RB1 gene expression has been reported to be upregulated in colorectal carcinomas (CRC) at both the mRNA and protein levels when compared to normal colonic mucosa. However, allelic loss at the genomic level has been detected in CRC with widely differing frequencies ranging from 11.5% to 50%. To determine whether there is indeed a correlation between RB1 allelic imbalance (AI) and expression, a consecutive series of 55 CRC from Singapore patients were analysed by microsatellite analysis, real-time RT-PCR and immunohistochemistry. Microsatellite analysis using 3 RB1 intragenic microsatellite markers and 2 markers flanking RB1 detected AI in 32.7% (18/55) of the cases, in at least 1 locus. The highest AI frequency (22.9%) was observed at the microsatellite marker D13S137 (Cu13), which maps 5 cM distal to RB1. AI was present in both early and late Dukes stages. Real-time RT-PCR revealed that all 40 cases analysed expressed RB1 mRNA, with mRNA overexpression in 37.5% (15/40) and pRB protein expression in 88.2% (30/ 34) of cases. Notably, a statistically significant correlation was found between AI of RB1 and mRNA overexpression of RB1 (p < 0.001, Fishers exact test). These findings provide evidence that despite AI, RB1 expression is not abrogated. Thus, our data suggests that RB1 may play a role in colorectal tumorigenesis through functional regulation of the transcript and protein rather than through its tumour suppressor role by gene inactivation. ' 2006 Wiley-Liss, Inc.Key words: RB1; colorectal cancer; allelic imbalance; expressionThe retinoblastoma gene, RB1, is a well-known tumour suppressor gene that encodes a nucleoprotein (pRB) with a critical role in cell cycle regulation, proliferation, differentiation and apoptosis. Numerous studies have shown frequent loss of RB1 function and either low or no pRb expression in different tumour types, such as pituitary adenomas, 1 glioblastomas, 2,3 hepatocellular carcinomas 4 and sporadic retinoblastomas. 5 In contrast, RB1 gene expression has been reported to be upregulated in colorectal carcinomas (CRC) at both the mRNA and protein levels when compared to normal colonic mucosa.6-13 Consistent with this increased expression, RB1 transcripts and pRb are not truncated or absent in colorectal cancers, suggesting that Rb is functional in CRC. 12,14 At the genomic level, allelic loss detected by loss of heterozygosity (LOH) has also been reported in the RB1 region in CRC with widely differing frequencies ranging from 11.5% 15 to as high as 50% of cases studied.16,17 These differences in frequencies may be because LOH assays actually determine allelic imbalance which may indicate either loss or gain. Increased allelic copy number at polymorphic loci of RB1 has also been documented by densitometric comparisons of Southern blots in 32% of colorectal tumours, 18 as well as by fluorescence in situ hybridization analysis in 44% of colorectal adenomas. 19 The presence of RB1 allelic loss would suggest that its expression would be decreased, contradictory to previous expression stud...

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AimsMultiple myeloma (MM) is a heterogeneous disease characterised by genetically complex abnormalities. The classical mutational spectrum includes recurrent chromosomal aberrations and gene-level mutations. Recurrent translocations involving the IGH gene such as t(11;14), t(4;14) and t(14;16) are well known. However, the presence of complex genetic abnormalities raises the possibility that fusions other than the recurrent IGH translocations exist. We therefore employed a targeted RNA-sequencing panel to identify novel putative fusions in a local cohort of MM.MethodsTargeted RNA-sequencing was performed on 21 patient samples using the Illumina TruSight RNA Pan-Cancer Panel (comprising 1385 genes). Fusion calls were generated from the Illumina RNA-Sequencing Alignment software (V.1.0.0). These samples had conventional cytogenetic and fluorescence in situ hybridisation data for the common recurrent chromosomal abnormalities (t(11;14), t(4;14), t(14;16) and 17p13 deletion). The MMRF CoMMpass dataset was analysed using the TopHat-fusion pipeline.ResultsA total of 10 novel fusions were identified by the TruSight RNA Pan-Cancer Panel. Two of these fusions, HGF/CACNA2D1 and SMC3/MXI1, were validated by reverse transcription PCR and Sanger sequencing as they involve genes that may have biological relevance in MM genesis. Four of these (MAP2K4/MAP2K4P1) are likely to be spurious secondary to misalignment of reads to a pseudogene. One record of the HGF/CACNA2D1 fusion was identified from the MMRF CoMMpass dataset.ConclusionsThe identification of novel fusions offers insights into the biology of MM and might have clinical relevance. Further functional studies are required to determine the biological and clinical relevance of these novel fusions.

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4792 Chromosomal abnormalities associated with unfavorable prognosis in multiple myeloma patients include involvement of the TP53, FGRFR3 and MAF genes. It is important to accurately detect these chromosomal abnormalities to aid clinicians in the decision of therapeutic plans. Chromosomal abnormalities are not detected by traditional karyotyping due to low proliferative rate of myeloma cells. Conventional Fluorescence In-situ Hybridization (FISH) enhances the sensitivity but lacks the specificity as it does not distinguish plasma cells (PC) from the other hematopoetic cells. This can be overcome by identification of PCs by cytoplasmic immunoglobulin staining followed by FISH (cIg-FISH). Currently, cIg-FISH is done by two methods – lysing red cells in the bone marrow aspirates and spinning the pellet onto charged slides using the Cytospin machine (Ahmann et al Cancer Genet Cytogenet 1998). The second technique uses cultured and fixed cells stored in Carnoy's fixative (Filkova et al 2006 http://www.myeloma.cz/res/file/archiv/2007-cytogen-sbornik-workshop.pdf). This second technique is more easily incorporated into the routine cytogenetic protocols used for chromosomal analysis. However, clumping and the small size of plasma cells seemed to be a major setback with the protocol. We have made minor modifications to this technique, with a different approach to fixing and dropping the cells on to the slides to give nicely separated plasma cells. These when subjected to immunostaining with kappa or/and lambda antibodies, followed by FISH, result in easily identifiable plasma cells with good bright signals under the fluorescence microscope. Twenty samples from patients with multiple myeloma were subjected to routine FISH, cIg-FISH, chromosomal karyotyping along with flow cytometry and the results were compared. Three FISH probes from Vysis for t(4;14), t(14;16) and deletion of TP53 were used. To test the utility of the technique for stored cell pellets, 4 fixed pellets stored from 2005–2008 were also tested with good results. Table 1. Comparative analysis of 20 patients with multiple myeloma by three different methods No % Plasma cells Conventional Karyotyping Conventional FISH Conventional cIg FISH 1 47 Normal Normal Normal 2 55 Monosomy 14 Monosomy 14 Monosomy 14 3 71 der(1;16) Monosomy 16 Monosomy 16 4 6 Normal Normal Normal 5 2 Normal Normal Normal 6 72 der(12;16) Monosomy 16 Monosomy 16 7 13 Normal Normal Trisomy 14 8 52 Normal Normal Normal 9 8 Monosomy 17 t(4;14),del17p t(4;14),monosomy 16, del17p 10 25 Normal Normal Normal 11 1 Normal Normal Normal 12 31 Normal Trisomy 4 Trisomy 4 13 Not done Not done Normal Normal 14 5 Normal Trisomy 17 Monosomy 14, Trisomy 17 15 19.5 Monosomy 14 t(4;14), monosomy 16 t(4;14), monosomy 16 16 34 Normal Normal Normal 17 50 Normal Normal Normal 18 1.8 Normal Normal Normal 19 28 Normal Normal Normal 20 47 Normal Normal Normal The percent of plasma cells seen in bone marrow aspirates ranged from 1.8–71%. Of 20 samples, 3 samples showed a positive cIg-FISH but a normal conventional FISH result. All samples showed a uniformly higher percent of abnormality with the cIg-FISH protocol due to selection of plasma cells as expected. Figure 1. Kappa/lambda positively stained plasma cells showing FGFR3/IGH translocation Figure 1. Kappa/lambda positively stained plasma cells showing FGFR3/IGH translocation In conclusion, this technique using fixed cells will be a major asset to all cytogenetic laboratories as It saves sample as the entire quantity of bone marrow aspirate can be used to set up cultures for chromosomal karyotyping and part of the fixed pellet can be used for cIg-FISH.There is no necessity of making Cytospin slides - saving time and effort.Stored pellets, especially if cells are well stored and preserved in fixative can be used for retrospective analysis. The technique can be easily incorporated as a routine targeted FISH test for MM samples, which is not practiced in most clinical cytogenetics laboratories at present. Disclosures: No relevant conflicts of interest to declare.