PRAME Antibody

What is PRAME and how does it contribute to melanoma diagnosis?

PRAME is a tumor-associated antigen first identified in melanoma patients and encoded by the PRAME gene. This protein is preferentially expressed in most melanomas, making it an excellent diagnostic biomarker. PRAME functions as a melanocyte differentiation antigen that is overexpressed in both solid and hematologic tumors . Its diagnostic value stems from the fact that PRAME protein expression is detected in most melanomas, while benign lesions typically show minimal or no expression. This differential expression pattern helps pathologists distinguish between malignant melanoma and benign nevi, particularly in challenging cases where traditional H&E staining may not provide a definitive diagnosis .

How prevalent is PRAME expression across different melanoma subtypes?

PRAME expression varies significantly across melanoma subtypes, creating important diagnostic implications. Research has demonstrated diffuse nuclear immunoreactivity for PRAME in 87% of metastatic melanomas and 83.2% of primary melanomas . Among specific subtypes, PRAME is diffusely expressed in:

The notably lower expression in desmoplastic melanomas (35%) represents an important limitation to consider in diagnostic applications . When both in situ and non-desmoplastic invasive melanoma components are present in the same specimen, PRAME expression is typically observed in both components.

How does PRAME expression in melanoma compare to that in benign lesions?

A key advantage of PRAME antibody in diagnosis is the stark contrast between expression patterns in malignant versus benign lesions. Most melanocytic nevi (86.4%) are completely negative for PRAME . When immunoreactivity is observed in benign nevi, it typically appears in only a minor subpopulation of lesional melanocytes and is seen in approximately 13.6% of cutaneous nevi, including dysplastic nevi, common acquired nevi, traumatized/recurrent nevi, and Spitz nevi . Researchers should also be aware that rare isolated junctional melanocytes with PRAME immunoreactivity can occasionally be observed in solar lentigines and even in benign non-lesional skin, which may present interpretive challenges in certain contexts.

What are the recommended protocols for PRAME immunohistochemistry detection?

Optimal PRAME antibody detection requires careful consideration of several technical parameters. Based on published protocols, an optimized assay typically includes:

• Heat-induced antigen retrieval for 30 minutes using high pH buffer (ER2, Leica)

• Incubation with monoclonal antibody EPR20330 at a dilution of 1:1000 (0.5 μg/ml) for 30 minutes

• Use of automated staining platforms such as Leica-Bond-3 (Leica Biosystems Inc)

For different applications, specific dilution ratios are recommended:

• Western Blot (WB): 1:500-1:1000

• Immunoprecipitation (IP): 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry (IHC): 1:50-1:500

Importantly, researchers should be aware that antigen retrieval can be performed with either TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0, though performance may vary between these methods .

What controls should be used when validating PRAME antibody performance?

Proper validation requires appropriate positive and negative controls. For positive controls, testis and seminoma tissues have been identified as reliable options due to their consistent PRAME expression . For cell line controls, K-562 cells show positive results in both Western Blot and Immunoprecipitation applications .

Validation should include concordance assessment between known PRAME mRNA expression levels and IHC staining patterns. Ideal validation includes:

• Cell lines with high mRNA expression should show homogeneous intense nuclear PRAME staining

• Cell lines with non-detectable PRAME mRNA should show absence of IHC staining

• Cell lines with intermediate PRAME mRNA levels should demonstrate patchy PRAME staining of variable intensity

How should researchers interpret PRAME immunostaining patterns in clinical specimens?

Interpretation of PRAME immunostaining requires understanding characteristic patterns associated with different lesion types. Key interpretive guidelines include:

• Melanoma pattern: Typically shows diffuse nuclear immunoreactivity throughout the lesion

• Nevus pattern: Most are completely negative; when positive, usually shows focal or patchy staining limited to a minor subset of lesional cells

• Ambiguous melanocytic lesions: Variable patterns that may not clearly align with either melanoma or nevus patterns

The subcellular localization of PRAME staining is primarily membranous and nuclear . When evaluating challenging cases, it's crucial to integrate PRAME staining results with morphological assessment and other diagnostic markers rather than relying on PRAME alone.

How does PRAME antibody perform in ambiguous melanocytic lesions (AMLs)?

The utility of PRAME antibody in ambiguous melanocytic lesions (those difficult to classify as either benign or malignant) remains somewhat controversial. Research indicates that PRAME positivity is significantly higher in melanoma compared to AML and nevus groups, but there is often no statistically significant difference between nevus and AML groups . This suggests potential limitations in using PRAME as a standalone marker for AMLs.

A statistical model incorporating multiple factors showed that high mitosis count, central pagetoid spread, and PRAME positivity together increased the probability of melanoma against an AML diagnosis . This highlights the importance of utilizing PRAME antibody as part of a comprehensive diagnostic approach rather than in isolation.

How does PRAME antibody compare with other diagnostic markers in melanoma detection?

PRAME antibody should be evaluated alongside other established markers for optimal diagnostic accuracy. Research has demonstrated the advantages of evaluating PRAME in conjunction with morphological features and other immunohistochemical markers, particularly Ki-67 and HMB-45 . While PRAME shows strong diagnostic utility, other markers provide complementary information:

• Ki-67: Provides valuable proliferation index data that helps distinguish between melanoma, AML, and nevus groups

• HMB-45: Staining pattern offers significant discriminatory value between the three groups

• P16: Shows limited utility in supporting differential diagnosis compared to other markers

It's important to note that in challenging melanocytic tumors, results of PRAME IHC and other ancillary tests often correlate well, but not always—the tests are not interchangeable and should be used in combination .

What is the utility of PRAME antibody in metastatic melanoma evaluation?

PRAME antibody demonstrates high value in the assessment of metastatic melanoma. Most metastatic melanomas are positive for PRAME, whereas nodal nevi are typically negative . This differential expression pattern makes PRAME antibody particularly useful for:

• Confirming melanoma metastasis in lymph nodes and other sites

• Distinguishing between metastatic melanoma and benign nodal nevi

• Potential use in margin assessment of a known PRAME-positive melanoma

What factors impact the variability of PRAME antibody staining results across laboratories?

Significant variability in PRAME immunoreactivity patterns can occur across different laboratories due to several factors:

• Antibody selection: While mAb EPR20330 is the most widely used primary anti-PRAME antibody for IHC, newer antibodies have become available with potentially different performance characteristics

• Protocol variations: Different laboratories employ variable antibody concentrations, incubation times, and antigen retrieval methods

• Staining platforms: Various automated staining platforms may yield different results even with identical protocols

• Storage and handling: PRAME antibody should be stored at -20°C and is stable for one year after shipment. Aliquoting is unnecessary for -20°C storage, and some formulations contain 0.1% BSA

Formal studies comparing performance profiles across antibodies and immunohistochemistry protocols would be valuable to inform comparability of results across studies .

How can researchers optimize PRAME antibody staining for challenging specimens?

For challenging specimens, several optimization strategies can improve PRAME antibody performance:

• Titration: It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as sample-dependent factors may affect performance

• Antigen retrieval modification: For difficult cases, comparing results using both high pH (TE buffer pH 9.0) and moderate pH (citrate buffer pH 6.0) antigen retrieval methods may yield improved results

• Detection system selection: PRAME antibody can be validated for use with multiple detection systems, including OptiView DAB IHC Detection, ultraView Universal DAB Detection, and ultraView Universal Alkaline Phosphatase Red Detection Kits, providing flexibility to laboratories and accommodating pathologists' preferences

What are the current limitations in PRAME antibody research that require further investigation?

Despite its utility, several key limitations and research gaps exist in PRAME antibody applications:

• Variability across platforms: Significant differences in immunoreactivity patterns observed using different platforms and/or antibodies require formal comparative studies

• Expression in benign lesions: The presence of PRAME expression, albeit usually only in a minor subpopulation of lesional melanocytes, in 13.6% of cutaneous nevi can create diagnostic challenges

• Utility in AMLs: The controversial utility of PRAME in ambiguous melanocytic lesions requires further investigation with larger cohorts and standardized assessment criteria

• Correlation with genomic data: Additional research correlating PRAME antibody expression patterns with genomic profiling of melanocytic lesions could enhance diagnostic precision