The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffm sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.

Ewing sarcoma family of tumors share recurrent translocations that fuse EWS from 22q12 to ®ve dierent members of transcription factors namely FLI-1, ERG, ETV1, E1AF and FEV. Dierent classes of DNA binding proteins, ATF1, WT1 and CHOP are fused to EWS generating distinct tumor phenotypes: clear cell sarcoma, desmoplastic small round cell tumor, and myxoid liposarcoma, respectively. We have cloned a novel gene located at 22q12 fused to EWS by a submicroscopic inversion of 22q in a small round cell sarcoma showing a translocation (t(1;22)(p36.1;q12). The gene, designated ZSG (Zinc ®nger Sarcoma Gene), is a putative Cys 2 -His 2 zinc ®nger protein which contains a POZ transcriptional repressor-like domain at the Nterminus. The rearrangement involves intron 8 of EWS and exon 1 of ZSG creating a chimeric sequence containing the transactivation domain of EWS fused to zinc ®nger domain of ZSG. This product lacks the transcriptional repressor domain at the N-terminus of ZSG. A rearrangement of the second ZSG allele was also found in tumor cells. This is the ®rst example of an intra-chromosomal rearrangement of chromosome 22, undetectable by cytogenetics, activating EWS in soft tissue sarcoma. Oncogene (2000) 19, 3799 ± 3804.Keywords: sarcoma; EWS; fusion; POZ/BTB domain; zinc ®ngerWe present here the ®rst report of an intrachromosomal rearrangement of chromosome 22 activating EWS by fusion with a novel zinc ®nger gene in a soft tissue sarcoma.The tumor occurred in a male boy aged 16 years, presenting with a pulmonary metastasis 2 years after a primary tumor of the chest wall. Histologically both primary and metastatic tumors were highly suggesting for pPNET, possibly Askin-Rosai type owing to the primary site. Immunophenotyping performed on primary and metastatic tumor showed reactivity for synaptophysin (AO10, Dako) and NSE (MIG-N3, Sambio) and the same small clusters and scattered cells decorated with desmin (D33, Dako) and lowweight cytocheratins (CAM 5.2, Becton Dickinson). A negative staining was observed with neuro®laments (RPN1105, Amersham) and MIC2 antigen (013, Signet). The latter ®nding, being MIC2 a hallmark of pPNET, prompted the diagnosis of a small round cell tumor with multidirectional dierentiation rather than of pPNET.Cytogenetic and¯uorescence in situ hybridization (FISH) analyses of the small round cell sarcoma under study revealed a translocation t(1;22)(p36.1;q12) (Figure 1a ± c). No structural involvement of chromosome 11, indicative of the translocation t(11;22) described in 90% of Ewing's tumors (Turc-Carel et al., 1983), (Sorensen et al., 1994) was observed.To rule out a molecular fusion of EWS with already known partners, we performed RT ± PCR on tumor RNA to assess the presence of EWS/FLI-1 (May et al., and Gerald, 1994;Gerald et al., 1995) fusion products. We could not detect PCR products with any primer set (data not shown).We tested the tumor DNA for the presence of EWS gene alterations by Southern blot analyses using as probe a partial EWS cDNA clone (Delattre et al., 1992). An abnormal r...

Background Two mutations in the MYBPC3 gene have been identified in Maine Coon (MCO) and Ragdoll (RD) cats with hypertrophic cardiomyopathy (HCM). Objective The present study examines the frequency of these mutations and of the A74T polymorphism to describe their worldwide distribution and correlation with echocardiography. Animals 1855 cats representing 28 breeds and random bred cats world-wide of which 446 underwent echocardiographic examination. Methods This is a prospective cross sectional study. Polymorphisms were genotyped using Illumina VeraCode GoldenGate or by direct sequencing. The disease status was defined by echocardiography according to established guidelines. Odds ratios for the joint probability of having HCM and the alleles were calculated by meta-analysis. Functional analysis was simulated. Results The MYBPC3 A31P and R820W were restricted to MCO and RD respectively. Both purebred and random bred cats had HCM and the incidence increased with age. The A74T polymorphism was not associated with any phenotype. HCM was most prevalent in MCO homozygote for the A31P mutation and the penetrance increased with age. The penetrance of the heterozygote genotype was lower (0.08) compared to the P/P genotype (0.58) in MCO. Conclusions and Clinical Importance A31P mutation occurs frequently in MCO cats. The high incidence of HCM in homozygotes for the mutation supports the causal nature of the A31P mutation. Penetrance is incomplete for heterozygotes at A31P locus, at least at a young age. The A74T variant does not appear to be correlated with HCM.

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.