;18)(p25;q21): A Case Report.

Complex chromosomal rearrangements in infertile males: complexity of rearrangement affects spermatogenesis

Many B-cell malignancies bear chromosomal translocations juxtaposing immunoglobulin (IG) genes with oncogenes, resulting in deregulated expression of the latter. Translocations affecting the IG heavy chain (IGH) locus in chromosomal region 14q32 are most prevalent. However, variant translocations involving the IG kappa (IGK) locus in 2p12 or the IG lambda (IGL) locus in 22q11 occur recurrently in B-cell neoplasias. No routine methods for the detection of all breakpoints involving IG light chain loci independently of the translocation partner have been described. For this reason, we have designed 2 novel interphase fluorescence in situ hybridization (FISH) assays using differentially labeled probes flanking the IGK and IGL locus, respectively. Based on extensive control studies, the diagnostic thresholds for the detection of breakpoints were set at 0.3% for IGK and 1.4% for IGL. Fifteen cases of B-cell malignancies with cytogenetically detectable chromosomal abnormalities in 2p11-14 were investigated with the FISH assay for IGK. Breakpoints affecting the IGK locus were detected in 7 cases including all 4 variant Burkitt's translocations t(2;8)(p12;q24) and a variant BCL2-associated translocation t(2;18)(p12;q21). Other translocation partners were chromosome bands 7q21 and 16q24. Ten cases with abnormalities in 22q11-12 were investigated with the FISH assay for IGL. Breakpoints in the IGL locus were diagnosed in 7 cases including both variant Burkitt's translocations t(8;22)(q24;q11) and a t(3;22)(q27;q11) involving the BCL6 locus. Other translocation partners were 2p13-14, 4q13 and 16p12. Our results show that these FISH assays provide flexible, simple and reliable tools in the diagnosis and characterization of genetic changes in B-cell malignancies.

To investigate the clinical use of array comparative genomic hybridization (aCGH) with fluorescence in situ hybridization (FISH) in preimplantion genetic diagnosis (PGD) for reciprocal and Robertsonian translocation carriers. From Jan. 2012 to Jun. 2013, a total of 220 PGD cycles from 151 reciprocal translocation and 62 Robertsonian translocation carrier couples, including 33 cycles for reciprocal translocation carriers and 22 cycles for Robertsonian translocation carriers performed using array CGH, and 119 cycles for reciprocal translocation carriers and 46 cycles for Robertsonian translocation carriers performed using FISH were retrospectively studied. The rate of accurate diagnosis was compared between two methods. Normal and/or balance rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 38.20% (123/322) and 67.20% (127/189), significantly higher than 15.39% (195/1 267) and 30.75% (202/657) by FISH (all P < 0.05). Abnormal rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 59.32% (191/322) and 30.69% (58/189), significantly lower than 83.03% (1 052/1 267) and 67.43% (443/657) by FISH (all P < 0.05). And the rate of aneu ploidy in non-translocated chromosome from reciprocal translocation embryos was 20.19% (65/322), which was significantly lower than 38.62% (13/189) from Robertsonian translocation embryos (P < 0.01). Normal and/or balance rates of the two translocated chromosomes detected by array CGH were significantly higher than FISH. And the rate of aneuploidy in non-translocated chromosomes from reciprocal translocation embryos was significantly lower than that from Robertsonian translocation embryos.

Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research

Individuals carrying balanced translocations have a high risk of birth defects, recurrent spontaneous abortions and infertility. Thus, the detection and characterization of balanced translocations is important to reveal the genetic background of the carriers and to provide proper genetic counseling. Next-generation sequencing (NGS), which has great advantages over other methods such as karyotyping and fluorescence in situ hybridization (FISH), has been used to detect disease-associated breakpoints. Herein, to evaluate the application of this technology to detect balanced translocations in the clinic, we performed a parental study for prenatal cases with unbalanced translocations. Eight candidate families with potential balanced translocations were investigated using two strategies in parallel, low-coverage whole-genome sequencing (WGS) followed-up by Sanger sequencing and G-banding karyotype coupled with FISH. G-banding analysis revealed three balanced translocations, and FISH detected two cryptic submicroscopic balanced translocations. Consistently, WGS detected five balanced translocations and mapped all the breakpoints by Sanger sequencing. Analysis of the breakpoints revealed that six genes were disrupted in the four apparently healthy carriers. In summary, our result suggested low-coverage WGS can detect balanced translocations reliably and can map breakpoints precisely compared with conventional procedures. WGS may replace cytogenetic methods in the diagnosis of balanced translocation carriers in the clinic.

Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation: relationship between segregation modes and high-magnification sperm morphology examination

Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos

Application of Multicolor Banding for Identification of Complex Chromosome 18 Rearrangements

TO THE EDITOR: We read an interesting paper by Palta et al. in a recent issue of the Korean Journal of Hematology titled, variant of acute promyelocytic leukemia with tuberculosis: a case report and review of literature [1]. We would like to add some comments to their article and suggest additional molecular methods to confirm variant translocations in acute promyelocytic leukemia (APL). Apart from its characteristic morphology, APL is strongly correlated with presence of the PML-RARA fusion gene due to a t(15;17) translocation. Patients displaying cryptic PML-RARA gene rearrangements or other translocations involving RARA are very rare. Since RARA generates chimeric fusion genes with various partners such as ZBTB16 at 11q23, NUMA1 at 11q13, NPM1 at 5q35, FIP1L1 at 4q12, and STAT5b at 17q11.2 (Fig. 1), confirmatory molecular methods to detect gene rearrangements are necessary when unique or unusual chromosomal translocations are found in APL cases. Palta et al. reported a case of APL accompanied by tuberculosis where cytogenetic study showed a variant chromosomal translocation, t(11;17)(q23;q21). First, although they describe this case as APL with t(11;17)(q23;q21) through bone marrow (BM), immunophenotyping, and cytogenetic studies, we noted an absence of results based on molecular methods. To demonstrate the existence of the ZBTB16-RARA fusion gene, a single specific reverse transcriptase-polymerase chain reaction (RT-PCR) with a specific primer to detect the gene rearrangement or multiplex RT-PCR (e.g. HemaVision) to detect ZBTB16-RARA could have provided an accurate molecular diagnostic result. Second, the authors' reference to a paper by Kang et al. was puzzling despite the fact that it was cited as a similar case with chromosomal translocations at a similar location, in cases of AML with chromosomal translocation t(11;17) [2]. In the first case, cytogenetic analysis from BM aspirate showed t(11;17)(q23;q21) in 18 out of 20 cells while the MLL probe in fluorescence in situ hybridization (FISH) showed split-out signals in 98.5% of interphase cells. The second case also showed t(11;17)(q23;q25) in the chromosome study while the FISH assay showed split-out signals in 79% of interphase cells. However, normal fluorescence patterns were found in 200 cells in additional FISH assays when a dual-color single-fusion PML-RARA probe was used for both cases, which led to the conclusion that the MLL gene was involved in the 11q23 breakpoint and RARA was not involved in the 17q breakpoint. Hence, the authors' citation of this study to explain the ZBTB16-RARA fusion gene appears inadequate, and a molecular study could unambiguously identify the involvement of ZBTB16 at 11q23 in their case. Fig. 1 Various RARA partner genes in acute promyelocytic leukemias. We suggest the authors conduct RT-PCR analysis of the ZBTB16-RARA fusion gene if any remnant RNA or cDNA samples from their APL patient are available. However, if only genomic DNA is available, analyzing the genomic breakpoints by multiplex long distance-polymerase chain reaction (LD-PCR) would be helpful. LD-PCR or long distance inverse (LDI)-PCR can identify known or unknown partner genes, monitor minimal residual disease (MRD), and identify multi-way chromosomal translocations such as three-way or four-way translocations [3]. We have recently used LD-PCR to confirm APL cases showing unusual karyotypes or discrepant results between conventional cytogenetics and molecular studies. More precisely, we identified a novel PML-ADAMTS17-RARA rearrangement and confirmed the genomic fusion breakpoints of cryptic PML-RARA rearrangement in 2 APL patients [3-5]. In conclusion, detection of leukemia-associated fusion genes is important due to their direct relevance to diagnosis, MRD assessment, and treatment response. Since APL patients with ZBTB16-RARA are resistant to all-trans-retinoic acid (ATRA), molecular methods with higher resolution than chromosome studies must be conducted to detect fusion genes and facilitate selection of the most appropriate treatment. We believe that molecular techniques such as FISH, RT-PCR, and LD-PCR should be used in addition to morphology and cytogenetic studies in APL diagnosis and evaluation so that atypical PML-RARA or RARA variant gene rearrangements are not missed.

The role of cytogenetic analysis in the diagnosis and management of haematological malignancies is undisputed.The accuracy of cytogenetic diagnosis has improved steadily over the past 20 years, primarily due to a series of technical developments. However, despite improvements in high-resolution banding and culture methods to detect the chromosomally abnormal cells, many haematological malignancies are retractable to conventional cytogenetic analysis. This may be due to the presence of multiple abnormal clones, complex rearrangements, a low mitotic index, or poor chromosome morphology. Since the late 1980s a range of techniques based around fluorescence in situ hybridization (FISH) have greatly enhanced cytogenetic analysis. These use a variety of nucleic acid sequences as probes to cellular DNA targets and serve to bridge the gap between molecular genetic and conventional cytogenetic methods. Virtually any genomic DNA can now be used as a probe with which to investigate a wide variety of DNA targets, from metaphase chromosomes to mechanically stretched DNA fibres. The simultaneous detection of multiple target regions is also possible, using differentially labelled probes detected by different colours. In research, FISH has played a pivotal role in the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukaemogenesis. Now, at the cutting edge, a new set of resources and technical innovations herald a new era for molecular cytogenetics, with colour karyotyping, comparative genomic hybridization (CGH) microarrays and mutation detection using padlock probes providing the promise of the future. The number of applications for FISH is almost unlimited (see Table I for some pertinent examples). This review will concentrate on the most recent developments in FISH which have had a considerable impact on the cytogenetic diagnosis and study of haematological malignancies, with some insight into the possible future roles for this flexible technology. The application of FISH to metaphase chromosomes provides unequivocal evidence of chromosome rearrangements. There are many different types of cloned or uncloned DNA which can be used for as a probe for FISH (reviewed in Buckle & Kearney, 1994). However, the most commonly used probes in cytogenetic analysis of haematological malignancy are: (i) repetitive sequence centromeric probes, (ii) whole chromosome paints, and (iii) locus-specific probes. Chromosome-specific centromeric probes which target tandemly repeated alpha (or beta) satellite sequences present in the heterochromatin of the chromosome centromeres are used to detect numerical chromosome abnormalities. Centromeric probes are commercially available for all human chromosomes and these provide a rapid and simple way of enumerating specific chromosome pairs, both in metaphase and interphase. This type of analysis is useful in many types of leukaemia where the chromosome morphology is poor and banding indistinct, such as in hyperdiploid acute lymphoblastic leukaemia (ALL). However, centromeric probes only give information on the number of centromeres of a particular type present; they cannot tell whether the chromosome is structurally abnormal. Whole chromosome painting probes are complex mixtures of sequences from the entire length of a specific chromosome. These are also available for all human chromosomes, and can be used to delineate chromosome pairs (Cremer et al, 1988; Pinkel et al, 1988). Whole chromosome painting probes (paints) can be derived from chromosome-specific libraries, PCR amplification of flow-sorted chromosome fractions, or microdissected DNA specific for each chromosome (Collins et al, 1991; Telenius et al, 1992; Vooijs et al, 1993; Guan et al, 1996). Chromosome paints are most useful for identifying the components of highly rearranged and marker chromosomes, where the banding pattern cannot be relied upon. However, their usefulness is limited to metaphase analysis, as the extended chromosome domains in interphase are often diffuse and difficult to quantitate. In addition, chromosome painting is a relatively insensitive technique and cannot detect small interstitial deletions, duplications or inversions. The resolution for the detection of small telomeric translocations is also limited. Single locus probes detect specific sequences present in only one copy. When using these probes the efficiency of hybridization needs to be considered; the larger the target sequence the more efficient the hybridization. Single-copy probes cloned in cosmid, YAC, P1, PAC and BAC vectors all give reliable FISH signals, with a fluorescent signal on both chromosome homologues in >90% of metaphases. Structural rearrangements detected using this type of probe include translocations, inversions and specific deletions (Dauwerse et al, 1990; Tkachuk et al, 1992; Sacchi et al, 1995; Jaju et al, 1998). The use of specific gene probes for chromosomal translocations has simplified the process of identifying known translocations, especially in complex or masked versions of the translocation (e.g. BCR/ABL, PML/RARα fusions), and has particular applications for interphase analysis. One of the greatest advances in cytogenetic analysis facilitated by FISH has been the ability to use non-dividing cells as DNA targets, referred to as interphase FISH (Cremer et al, 1986). This enables the screening of large numbers of cells and provides access to a variety of sources of haemopoietic cells including blood and bone marrow smears and haemopoietic progenitor cells from colony assays (Bentz et al, 1993; Poddighe et al, 1993; Mühlmann et al, 1998). This has considerable advantages for some haemopoietic malignancies, where the proliferative activity is low, or when the mitotic cells do not represent the neoplastic clone, for example chronic lymphoblastic leukaemia (CLL), Hodgkin's disease, multiple myeloma. Interphase FISH permits the identification of both numerical and structural chromosome abnormalities both as an aid to cyto-genetic diagnosis and for monitoring disease progression. Interphase FISH has had a major impact on the cytogenetic analysis of B-CLL, revealing a much higher incidence of trisomy 12 than found by conventional cytogenetic analysis (Anastasi et al, 1992; Garcia-Marco et al, 1997). An examination of the relationship between clinical stage and trisomy 12 showed an association with atypical morphology, advanced stage of disease and low proliferative activity. In addition, immunophenotyping and FISH showed that the +12 is present in only a proportion of clonal B cells (Garcia-Marco et al, 1997). All of this data suggests that trisomy 12 is a secondary event in the development of CLL. For chromosome deletions, specific locus or region-specific probes have been used to demonstrate a high frequency of mono-allelic deletions of the RB1 and p53 genes in B-cell malignancies (Stilgenbauer et al, 1993, 1995; Döhner et al, 1995; Cano et al, 1996). Interphase FISH was also instrumental in identifying the critical region of deletion on 11q13 associated with B-cell lymphoid malignancy, which consequently identified mutations of the ATM gene in T-prolymphocytic leukaemia (PLL) (Stilgenbauer et al, 1997). DNA probes for the fusion genes involved most specific chromosomal translocations and inversions in leukaemia are now commercially available. The differential labelling and detection of these probes in different colours enables a direct visualization of the fusion gene. The simplest scheme is to use two probes (one from each of the fusion genes), differentially labelled and detected with two different-coloured fluorochromes (see Fig 1A). An interphase cell positive for the translocation will exhibit a red–green fusion signal representing the translocation, and a single red and green signal corresponding to the normal chromosome homologues. However, the false positive rate using this approach is quite high (approximately 5%). In addition, the presence of variant translocations or translocations in which the breakpoints are spread over a large distance (e.g. Burkitt's lymphoma), means that the false negative rate can also be quite high. There are several more complex strategies to overcome this (see Figs 1B and 1C). Firstly, if a series of probes spanning both translocation breakpoints are used, this will result in splitting of both fluorescent signals, and the presence of two red–green fusions. Another, more complex, strategy is to employ three or even four different colours, so that the incidence of false positives and false negatives is reduced (Ried et al, 1993; Sinclair et al, 1997). However, the more complicated the colour scheme, the more difficult and complex the analysis. At present, this analysis is done manually, so this is a serious consideration. . Schematic representation of the detection, by FISH, of the Philadelphia translocation in interphase nuclei. In each case the left-hand panel shows the location of the FISH signals on metaphase chromosomes (partial karyotype), and the right-hand panel the interphase FISH signals. In (A) two probes from the flanking regions of the BCR and ABL genes are labelled and detected in different colours: BCR in red and ABL in green. The BCR/ABL fusion results in co-localization of the red and green signals on the der(22) (Philadelphia) chromosome, with a single red and green signal separated, corresponding to the normal chromosomes 22 and 9, respectively. A BCR/ABL negative cell would show two separate red and two green signals. The scheme in (B) uses two probes, this time spanning both the BCR and ABL breakpoint regions. In this case, two red/green fusion signals are formed: one corresponding to the der(9), and the second to the der(22). A positive cell would therefore exhibit one red, one green and two red/green fusions (from Dewald et al, 1998). In (C), a third probe from the region just proximal to ABL on 9q34 is used, labelled in a different colour (represented here in yellow). A translocation positive cell exhibits one green/yellow doublet, one red/green and a single red and yellow signal (from Sinclair et al, 1997). The possibility of using interphase FISH as screening test for specific abnormalities found in acute myeloid leukaemia (AML) subtypes was recently described by Fischer et al (1996). This study used 23 different probes and six to eight hybridizations per patient. They found that interphase FISH was more sensitive for the detection of t(8;21), inv(16), +8q, +11q, +21q, +22q and −Y, and obtained a cytogenetic result in a proportion of cases with no evaluable metaphases. However, this kind of analysis may eventually be replaced by disease-specific DNA chips (see Matrix-CGH below). The detection of residual Philadelphia-positive cells is important after allogeneic bone marrow transplant or interferon (IFN) treatment. In particular, the degree of response to IFN treatment has been shown to be an independent prognostic indicator. The sensitivity of conventional cytogenetics is around 5%, and may be difficult due to low mitotic rate of cells after treatment. RT-PCR is the most sensitive method for detection of BCR-ABL (approximately 10−6) but quantification is difficult. Interphase FISH offers the prospect of using peripheral blood samples, reducing the need for frequent bone marrow aspirates. However, 'in house' cut-off levels must be established for each probe set. Conventional FISH probes for the detection of BCR-ABL gene fusion in interphase cells have suffered from a high false positive rate (Tkachuk et al, 1992). The development of three-colour/three-probe FISH protocols for BCR-ABL detection has significantly lowered the false positive rate, and also increased the sensitivity of detection (Sinclair et al, 1997; Dewald et al, 1998). Sinclair et al (1997) used a third probe (for the ASS gene) 200 kb proximal to ABL, such that when a true BCR-ABL fusion was present, there was one co-localization for BCR-ABL, and a separate ASS signal corresponding to the der(9). In cells where the BCR and ABL signals co-localized due to chance, the ASS signal co-localized with the red ABL signal on both chromosomes 9 (see Fig 1C). This three-colour approach resulted in a low false positive and false negative rate. Dewald et al (1998) used a similar strategy, with probes spanning both the BCR and ABL breakpoints. This resulted in two different co-localizations: one representing the der(22) and the other the der(9) chromosomes (see Fig 1B). Strict scoring criteria, experienced operators and scoring of >3000 cells all enabled the detection of residual disease in 0.079% of cells. This skilled and time-consuming approach was also successful in detecting variant translocations. Although the sensitivity of dual-colour interphase FISH is less than for RT-PCR, PCR is not a possibility in a number of cases, for example for the detection of deletions, monosomy or trisomy. Interphase FISH has been used for the detection of residual disease after allogeneic bone marrow transplantation (Anastasi et al, 1991; Wessman et al, 1993). Kasprzyk & Secker-Walker (1997) studied hyperdiploid karyotypes in ALL to detect minimal residual disease. Using three-colour interphase FISH, targeting three chromosomes simultaneously, they were able to achieve a sensitivity of 10−4, and predict relapse in a number of cases. The ability to combine interphase FISH analysis with immunological staining for cell surface antigens provides a powerful method to combine cell by cell analysis with morphology or immunophenotype. Simultaneous immunophenotyping and FISH analysis has been used to investigate lineage involvement in myelodysplastic syndrome (MDS), chronic myeloid leukaemia (CML) and other myeloproliferative syndromes (Price et al, 1992; Nylund et al, 1993; Torlakovic et al, 1994; Soenen et al, 1995; Haferlach et al, 1997, reviewed in Knuutila, 1997). Concurrent immunophenotype and FISH analysis has also been used to demonstrate that the leukaemia which emerged 5 years after sex-mismatched allogeneic bone marrow transplant occurred in donor cells (Katz et al, 1993). In CML, three-colour detection of the Philadelphia translocation and immunophenotype enabled the identification of the translocation in CD20-positive B cells (Torlakovic et al, 1994) and more recently CD3-positive T cells and CD34-positive precursor cells (Haferlach et al, 1997). This supports the belief that CML is a disorder of an early progenitor cell, capable of differentiating into myeloid and some lymphoid lineages (reviewed in Knuutila, 1997). There are also reports of the clonal involvement of B cells in MDS, using del(20q) and monosomy 7 as clonal markers (White et al, 1994; van Lom et al, 1995). In Hodgkin's disease the low percentage of Hodgkin and Reed-Sternberg (HRS) cells means that even interphase FISH may not detect clonal abnormalities. In a recent study the combination of CD30+ staining and FISH with pairs of centromeric probes revealed numerical abnormalities in 100% of HRS cells (Weber-Matthiesen et al, 1995). Surprisingly, clonal abnormalities found in metaphase analysis were not consistent with the interphase FISH analysis, indicating that metaphase analysis of Hodgkin's disease may not be informative. FISH has proved an invaluable aid in the mapping of translocation breakpoints, resulting in the identification of many fusion genes (reviewed in Rabbitts, 1994). A recent addition to the repertoire of FISH techniques now provides significant advantages over other molecular methods for mapping breakpoints which are dispersed over large distances. The term Fibre-FISH is used to describe a collection of methods for performing FISH to extended DNA stretched out on a glass slide (Wiegant et al, 1992; Parra & Windle, 1993; Bensimon et al, 1994; reviewed in Raap, 1998). vandraager et al (1996) have demonstrated the usefulness of this technique for mapping breakpoints of the cyclin D1 gene in mantle cell lymphomas. Using a series of overlapping probes from the 11q13 breakpoint region labelled in alternating red and green fluorochromes creates a colour bar code for the region. Translocations are recognized by the disruption of this bar code into its two complementary parts. The advantages of this method over Southern blotting or pulsed field gel electrophoresis are its simplicity and speed: only a few images need to be examined, and chromosomal breaks over a distance of 250 kb can be visualized. However, the parameters underlying the technique are poorly understood, and at present it remains a research rather than diagnostic tool, confined to a few specialist laboratories. The strength of conventional (G-banded) cytogenetic analysis has always been the ability to survey the entire genome for clues to pathogenesis. However, the poor chromosome morphology and low mitotic index of many leukaemias and lymphomas means that conventional cytogenetic analysis is often limited. In addition, the analysis of banding pattern in highly rearranged karyotypes is difficult and unreliable. One of the remaining challenges for the new FISH techniques is to identify cryptic rearrangements, particularly involving telomeric regions, in apparently normal karyotypes. A significant proportion (15–20%) of bone marrow karyotypes in leukaemia are reported as normal by conventional (G-banded) cytogenetic analysis. Despite significant improvements in the quality of leukaemic metaphase preparations over the past decade, the abnormality rate has not improved. The t(12;21)(p13;q22) remained undetected until 1994, despite the fact that it accounts for 25% of childhood B-cell ALL cases (Romana et al, 1994). This translocation still remains undetectable by conventional cytogenetic analysis. The difficulty in detecting chromosome abnormalities such as this in the fact that there is a of staining regions of a similar The recent development of whole chromosome painting provides the promise of identifying cryptic chromosome rearrangements, a of all chromosome abnormalities in a single FISH using the method of probe labelling was described by et al In this probes are labelled with mixtures of fluorochromes such that no two probes have the The number of targets which can be in this is where number of fluorochromes available. FISH with to different colours has been available for a number of years, using probes labelled with three fluorochromes (Dauwerse et al, 1992; et al, 1992). the number of fluorochromes to enables the identification of all pairs of human The has been due in to the of new fluorochromes in the and and to two detection methods to mixtures of fluorochromes et al, et al, 1996). of these used a set of whole chromosome paints, labelled with different mixtures of The detection FISH relied on separate images for each of using et al, 1996). The labelling combination for each chromosome was and in using The second used a single of the and a combination of and et al, 1996). An was used to the at each of the of these techniques have demonstrated chromosome rearrangements in complex karyotypes in cell and in haematological malignancies et al, et al, 1997; also Fig However, the sensitivity of both or remains to be The of this are the on metaphase analysis, and the resolution of painting probes. All of the available whole chromosome paints are in some of the particularly the telomeric regions. that the sensitivity of painting for the detection of translocations involving regions may be as low as also Fig In addition, whole chromosome painting will not detect deletions, duplications or inversions. In both and still to the and a combination of FISH is still to identify all abnormalities in complex karyotypes. . FISH to the analysis of a complex in the myeloid cell (A) after analysis. The structural abnormalities identified are: A cryptic was also present, but difficult to identify by analysis. (B) A metaphase after analysis. of amplification are in green and deletions in of the genome which are regions were identified the entire chromosome of and A deletion of due to the of an The analysis identified the of a marker chromosome, as as revealing several cryptic translocations in The of the analysis was to the of the with large deletions translocations in most cases. et al (1997) have recently described an approach which use of the regions of between different to approach a of colour of The of a colour for each chromosome was described by et al using a series of from the length of the chromosome, labelled differentially and detected in a different banding on the of between and has been useful in comparative of regions (reviewed in et al, 1997). The by and have now a set of paints derived from cell Chromosome-specific painting probes were derived from and by chromosome and When used for FISH to human metaphase chromosomes, this resulted in the of each chromosome into between two and six labelling using three fluorochromes resulted in a colour banding pattern for each chromosome. In with specific an colour can be Although at present the number of colour is the of this approach is with the to identify chromosomal inversions and colour banding has been used to identify cryptic translocations in CML A set of chromosome-specific probes which identify the of all human chromosomes the of the is now available for FISH of and of 1996). These contain DNA sequences cloned in cosmid, and PAC clones, the of which have been to between and kb from human chromosome These probes have been in a FISH for rearrangements on a series of with cryptic chromosome rearrangements et al, 1997). This is dual-colour an of all chromosome regions on a single However, the approach a high mitotic index and is most for the analysis of which on peripheral blood or where a cell is available. In the of these probes for leukaemic karyotypes has been to identify the specific region in rearrangements found by painting (see Fig An a FISH would the of all chromosome regions in a single However, the of labelling and multiple colour detection methods for cosmid, or even and PAC a series of developments. Firstly, the simultaneous analysis of all chromosome in a different colour the number of targets with fluorochromes is the of such an would on it is not whether the targets of such small probes labelled with several different fluorochromes can be due to of resolution of the is that the development of fluorochromes will this type of analysis . The use of chromosome-specific probes to identify the of chromosome on two In each case dual-colour hybridization was out with the probe labelled in and detected in red fluorescent and the probe labelled with and detected with fluorescent In (A) probes for and identified the on the as derived from In (B) the on was identified as from Although these abnormalities were detected by painting no information of the chromosomal region is also important to that the abnormality in (A) was described by as The of all of the new is that they still metaphase The advantages of are that it the need for cells and not any of the chromosome The is genomic and DNA are labelled with different in and normal metaphase The in number between the normal and is by in red and green fluorescence the length of the chromosome (see Fig the 5 years its et al, has a of identifying new regions of amplification and deletion in a wide variety of types (reviewed in et al, 1997). The use of for haematological malignancies is more limited (Bentz et al, et al, et al, et al, 1997). The of for haematological malignancies are the to detect rearrangements, and the for cells with the clonal However, and some lymphomas have from the application of (Bentz et al, et al, et al, 1996). One study of identified and not identified or not detected by clonal were identified in six out of cases with a normal (Bentz et al, The for results between and were a complex the or a of metaphases. This study that banding analysis may abnormalities and may important chromosome have not been identified in CLL. In a study of myeloid leukaemias found a between and results (Bentz et al, The only were a of to detect and The major of is its due to the on metaphase For deletions, the resolution of has been at (Bentz et al, 1998). the most future for in to cloned DNA (see below). This to overcome the of using metaphase chromosomes as a target for by metaphase chromosomes with cloned DNA in small and to the surface of a glass et al (1997) used for the detection of high number amplification using as For low number larger cloned probes or were For deletions, a resolution of it not between and The other of metaphase also at of clonal cells and will not detect translocations. One types of (i) disease-specific probe (ii) or (iii) DNA for specific regions, at over the whole probes are DNA in which the has been replaced by The rapid and of with complementary DNA sequences for a number of including FISH probes have been for the human telomeric the fluorescent detection of all in a single These signals to be a for fluorescence than conventional FISH signals, an of length et al, 1996). This may also be extended to other sequences such as centromeric may also be to combine telomeric probes with the chromosome-specific DNA probes to provide a of and specific chromosome This new the promise of detecting single in cells. probes of two different each 20 by a When the probe sequence the the and of the probe are and the probe is et al (1997) used two different probes, each labelled with a different to detect single in a centromeric The sensitivity of this may be improved by the of new sensitive labelling techniques such as the use of fluorescent signal et al, 1995; et al, 1995). to the sensitivity is amplification of the This to the would fluorescent detection of mutations in nuclei. amplification has been with some using extended DNA from but at this stage not or on et al, 1998). In the relatively time its FISH has had a major impact on cytogenetic analysis, due to the sensitivity and of its Although some of the applications will research the and probes for most cytogenetic abnormalities are now the of most clinical laboratories. However, conventional FISH can only provide to the specific and some of the The recent of FISH to the visualization of the entire human genome in different colours has the of and The of this approach is the ability to the whole genome in a single hybridization the screening of cytogenetics with the accuracy of molecular The belief that cytogenetics is more an than a has been from the the aid of new colour techniques and cytogenetic analysis now a molecular of The major impact of this development in field of haematological is to be the identification of new and non-random chromosome rearrangements and clinical of the most recent innovations to The most of fluorescent metaphase is now by and and will not only the of such but the sensitivity of interphase FISH analysis. many of the of cytogenetic analysis by the future for cytogenetics has The all of the of the particularly for the and analysis of the cell of the described here was by the and the

The management of patients with leukaemia: the role of cytogenetics in this molecular era.

To study the genetic aberrations and their pathologic significance in follicular lymphoma (FL). Paraffin-embedded tissue samples of 55 cases of FL, 28 cases of other small B-cell lymphomas and 10 cases of reactive follicular hyperplasia were retrieved. Nested polymerase chain reaction (PCR) was used to detect clonal rearrangement of immunoglobulin heavy chain gene (IgH) in FL and other small B-cell lymphomas. The translocation t (14; 18) was studied by PCR and dual-color fluorescence in-situ hybridization (FISH) in FL. Cases of reactive follicular hyperplasia were used as controls. Amongst the 55 cases studied, 49 cases were nodal and 6 cases were extranodal. There were 33 males and 22 females. The male-to-female ratio was 1.5:1. The median age of the patients was 57 years. Twenty-five cases belonged to histologic grade 1, while 19 cases were grade 2 and 11 cases were grade 3. Beta-actin DNA was detected in 50 cases of FL. Amongst those 50 cases, clonal IgH rearrangement was present in 34 (68%). Twenty-four cases (48%) and 25 cases (50%) were positive for FR3A and FR2 respectively. Fifteen cases (30%) showed dual positivity for both FR3A and FR2. Thirty-four cases (68%) demonstrated clonal IgH rearrangement. As for other small B-cell lymphomas, 25 cases were positive for beta-actin. FR3A and FR2 were detected in 18 and 17 cases respectively. Clonal IgH rearrangement was demonstrated in 24 cases. In contrast, none of the 4 cases of reactive follicular hyperplasia showed the clonal rearrangement pattern. Amongst the 44 cases of nodal FL analyzed, t (14; 18) was detected in 15 cases (with 14 cases in MBR and 1 case in mcr). In general, FISH was superior to PCR in detecting t (14; 18) using paraffin-embedded tissue samples. The detection rate of clonal IgH rearrangement in FL is lower than that in other small B-cell lymphomas. Demonstration of t (14; 18) in paraffin-embedded tissue samples by FISH helps in diagnosis of FL. FISH is superior to PCR, as the technique is more sensitive and less labor intensive.

The principle of the fluorescence in situ hybridization (FISH) method is in the base pairing of the DNA probe to complementary sequences in the studied specimen. The hybridization of specific DNA or RNA probes to the cellular targets attached to the microscopic slides is widely used for the identification of chromosomal translocations, deletions, amplifications of specific genes, and chromosome number changes in mitotic and/or interphase cells. The use of FISH with the modifications of the basic method meant a breakthrough in detection and diagnosis of human malignancies. During the last tow years FISH was used in our laboratory for: (a) identification of constitutive and acquired numerical and structural chromosomal abnormalities; (b) detection of minimal residual disease or early relapse in patients treated for leukemia by bone marrow transplantation (BMT) and/or chemotherapy; (c) determination of the cytogenetic pattern of non-dividing or terminally differentiated cells. To confirm the structural rearrangements found by the classical G-banding technique, the whole chromosome painting probes which hybridize to multiple chromosomal sequences were used. The alpha-satellite DNA probes which detect centromeric repetitive sequences were utilized for determining the numerical and sex chromosome changes. Specific unique chromosomal sequences which can confirm all chromosomal rearrangements, i.e., deletions, translocations or inversions with the corresponding breakpoints were introduced for specific cases. Recently, every chromosomal translocation, deletion and any other structural or numerical change found by conventional cytogenetic analysis in the bone marrow cells of the patients with leukemia has been verified in our laboratory by FISH. The results of this study showed that FISH is more efficient than conventional cytogenetics in detecting residual malignant cells. For chromosomal rearrangements FISH is an extremely sensitive method which not only verifies but also interprets with more precision the findings of classical cytogenetics.

Objective To evaluate the residual risk (i.e. failure risk in detecting aneuploidies abnormalities except for chromosome 13, 18, 21, X and Y) of cytogenetic abnormalities using interphase fluorescence in situ hybridization (FISH) for the second-trimester amniocytes. Methods The results of interphase FISH and conventional karyotyping of 2 837 consecutive amniotic fluid specimens were analyzed retrospectively. Probes for chromosomes 13, 18, 21, X and Y were used. The detection rate and residual risk for interphase FISH were calculated for the following three major clinical indications for prenatal diagnosis (advanced maternal age, abnormal maternal serum screening indicating an increased risk for trisomy 18 or trisomy 21, and ultrasound abnormalities). Results Consecutive interphase FISH and karyotyping of second-trimester amniocytes for prenatal diagnosis were performed from January 1, 2010 to July 31, 2013. Among the 2 837 cases, 85 (3.0%) cases with abnormal karyotypes were found, including 73 cases of aneuploidies involving chromosome 13, 18, 21, X and Y, which were considered detectable by interphase FISH; 12 cases of chromosomal anomalies, other than aneuploidies of chromosome 13, 18, 21, X and Y, were diagnosed after karyotyping and were not detected by interphase FISH, including six cases of balanced rearrangements, five cases of imbalanced rearrangements, and one case of pseudomosaic of trisomy 20. Of these 12 chromosomal anomalies, three cases of imbalanced rearrangements involving chromosome 21 showed positive FISH results, and the other nine cases showed negative FISH results among which four case of hereditary balanced rearrangemerts and two cases of novel balanced rearrangements. The total detection rate for interphase FISH was 89.4% (76/85), the misdiagnosis rate of chromosome abnormalities was 14.1%(12/85), and the residual risk was 0.43% (12/2 761) following interphase FISH of the second-trimester amniocytes. Conclusions Interphase FISH is a useful adjunct to conventional karyotyping, but should not be regarded as a replacement for karyotyping as too many structural chromosomal abnormalities will be missed. Providing patients with a detection rate and residual risk during counselling may help them understand the advantages and limitations of interphase FISH in their prenatal diagnostic evaluation. Key words: In situ hybridization, fluorescence; Chromosome aberrations; Prenatal diagnosis; Pregnancy trimester, second; Amniotic fluid

X-autosomal translocation — a distraction or a cause of primary ovarian insufficiency?