The comparative utility of fluorescence in situ hybridization and reverse transcription-polymerase chain reaction in the diagnosis of alveolar rhabdomyosarcoma.

Fluorescence in situ hybridization (FISH) for FOXO1 gene rearrangement and reverse transcription-polymerase chain reaction (PCR) for PAX3/7-FOXO1 fusion transcripts have become routine ancillary tools for the diagnosis of alveolar rhabdomyosarcomas (ARMS). Here we summarize our experience of these adjunct diagnostic modalities at a tertiary center, presenting the largest comparative series of FISH and PCR for suspected or possible ARMS to date. All suspected or possible ARMS tested by FISH or PCR for FOXO1 rearrangement or PAX3/7-FOXO1 fusion transcripts over a 7-year period were included. FISH and PCR results were correlated with clinical and histologic findings. One hundred samples from 95 patients had FISH and/or PCR performed. FISH had higher rates of technical success (96.8 %) compared with PCR (88 %). Where both tests were utilized successfully, there was high concordance rate between them (94.9 %). In 24 histologic ARMS tested for FISH or PCR, 83.3 % were translocation-positive (all for PAX3-FOXO1 by PCR) and included 3 histologic solid variants. In 76 cases where ARMS was excluded, there were 3 potential false-positive cases with FISH but none with PCR. PCR had similar sensitivity (85.7 %) and better specificity (100 %) in aiding the diagnosis of ARMS, compared with FISH (85 and 95.8 %, respectively). All solid variant ARMS harbored FOXO1 gene rearrangements and PAX3-FOXO1 ARMS were detected to the exclusion of PAX7-FOXO1. In comparative analysis, both FISH and PCR are useful in aiding the diagnosis of ARMS and excluding its sarcomatous mimics. FISH is more reliable technically but has less specificity than PCR. In cases where ARMS is in the differential diagnosis, it is optimal to perform both PCR and FISH: both have similar sensitivities for detecting ARMS, but FISH may confirm or exclude cases that are technically unsuccessful with PCR, while PCR can detect specific fusion transcripts that may be useful prognostically.

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity.

We present clinicopathologic data on 10 pulmonary myxoid sarcomas, which are defined by distinctive histomorphologic features and characterized by a recurrent fusion gene, that appear to represent a distinct tumor entity at this site. The patients [7 female, 3 male; aged 27 to 67 y (mean, 45 y)] presented with local or systemic symptoms (n=5), symptoms from cerebral metastasis (1), or incidentally (2). Follow-up of 6 patients showed that 1 with brain metastasis died shortly after primary tumor resection, 1 developed a renal metastasis but is alive and well, and 4 are disease free after 1 to 15 years. All tumors involved pulmonary parenchyma, with a predominant endobronchial component in 8 and ranged from 1.5 to 4 cm. Microscopically, they were lobulated and composed of cords of polygonal, spindle, or stellate cells within myxoid stroma, morphologically reminiscent of extraskeletal myxoid chondrosarcoma. Four cases showed no or minimal atypia, 6 showed focal pleomorphism, and 5 had necrosis. Mitotic indices varied, with most tumors not exceeding 5/10 high-power fields. Tumors were immunoreactive for only vimentin and weakly focal for epithelial membrane antigen. Of 9 tumors, 7 were shown to harbor a specific EWSR1-CREB1 fusion by reverse transcription-polymerase chain reaction and direct sequencing, with 7 of 10 showing EWSR1 rearrangement by fluorescence in situ hybridization. This gene fusion has been described previously in 2 histologically and behaviorally different sarcomas: clear cell sarcoma-like tumors of the gastrointestinal tract and angiomatoid fibrous histiocytomas; however, this is a novel finding in tumors with the morphology we describe and that occur in the pulmonary region.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin's lymphomas with high rates of somatic hypermutation.

BACKGROUND: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR. AIMS: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies. METHODS: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1. RESULTS: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% (2.3-11.9)) with a clonal rearrangement. CONCLUSIONS: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.

Immunoglobulin heavy chain sequence analysis in Waldenstrom's macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance.

In this study, we used IGH sequence analysis to assess the maturational status of Waldenstrom's (WM) macroglobulinemia and its putative precursor immunoglobulin (Ig)-M monoclonal gammopathy of undetermined significance (MGUS). IGH sequence analysis was performed using standard methods in 23 cases (20 WM and 3 IgM MGUS as defined by consensus panel criteria). Waldenstrom's macroglobulinemia cases were characterized by heavily mutated IGH genes (median, 6.3%; range, 3.8%-13.9%) but without intraclonal variation (ICV). IgM MGUS was similarly characterized by somatic hypermutation (median, 7.5%; range, 7%-7.7%), but ICV was evident in 1 of the 3 cases. We would therefore conclude that WM is characterized by somatic hypermutation without ICV, which supports a derivation from postgerminal center/memory B cells. IgM MGUS is also characterized by somatic hypermutation but, in a manner similar to IgA/IgG MGUS, can be associated with ICV, although the significance of this remains unclear.

Incomplete DJH rearrangements.

Analysis of Ig genes in B-cell malignancies has become an essential method in molecular diagnosis, and polymerase chain reaction (PCR) amplification of Ig heavy chain gene (IgH) rearrangements is now widely used for detection of clonality and minimal residual disease (MRD). Although several different sensitive protocols are now available for PCR analysis of IgH genes, they are frequently hampered owing to the high rate of somatic hypermutation present in multiple myeloma (MM). We recently described a new approach using incomplete DJH rearrangements as an alternative target. About 60% of MM samples contain an incomplete DJH rearrangement, 90% of them lacking on somatic mutations. This approach allows resolution of problems derived from primer mismatches, making DJH rearrangement a reliable and sensitive target for detection of clonality and MRD investigation in MM.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.

An assessment of the geographical scale of recurrent gene flow in wild populations of two species of Mekong River carps (Henicorhynchus spp.)

The Mekong is the most productive river fishery in the world, and such as, the Mekong River Basin (MRB) is very important to very large human populations across the region as a source of revenue (through fishing and marketing of aquatic resources products) and as the major source for local animal protein. Threats to biodiversity in the MRB, either to the fishery sector itself or to other sectors are a major concern, even though currently, fisheries across this region are still very productive. If not managed properly however, fish population declines will cause significant economic impact and affect livelihoods of local people and will have a major impact on food security and nutrition. Biodiversity declines will undoubtedly affect food security, income and socio-economic status of people in the MRB that depend on aquatic resources. This is an indicator of unsustainable development and hence should be avoided. Genetic diversity (biodiversity) that can be measured using techniques based on DNA markers; refers to variation within and among populations within the same species or reproductive units. In a population, new genetic variation is generated by sexual recombination contributed by individuals with mutations in genes and chromosomes. Over time, populations of a species that are not reproducing together will diverge as differential impacts of selection and genetic drift change their genetic attributes. For mud carp (Henicorhynchus spp.), understanding the status of breeding units in the MRB will be important for their long term persistence, sustainability and for implementing effective management strategies. Earlier analysis of stock structure in two economically important mud carp species (Henicorhynchus siamensis and H. lobatus) in the MRB completed with mtDNA markers identified a number of populations of both species where gene flow had apparently been interrupted or reduced but applying these data directly to management unit identification is potentially compromised because information was only available about female dispersal patterns. The current study aimed to address this problem and to fully assess the extent of current gene flow (nDNA) and reproductive exchange among selected wild populations of two species of carp (Henicorhynchus spp.) of high economic importance in the MRB using combined mtDNA and nDNA markers. In combination, the data can be used to define effective management units for each species. In general, nDNA diversity for H. lobatus (with average allelic richness (A) 7.56 and average heterozygosity (Ho) 0.61) was very similar to that identified for H. siamensis (A = 6.81 and Ho = 0.75). Both mud carp species show significant but low FST estimates among populations as a result of lower genetic diversity among sampled populations compared with genetic diversity within populations that may potentially mask any 'real' population structure. Overall, population genetic structure patterns from mtDNA and nDNA in both Henicorhynchus species were largely congruent. Different population structures however, were identified for the two Henicorhynchus species across the same geographical area. Apparent co-similarity in morphology and co-distribution of these two relatively closely related species does not apparently imply parallel evolutionary histories. Differences in each species population structure likely reflect historical drainage rearrangement of the Mekong River. The data indicate that H. siamensis is likely to have occupied the Mekong system for much longer than has H. lobatus in the past. Two divergent stocks were identified for H. lobatus in the MRB below the Khone Falls while a single stock had been evident in the earlier mtDNA study. This suggests that the two Henicorhynchus species may possess different life history traits and that different patterns of gene flow has likely influenced modern genetic structure in these close congeners. In combination, results of the earlier mtDNA and the current study have implications for effective management of both Henicorhynchus species across the MRB. Currently, both species are essentially treated as a single management unit in this region. This strategy may be appropriate for H. lobatus as a single stock was evident in the main stream of the MRB, but may not be appropriate for H. siamensis as more than a single stock was identified across the same range for this species. Management strategies should consider this difference to conserve overall biodiversity (local discrete populations) and this will include maintaining natural habitat and migration pathways, provision of fish sanctuaries (refuges) and may also require close monitoring of any stock declines, a signal that may require effective recovery strategies.