Expression of multiple myeloma associated markers in bone marrow spicules using a novel immunohistochemical technique

Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, 72205, USA.
Biotechnic & Histochemistry (Impact Factor: 1.44). 05/2010; 86(2):119-23. DOI: 10.3109/10520290903565978
Source: PubMed


Immunohistochemistry (IHC) is an important tool used for diagnosis and prognosis of several hematological malignancies, and it frequently is used for quantitative and qualitative analysis of expression of different protein biomarkers in tissue sections. To understand the histopathological alterations in multiple myeloma (MM), IHC analysis of bone marrow (BM) biopsy is commonly used. Owing to the harsh decalcification process generally used for processing of bone marrow biopsies, however, protein epitopes occasionally are rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin clot matrix that allows IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Using paired BM spicule-clot and BM core biopsies from patients diagnosed with multiple myeloma, we studied six MM related antibodies including kappa and lambda immunoglobulin light chains, CD56, CD138, CYR61 and DKK1.

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Available from: Madhumita Santra, May 29, 2014
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    • "CCN1 CCN1 is a biomarker and possible therapeutic target in multiple myeloma. Immunohistochemistry on bone marrow spicules or from bone marrow core biopsies from multiple myeloma patients found CCN1 to be expressed strongly in the cytoplasm of lymphoid and myeloid cells but not plasma cells (Santra et al. 2011). A possible role of CCN1 in the MSC response to multiple myeloma indicated the protein could reduce bone lesions (Li et al. 2012). "
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    • "It is expressed in regenerating/ proliferating MSC and osteoblast precursors but not in mature bone cells [7,8]. Earlier and recent reports have demonstrated that CCN1 is expressed in MM myeloid and lymphoid cells, as well as other tumor cells such as breast and prostate cancer, and may contribute to tumor cell survival by providing signals stimulating angiogenesis and survival through acquire anoikis resistance [14,44,46-48]. We observed CCN1 intron splicing in INA-6 myeloma cells for all four introns of the CCN1 mRNA and enhanced transcription of CCN1 after co-culture with MSC. "
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    ABSTRACT: CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.
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