ABSTRACT
MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.
Subject(s)
Biomarkers, Tumor/analysis , Myeloid Differentiation Factor 88/analysis , Receptors, CXCR4/analysis , Waldenstrom Macroglobulinemia/diagnosis , Aged , Aged, 80 and over , B-Lymphocytes/chemistry , B-Lymphocytes/pathology , Clone Cells , Female , Humans , Male , Middle Aged , Mutation , Myeloid Differentiation Factor 88/genetics , Plasma Cells/chemistry , Plasma Cells/pathology , Polymerase Chain Reaction , Receptors, CXCR4/genetics , Sensitivity and Specificity , Tumor Cells, Cultured , Waldenstrom Macroglobulinemia/pathologyABSTRACT
BACKGROUND: Mammaglobin mRNA expression is found in 70-80% of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P, and gamma-aminobutyrate type A receptor pi subunit (GABApi) complement the expression of mammaglobin. Collectively the expression profile of these four genes could be used as a diagnostic and prognostic indicator for breast cancer. METHODS: A multigene reverse transcription-PCR (RT-PCR) assay was established to detect the expression of mammaglobin, GABApi, B305D, and B726P simultaneously. Specific primers and TaqMan probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens. RESULTS: The multigene RT-PCR assay detected substantial expression signals in 27 of 27 primary tumor and 50 of 50 metastatic breast lymph node samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various healthy tissue samples, or in 14 colon tumor samples. CONCLUSION: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Lymph Nodes/chemistry , Neoplasm Proteins/analysis , Uteroglobin/analysis , Amino Acid Substitution , Breast Neoplasms/pathology , Colonic Neoplasms/chemistry , Female , Gene Expression Profiling , Humans , Lymphatic Metastasis , Mammaglobin A , Neoplasm Proteins/genetics , Protein Subunits , Receptors, GABA-A/analysis , Receptors, GABA-A/genetics , Sensitivity and Specificity , Uteroglobin/geneticsABSTRACT
Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p < 0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of approximately 3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small alpha-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.
