Comparative Genomic Features of Streptococcus canis Based on Pan-Genome Orthologous Group Analysis According to Sequence Type.

Genomic characteristics of Streptococcus agalactiae based on the pan-genome orthologous group analysis according to invasiveness and capsular genotype

Can Whole Genome and Whole Transcriptome Sequencing Replace Standard Procedures in CLL Diagnostics?

Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Listeria monocytogenes (L. monocytogenes) is a ubiquitous organism that can easily enter the food chain. Infection with L. monocytogenes can cause invasive listeriosis. Since 2014, in Austria, L. monocytogenes isolates from human and food/food-associated samples have been provided on a mandatory basis by food producers and laboratories to the National Reference Laboratory. Since 2017, isolates undergo routinely whole genome sequencing (WGS) and core genome Multilocus Sequence Typing (cgMLST) for cluster analyses. Aims of this study were to characterize isolates and clusters of 2017 by using WGS data and to assess the usefulness of this isolate-based surveillance for generating hypotheses on sources of invasive listeriosis in real-time. WGS data from 31 human and 1744 non-human isolates originating from 2017, were eligible for the study. A cgMLST-cluster was defined as two or more isolates differing by ≤10 alleles. We extracted the sequence types (STs) from the WGS data and analyzed the food subcategories meat, fish, vegetable and diary for associations with the ten most prevalent STs among food, through calculating prevalence ratios (PR) with 95% confidence intervals (CI). The three most frequent STs among the human isolates were ST1 (7/31; 22.6%), ST155 (4/31; 12.9%) and ST451 (3/31; 9.7%) and among the non-human isolates ST451 (614/1744; 35.2%), ST8 (173/1744, 10.0%) and ST9 (117/1744; 6.7%). We found ST21 associated with vegetables (PR: 11.39, 95% CI: 8.32–15.59), ST121 and ST155 with fish (PR: 7.05, 95% CI: 4.88–10.17, PR: 3.29, 95% CI: 1.86–5.82), and ST511, ST7 and ST451 with dairy products (PR: 8.55, 95% CI: 6.65–10.99; PR: 5.05, 95% CI: 3.83–6.66, PR: 3.03, 95% CI: 2.02–4.55). We identified 132 cgMLST-clusters. Six clusters contained human isolates (ST155, ST1, ST101, ST177, ST37 and ST7) and for five of those cgMLST-based cluster analyses solely was able to hypothesize the source: an Austrian meat processing company, two Austrian cheese manufacturers and two vegetable processing companies, one based in Austria and the other in Belgium. Determining routinely STs in food isolates by WGS allows to associate STs with food products. Real-time WGS of L. monocytogenes isolates provided mandatorily, proved to be useful in promptly generating hypotheses on sources of invasive listeriosis.

Candida glabrata is a pathogen with reduced susceptibility to azoles and echinocandins. Analysis by traditional multilocus sequence typing (MLST) has recognized an increasing number of sequence types (STs), which vary with geography. Little is known about STs of C. glabrata in Australia. Here, we utilized whole genome sequencing (WGS) to study the genetic diversity of 51 Australian C. glabrata isolates and sought associations between STs over two time periods (2002–2004, 2010–2017), and with susceptibility to fluconazole by principal component analysis (PCA). Antifungal susceptibility was determined using Sensititre YeastOneTM Y010 methodology and WGS performed on the NextSeq 500 platform (Illumina) with in silico MLST STs inferred by WGS data. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-fluorocytosine resistance were analyzed. Of 51 isolates, WGS identified 18 distinct STs including four novel STs (ST123, ST124, ST126, and ST127). Four STs accounted for 49% of isolates (ST3, 15.7%; ST83, 13.7%; ST7, 9.8%; ST26, 9.8%). Split-tree network analysis resolved isolates to terminal branches; many of these comprised multiple isolates from disparate geographic settings but four branches contained Australian isolates only. ST3 isolates were common in Europe, United States and now Australia, whilst ST8 and ST19, relatively frequent in the United States, were rare/absent amongst our isolates. There was no association between ST distribution (genomic similarity) and the two time periods or with fluconazole susceptibility. WGS identified mutations in the FKS1 (S629P) and FKS2 (S663P) genes in three, and one, echinocandin-resistant isolate(s), respectively. Both mutations confer phenotypic drug resistance. Twenty-five percent (13/51) of isolates were fluconazole-resistant (MIC ≥ 64 μg/ml) of which 9 (18%) had non wild-type MICs to voriconazole and posaconazole. Multiple SNPs were present in genes linked to azole resistance such as CgPDR1 and CgCDR1, as well as several in MSH2; however, SNPs occurred in both azole-susceptible and azole-resistant isolates. Although no particular SNP in these genes was definitively associated with resistance, azole-resistant/non-wild type isolates had a propensity to harbor SNPs resulting in amino acid substitutions in Pdr1 beyond the first 250 amino acid positions. The presence of SNPs may be markers of STs. Our study shows the value of WGS for high-resolution sequence typing of C. glabrata, discovery of novel STs and potential to monitor trends in genetic diversity. WGS assessment for echinocandin resistance augments phenotypic susceptibility testing.

Expanding the Diagnostic Capabilities of Molecular Microbiology by Genomic Methods

BackgroundAntimicrobial resistance (AMR) is a major global health threat, with the greatest burden observed in resource-limited settings. Prior work by the Antibiotic Resistance in Communities and Hospitals (ARCH) research consortium revealed a high burden of multidrug-resistant Escherichia coli (MDRE) colonization in both community and healthcare settings in 6 low- and middle-income countries. We aimed to characterize the genomic diversity of MDRE colonizing strains recovered in ARCH studies to provide a high-resolution picture of AMR in community and healthcare networks on a global scale.Table 1.Distribution of most frequent major colonizing multidrug-resistant Escherichia coli sequence types across community and healthcare settings recovered from the Bangladesh, Botswana and Chile Antibiotic Resistance in Communities and Hospitals study sites (N=2874)*The five most frequently observed sequence types from each site were includedFigure 1.Distribution of most frequent major colonizing multidrug-resistant Escherichia coli sequence types across community and healthcare settings recovered from the Bangladesh, Botswana and Chile Antibiotic Resistance in Communities and Hospitals study sitesFor data visualization purposes, the five most frequently observed sequence types from each site were included.MethodsA cross-sectional, population-based prevalence survey was conducted between December 2018 and March 2022. Fecal and perirectal specimens were collected from hospitalized patients and community participants and plated to selective agars. Target phenotypes included extended-spectrum cephalosporin-resistant (all sites), carbapenem-resistant (all sites) and fluoroquinolone-resistant E. coli (Chile) confirmed through susceptibility testing. MDRE isolates underwent whole genome sequencing (WGS). WGS data from three sites (Bangladesh, Botswana and Chile) underwent quality check (QC), trimming, assembly, species and sequence type (ST) calling. E. coli STs with ≥10 isolates were classified as major STs and those with < 10 isolates were classified as minor.Figure 2.Venn Diagram of unique and shared colonizing multidrug-resistant Escherichia coli sequence types recovered from the Bangladesh, Botswana and Chile Antibiotic Resistance in Communities and Hospitals study sitesResultsA total of 2874 (Bangladesh:1180, Botswana:756, Chile:933) MDRE isolates passed QC and were included. Among recovered MDRE isolates, 306 unique STs (42 major, 264 minor) were observed, 222 in Bangladesh, 107 in Chile and 89 in Botswana. Overall, the five most frequent STs were ST131 (N=407), ST1193 (N=217), ST10 (N=172), ST69 (N=124) and ST 38 (N=99)(Table 1, Figure 1). Only 29 (20 major, 9 minor) STs were shared across all three site (Figure 2). Across healthcare and community settings 127 STs were shared, 73 STs were detected exclusively in the healthcare setting and 106 ST were detected exclusively in the community setting.ConclusionColonizing MDRE strains from global low-resource settings exhibited considerable diversity across disparate global sites with ST overlap between community and healthcare settings.DisclosuresAhmed Babiker, MBBS, MSc, Beckman Coulter Inc.: Advisor/Consultant Jose M. Munita, MD, MSD: Grant/Research Support|Pfizer: Grant/Research Support

Comparative genomic hybridization (CGH) analysis of microscopic tumor samples is allowed by universal DNA amplification using degenerate oligonucleotide primed‐PCR (DOP‐PCR). To evaluate the reliablity of DOP‐PCR CGH, we performed DOP‐PCR CGH and standard CGH in parallel using DNAs extracted from 10 malignant tumors of the hepatobiliary tract and pancreas. Similar results were obtained by both methods with a few exceptions, indicating that DOP‐PCR CGH provides cytogenetic information equivalent to that obtained from standard CGH. We also investigated the sensitivity of DOP‐PCR CGH using sequential dilutions of DNA from microdissected tumor cells. DOP‐PCR using 100 to 800 pg of template DNA yielded successful CGH results. However, less than 50 pg of template DNA was not suitable because of the small amount of generated DNA. These findings suggest that DOP‐PCR CGH is applicable for CGH analysis of tiny specimens which are too small for standard CGH. Accordingly, DOP‐PCR CGH analysis may become a useful method in clinical laboratory examination.

Using different cytogenetic techniques in combination is crucial to studying the high complexity of genetic rearrangements in tumor cells. The 8 clear cell (cc) and 5 papillary (p) renal cell carcinomas (RCC) were analyzed using multicolor fluorescence in situ hybridization (multicolor-FISH), conventional Giemsa banding (G-banding) and comparative genomic hybridization (CGH) analysis. CGH analysis was carried out with DNA from frozen tissue sections and short-term cultures of primary tumors. Using CGH analysis, both tissue sections and cell cultures of ccRCC showed the typical chromosomal changes such as the loss of 3p, 4q, 6q, 8p, 9q, 14 and a gain of 5q and 7. Most imbalances detected by CGH in cell culture could be deciphered by multicolor-FISH and G-banding analysis as unbalanced trans-locations t(3;6)(p11.1;p11.1), t(8;14)(p11.1;q11.1), t(3;5) (p14;q21-22), t(1;15)(p11;q11.1), t(3;15)(p11;q11.1)t(8;17) (p11.1;q11.1), t(8;17)(q22;p11.1). Only one balanced trans-location t(9;18)(q34;q11.1) was shown in ccRCC. CGH of papillary RCC displayed mostly gains of whole chromosomes 7, 12, 16 and 17 and a loss of chromosome Y. There was 1 papillary RCC that displayed a partial gain of chromosome 7, showing an unbalanced translocation t(7;11)(q11.1;q25). The balanced translocations t(2;9)(q11.1;q34) and t(7;15) (q22 approximately 31;q21-22) were registered in pRCC. The combined analysis of RCC by different methods allowed a more accurate characterization of the complex karyotypes of tumor tissue, and offered a comprehensive description of given tumors.

IntroductionGastroenteritis caused by non-O1/non-O139 Vibrio cholerae exhibited an increasing trend in recent years in China. Whole genome sequence (WGS) data could play an important role both in the identification of the outbreaks and in the determination of the serogroup. Here, we present the employment of WGS data in the investigation of two outbreaks caused by non-O1/non-O139 V. cholerae in Guangdong, China, 2020–2021.MethodsWe obtained the whole genome sequence of 66 V. cholerae strains isolated in two outbreaks with next generation sequencing technology. We retrieved the publicly available WGS data of non-O1/non-O139 V. cholerae from public database. We used a pipeline integrated in China Pathogen Identification Net (PIN) to complete the phylogenetic analysis.ResultsTwo outbreaks caused by non-O1/non-O139 V. cholerae were identified using WGS data. These V. cholerae strains were determined as serogroup O5. Type 3 and 6 secretion systems were detected in these serogroup O5 strains. These serogroup O5 strains belonged to sequence type (ST) 88.ConclusionsOur analysis indicated the risk of non-O1/non-O139 V. cholerae leading to outbreaks of diarrheal diseases. The application of genomic data played an important role in the identification of the serogroup of non-O1/non-O139 V. cholerae in the lack of antiserum, which gave an example of the application of genome data in disease surveillance and public health emergency response.

Identification of Four Distinct Regions of Allelic Imbalances on Chromosome 1 by the Combined Comparative Genomic Hybridization and Microsatellite Analysis on Hepatocellular Carcinoma

This study examined the phylogenetic structure of serotype a Haemophilus influenzae (Hia) isolates recovered from patients in Canada. Hia isolates from 490 separate patients and an American Type Culture Collection (ATCC) strain were analyzed by multilocus sequence typing (MLST), with 18 different sequence types (STs) identified. Most (85.7%) Hia patient isolates were typed as ST-23 and another 12.7% belonged to 14 different STs with 6, 5, or 4 MLST gene loci related to ST-23 (ST-23 complex). Core genome single-nucleotide variation phylogeny (SNVPhyl) on whole genome sequence (WGS) data of 121 Hia patient isolates representing all identified STs and the ATCC strain revealed 2 phylogenetic populations, with all the ST-23 complex isolates within 1 population. The other phylogenetic population contained only the ATCC strain and 3 patient isolates. Concatenated hitABC sequences retrieved from WGS data and analyzed by MEGA (Molecular Evolutionary Genetic Analysis) alignment confirmed the phylogeny obtained by SNVPhyl. The sodC gene was found only in isolates in the minor phylogenetic population. The 2 phylogenetic populations of the Canadian Hia isolates are similar to the 2 clonal divisions described for serotype b H. influenzae. Combining MLST, core SNVPhyl, and hitABC gene sequence alignment showed that most (99.4%) Canadian Hia patient isolates belonged to 1 major phylogenetic population.

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

Flat urothelial hyperplasia, defined as markedly thickened urothelium without cytological atypia, is regarded in the new WHO classification as a urothelial lesion without malignant potential. Frequent deletions of chromosome 9 detected by fluorescence in situ hybridization (FISH) have been previously reported in flat urothelial hyperplasias found in patients with papillary bladder cancer. Using comparative genomic hybridization (CGH) and microsatellite analysis, these hyperplasias and concomitant papillary tumours of the same patients were screened for other genetic alterations to validate and extend the previous findings. Eleven flat hyperplasias detected by 5-ALA-induced fluorescence endoscopy and ten papillary urothelial carcinomas (pTaG1-G2) from ten patients were investigated. After microdissection, the DNA of the lesions was pre-amplified using whole genome amplification (I-PEP-PCR). Loss of heterozygosity (LOH) analyses were performed with five microsatellite markers at chromosomes 9p, 9q, and 17p. CGH was performed using standard protocols. In 6 of 11 hyperplasias and 7 of 10 papillary tumours, deletions at chromosome 9 were simultaneously shown by FISH, LOH, and CGH analyses. There was a good correlation between FISH, LOH, and CGH analyses, with identical results in 6 of 10 patients. In addition to deletions at chromosome 9, further genetic alterations were detected by CGH in 9 of 10 investigated hyperplasias, including changes frequently found in invasive papillary bladder cancer (loss of chromosomes 2q, 4, 8p, and 11p; gain of chromosome 17; and amplification at 11q12q13). There was considerable genetic heterogeneity between hyperplasias and papillary tumours, but a clonal relationship was suggested by LOH and/or CGH analyses in 5 of 10 cases. These data support the hypothesis that flat urothelial hyperplasias can display many genetic alterations commonly found in bladder cancer and could therefore be an early neoplastic lesion in the multistep development of invasive urothelial carcinoma.