MUM1 Antibody

What is MUM1 and why is it important in research?

MUM1 (Multiple Myeloma Oncogene 1) is a transcription factor also known as Interferon Regulatory Factor 4 (IRF4). It plays crucial roles in multiple stages of lymphocyte development, particularly B-cell differentiation and T-cell function. The human MUM1 protein has a canonical length of 451 amino acid residues with a molecular weight of approximately 51.8 kilodaltons, although two isoforms have been identified .

MUM1 is primarily localized in the nucleus and is notably expressed in the rectum, lymph node, colon, bone marrow, and appendix . Its importance lies in its role in:

• Myeloid dendritic cell differentiation

• Regulation of transcription

• B-cell development, especially in the late stages of germinal center (GC) B-cell differentiation

• Differentiation of mature B cells into plasma cells

• Th2 and Th17 T-cell differentiation and T-cell cytotoxic function

Due to its distinctive expression patterns in normal and neoplastic tissues, MUM1 antibodies serve as valuable diagnostic markers in hematopathology and cancer research.

How does MUM1 expression correlate with B-cell differentiation stages?

MUM1 expression follows a specific pattern during B-cell development that makes it particularly useful for understanding B-cell maturation and the histogenesis of B-cell lymphomas:

• MUM1 is absent in mantle zone B cells (pre-GC B cells)

• It begins expression only at the centrocyte stage in the light zone of germinal centers

• Expression continues and increases during post-germinal center maturation toward plasma cells

• Fully differentiated plasma cells show strong MUM1 expression

This pattern contrasts with BCL-6, which is expressed when B cells enter the germinal center and maintained until GC exit. Thus, MUM1 serves as a marker for the transition from BCL-6 positivity (GC B cells) to CD138 expression (immunoblasts and plasma cells) .

Researchers can use this differential expression pattern to study B-cell development and classify lymphoid neoplasms according to their cell of origin.

How is MUM1 antibody used in the classification of Diffuse Large B-Cell Lymphoma (DLBCL)?

MUM1 antibody is a critical component of the Hans algorithm used to classify DLBCL into clinically relevant subtypes:

• Classification procedure: DLBCL cases are categorized as either Germinal Center B-cell-like (GCB) or non-GCB subtype, with the latter generally having a poorer prognosis

• Cut-off value: According to the Hans algorithm, ≥30% MUM1-positive neoplastic B-cells (in the absence of CD10 and presence of BCL6) characterizes the non-GCB phenotype

• Technical considerations: Precise calibration of antibody concentration and proper heat-induced epitope retrieval (HIER) are essential for accurate classification

In research and diagnostic settings, MUM1 immunohistochemistry should be performed in conjunction with CD10 and BCL6 staining to accurately determine DLBCL subtype, which can influence therapeutic decisions and prognosis assessment.

What is the relationship between MUM1 and AID expression in lymphomas?

Activation-induced cytidine deaminase (AID) and MUM1 expression show significant association in DLBCL, providing insight into lymphoma pathogenesis:

• A retrospective cohort study of 20 cases of DLBCL demonstrated a concordant expression rate of AID and MUM1 of 80% with a Cohen's kappa of 0.578 (p=0.004)

• A significant association between AID and MUM1 expression was observed with a prevalence ratio of 2.25 (95% CI: 1.08-4.67; p=0.008)

• MUM1 is known to upregulate AID expression in both normal and pathological conditions

This association is biologically significant because:

• AID enzyme plays a vital role in somatic hypermutation (SHM) and class switch recombination (CSR), which can contribute to lymphomagenesis when dysregulated

• The concordant expression suggests that MUM1 may influence the aberrant activation of AID in DLBCL

• This relationship may reflect additional molecular heterogeneity within DLBCL that could influence prognosis or treatment response

How can MUM1 antibody staining differentiate between AITL with HRS-like cells and classical Hodgkin Lymphoma?

MUM1 staining patterns can be used to differentiate between Angioimmunoblastic T-cell Lymphoma (AITL) with Hodgkin/Reed-Sternberg (HRS)-like cells and classical Hodgkin Lymphoma (cHL):

This distinctive rosetting pattern in AITL is thought to occur because MUM1 is expressed not only in HRS-like cells but also in the neoplastic T-cells surrounding them . One hypothesis suggests that rosetting by neoplastic T-cells derived from follicular T helper cells (TFH) might protect aberrant B-cell clones from immune surveillance, leading to the emergence of HRS-like cells .

This pattern provides a useful diagnostic marker for differentiating these entities when morphologic and immunohistochemical overlap exists.

What are the optimal conditions for MUM1 immunohistochemistry?

For optimal MUM1 immunohistochemistry results, the following technical conditions are recommended based on the Nordic Immunohistochemical Quality Control (NordiQC) assessment:

• Antibody clones: The monoclonal antibody clones MUM1p, EAU32, and the recombinant monoclonal antibody clone EP190 are all recommended for MUM1 detection

• Heat-induced epitope retrieval (HIER):

  • Use of alkaline buffer (pH 9.0) is preferred

  • Optimal HIER time averages 40 minutes (compared to 33 minutes for protocols with insufficient results)

• Detection systems:

  • 3-step polymer or multimer-based detection systems yield superior results

  • For example, BOND Refine (Leica) has been used successfully with mAb EAU32 at dilutions of 1:300-1:400

• Controls:

  • Positive control: Tonsil tissue is recommended, where plasma cells and late-stage germinal center B-cells should show moderate to strong nuclear staining, and dispersed lymphocytes in mantle zones should display at least weak nuclear staining

  • Negative control: Colon tissue, where epithelial, endothelial, and smooth muscle cells should not show staining

The pass rate for MUM1 immunohistochemistry has improved over time in NordiQC assessments: 58% (2011), 60% (2016), and 73% (2020), indicating progressive standardization and improvement in methodology .

What are common technical issues with MUM1 immunohistochemistry and how can they be resolved?

Common technical issues affecting MUM1 immunohistochemistry results include:

• Insufficient heat-induced epitope retrieval (HIER):

  • Problem: Too short heating time or use of inappropriate buffer

  • Solution: Use alkaline buffer (pH 9.0) with minimum 40 minutes heating time

• Poor antibody selection:

  • Problem: Some clones consistently produce inferior results (e.g., rmAb MRQ-43 and mAb BC5)

  • Solution: Use recommended clones like MUM1p, EAU32, or EP190

• Inadequate detection systems:

  • Problem: Less sensitive detection systems yield weak staining

  • Solution: Implement 3-step polymer or multimer-based detection systems

• Improper antibody dilution:

  • Problem: Poor signal-to-noise ratio

  • Solution: Precise calibration of primary antibody concentration (e.g., 1:300-1:400 for mAb EAU32)

• Interpretation challenges:

  • Problem: Difficulty distinguishing true positive staining from background

  • Solution: Use appropriate controls (tonsil as positive control, colon as negative control) and establish clear criteria for nuclear versus cytoplasmic staining

Resolution of these issues can significantly improve staining quality, as evidenced by the improvement in pass rates from 60% in 2016 to 73% in 2020 in NordiQC assessments .

How reliable is MUM1 antibody for identifying melanocytic lesions?

MUM1 antibody has shown considerable utility in identifying melanocytic lesions, with comparable or superior sensitivity to conventional melanocytic markers:

Key findings regarding MUM1 in melanocytic lesions:

• MUM1 demonstrated strong positivity in four cases of conventional melanomas that were negative or weakly positive with HMB45 and anti-MelanA

• MUM1 showed more diffuse and strong staining of benign melanocytic nevi than either HMB45 or anti-MelanA, strongly staining 75% of benign nevi compared with 13% and 63%, respectively

• MUM1 showed particular utility in clear cell sarcoma (malignant melanoma of soft parts), strongly staining 2/2 cases along with S100, while only one case was strongly positive with HMB45, and neither was strongly positive with anti-MelanA

• Importantly, MUM1 did not stain any nerve sheath lesions (neurofibromas, schwannomas, and malignant peripheral nerve sheath tumors) in contrast to S100, making it useful for distinguishing between nerve sheath and melanocytic lesions

These findings suggest that MUM1 can be a valuable addition to melanocytic marker panels, especially for challenging cases where conventional markers may yield equivocal results.

How can single-cell analysis of MUM1-positive germinal center B cells inform lymphomagenesis theories?

Advanced research into MUM1-positive cells in germinal centers has provided insights into B-cell maturation and lymphomagenesis:

PCR analysis of single MUM1-positive cells isolated from germinal centers has revealed that these cells:

• Contain rearranged immunoglobulin heavy chain genes with varying numbers of VH somatic mutations

• Display a distinct phenotype (MUM1+/Bcl-6−/Ki67−) different from that of most germinal center B cells (MUM1−/Bcl-6+/Ki67+) and mantle B cells (MUM1−/Bcl-6−/Ki67−)

• Represent a morphologic spectrum ranging from centrocytes to plasmablasts/plasma cells

These findings suggest that MUM1-positive germinal center B cells represent surviving centrocytes and their progeny committed to exiting the germinal center and differentiating into plasma cells .

For researchers studying lymphomagenesis, these observations provide a framework for understanding the cell of origin of various lymphomas:

• Unlike normal germinal center B cells, where MUM1 and Bcl-6 expression are mutually exclusive, tumor cells in approximately 50% of MUM1-positive diffuse large B-cell lymphomas co-express MUM1 and Bcl-6

• This aberrant co-expression suggests deregulation of these proteins in lymphomagenesis

• Understanding this deregulation may provide insights into lymphoma development and potential therapeutic targets

How do technical factors in MUM1 antibody production affect experimental outcomes?

The technical aspects of MUM1 antibody production can significantly impact experimental outcomes in research:

• Epitope selection is critical:

  • The monoclonal antibody MUM1p generated against amino acids 144-451 of human MUM1 protein avoids N-terminal sequences encoding DNA-binding motifs (which have high homology with other family proteins)

  • This specificity helps prevent cross-reactivity with related interferon regulatory factors

• Antibody clone selection affects staining patterns:

  • Multiple clones are available (MUM1p, EAU32, EP190, MRQ-43, BC5) with variable performance

  • Western blotting studies have confirmed that MUM1p specifically recognizes a 50-52 kDa band corresponding to MUM1 protein in both transfected cells and natural expression systems

• Validation methods ensure reliability:

  • Proper validation includes immunoprecipitation followed by Western blotting with different antibodies

  • In one study, lysates of MUM1p-immunoprecipitated IM9 myeloma cells revealed by Western blotting with an anti-MUM1 polyclonal antibody gave the expected 50-kD band of MUM1

For researchers designing experiments involving MUM1 detection, consideration of these technical factors is essential for obtaining reliable and reproducible results. The choice between monoclonal antibodies (like MUM1p) and recombinant monoclonal antibodies (like EP190) should be based on the specific application and validation requirements of the experiment.

What are the implications of MUM1 and BCL6 co-expression patterns for understanding lymphoma pathogenesis?

The relationship between MUM1 and BCL6 expression provides significant insights into lymphoma pathogenesis:

• Normal B-cell differentiation:

  • In normal germinal centers, MUM1 and BCL6 expression are mutually exclusive

  • BCL6 expression occurs immediately after a B cell enters the germinal center and is maintained until GC exit

  • MUM1 positivity begins only at the centrocyte stage and continues during post-GC maturation

• Deregulation in lymphomas:

  • Unlike normal GC B cells, tumor cells in approximately 50% of MUM1-positive diffuse large B-cell lymphomas co-express MUM1 and BCL6

  • This aberrant co-expression suggests dysregulation of the normal sequential expression pattern

  • The persistence of BCL6 expression in cells that have initiated plasma cell differentiation (indicated by MUM1 expression) represents a pathological state

• Diagnostic and prognostic implications:

  • The co-expression pattern can help identify specific disease subsets within lymphoma categories

  • In B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, MUM1 expression reinforces evidence that this disorder is histogenetically heterogeneous

  • A proportion of cases reflect post-GC B cells, and this histogenetic classification may influence prognosis

• Research applications:

  • Double immunofluorescence labeling for MUM1/BCL6 on tonsil frozen sections allows visualization of their relationship in normal tissues

  • Combination of MUM1, BCL6, CD138/syndecan-1, and analyses of Ig and BCL6 gene mutations provides a powerful tool for understanding lymphoma histogenesis

These findings suggest that disruption of the normal MUM1/BCL6 expression sequence may be a key event in lymphomagenesis, offering potential targets for therapeutic intervention and providing valuable markers for lymphoma classification.