Demonstration of 23-Gene Expression Profile Test Utility Within PRAME Immunohistochemistry Results: A Case Series
Authors: Gregory A. Hosler, Matthew S. Goldberg, Etan Marks
Published: 2026

ABSTRACT
Abstract:Histopathological examination using hematoxylin and eosin (H&E)-stained tissue is sufficient for diagnosing most melanocytic neoplasms as benign or malignant; however, diagnostic tools such as PRAME (preferentially expressed antigen in melanoma) immunohistochemistry (IHC) and the 23-gene expression profile (23-GEP) test can aid in the analysis of histopathologically ambiguous lesions, which can be referred to as diagnostically challenging, equivocal, indeterminate, or uncertain. When compared with the gold standard of H&E diagnosis by a dermatopathologist, PRAME IHC showed 83.2% sensitivity and 99.3% specificity in an initial validation cohort and 23-GEP showed 91.5% sensitivity and 92.7% specificity in an independent validation cohort. In addition, 23-GEP has been evaluated in 3 studies with patients with known outcomes (eg, metastasis) and has reported metrics of 90.4%–96.3% sensitivity and 87.3%–96.2% specificity in distinguishing benign versus malignant melanocytic neoplasms. In this study, we present the utility of the 23-GEP test within 8 equivocal cases with various PRAME IHC staining patterns. Cases underwent histopathological review and received at least 4 and up to 5 diagnoses each. In samples where PRAME IHC is inconclusive, conflicting, or ambiguous regarding a final diagnosis, the 23-GEP can provide additional diagnostic information to upgrade or downgrade the overall malignant potential of the lesion in question.

INTRODUCTION

Histopathological review through hematoxylin and eosin (H&E)-stained tissue is the gold standard for melanoma diagnosis. Nevertheless, diagnostic concordance among dermatopathologists based on histopathology alone can be lower than 50% when the melanocytic proliferations are in the ambiguous region of the histopathological spectrum.1 This lack of diagnostic concordance has led to a significant increase in the utilization of additional diagnostic tools, including immunohistochemistry (IHC) and gene expression profile (GEP) tests, to aid in melanoma diagnosis.2

Dermatopathologists have a preferred system to use additional diagnostic testing, including IHC testing and use of 23-GEP, to resolve diagnostic ambiguity. IHC is usually performed in-house and can be available for interpretation the same or next day. In fact, according to data from the United States SEER-Medicare linked database, use of IHC has increased from 11% of melanoma cases in 2000 to 51% in 2017, with the increase attributed to differences in training, billing patterns, and defensive medicine.3 PRAME (preferentially expressed antigen in melanoma) IHC was introduced in 2018 and has quickly become a common in-house stain.4 PRAME IHC performance has been measured against lesions with consensus histopathology review with varying results. Positive PRAME expression in melanomas has been reported between 70%–100% and completely negative PRAME expression in 75%–100% of nevi.5 This performance variability may be partially due to the relative subjectivity in PRAME interpretation, which can vary from observer to observer. In addition to some variability in the reported performance of PRAME, there are additional limitations of PRAME IHC, including a lack of validation in patients with known outcomes.6,7 Studies have also indicated that PRAME IHC is uninformative for certain subtypes, such as desmoplastic melanoma and spitzoid neoplasms.5,8 In addition, PRAME-negative melanomas pose a diagnostic dilemma and risk, especially if histopathology is concerning. Conversely, PRAME-positive nevi could lead to overdiagnosis if the PRAME IHC result is indiscriminately incorporated diagnostically. Lesions with heterogeneous PRAME IHC presentation also pose an issue where using traditional PRAME IHC cutoffs could yield a false negative result. Alternative cutoffs which slightly increase the accuracy of PRAME IHC have been proposed in a meta-analysis.9

Molecular tests, including 23-GEP, have proven to be valuable in determining the malignant potential of melanocytic lesions and are often recommended by guidelines.10–13 The 23-GEP test is a diagnostic tool that uses qRT-PCR evaluation of 14 discriminant and 9 control genes in a logistic regression algorithm to provide an objective test result.14 Performance of the 23-GEP has been reported in lesions with histopathological assessment (91.5% sensitivity, 92.7% specificity)15 and lesions with known outcomes (90.4%–96.3% sensitivity, 87.3%–96.2% specificity).16–18 An independent single-institution cohort of 369 patients demonstrated real-world performance of 23-GEP, reporting 92.1% sensitivity and 94.7% specificity.19

Recently, a single-institution study of ambiguous melanocytic lesions (n = 56) sought to compare ancillary testing including PRAME IHC, 23-GEP, and fluorescence in situ hybridization (FISH). Agreement with the final diagnosis was reported at 83.9%, 82.1%, and 77.1%, respectively. However, 23-GEP intermediate results were categorized as discordant.20 Classification of intermediate results as discordant for both benign and malignant final diagnoses does not correlate with previous studies or follow best practices.19 Reporting intermediate results separately for statistical analyses is considered best practice for cross-study comparisons. If intermediate results were considered separately, the agreement with the final diagnosis of 23-GEP would be calculated to be 90.9%.19

The diagnosis of cutaneous melanoma fundamentally remains a histopathological diagnosis that most often does not require the use of ancillary tests. When routine H&E does not yield enough information for the pathologist to determine the lesion's overall malignant potential, the use of more than 1 type of ancillary test has previously been shown to be synergistic in achieving a confident diagnosis.21 In this study, we present the utility of the 23-GEP test in 8 lesions with histopathologically discordant diagnostic designations with PRAME IHC results. Examples of PRAME IHC inconclusive staining, PRAME-negative melanoma, and PRAME-positive nevi are discussed. Of note, it is not the intention of these cases to imply that all melanomas are PRAME IHC-positive, or that inconclusive PRAME IHC staining in an unequivocal melanoma or nevus necessitates additional testing.

MATERIALS AND METHODS

Cohort and Dermatopathology Review

Melanocytic neoplasms were acquired from a single institution through an IRB-approved protocol. For each sample, 9 FFPE unstained slides, 1 H&E slide, and 1 PRAME IHC slide were received along with associated demographic patient information. The H&E slides were electronically scanned at 20× with a Leica Biosystems Aperio AT2 and reviewed digitally with 4–40× magnification using Aperio eSlide Manager 12.4.3.5008. Magnification was captured at 2× and 10×. H&E whole slide images were available for histopathological review along with patient age and lesion location; diagnostic designations of benign, unknown malignant potential (UMP), or malignant were available for selection. PRAME IHC and/or 23-GEP test results were not included during the H&E review process. Each lesion had at least 3 and up to 4 reviews performed by 8 independent board-certified dermatopathologists. Taken together with the submitted lesion's initial diagnostic information, lesions received a minimum of 4 and a maximum of 5 diagnoses for comparison. Lesions were included in the study if they were diagnostically discordant, indicating they had some level of ambiguity.

23-GEP

23-GEP was performed as described previously.14,22 In brief, 9 unstained FFPE slides with at least 10% tumor burden were macro-dissected. RNA was extracted, and qRT-PCR was performed. A logistic regression algorithm was used to produce an aggregated score and binned test result of: suggestive of benign (−16.7 to −2.1), suggestive of malignant (0.0 to 11.1), or intermediate (cannot exclude malignancy) (−2.0 to −0.1).

PRAME IHC

PRAME IHC was performed according to the manufacturers' instructions. In brief, the slides were prepared by using a Biocare Intellipath FLEX stainer. The slides were incubated with the anti-PRAME antibody (Biocare, 1:400 dilution) at room temperature for 50 minutes. The slides were washed with Biocare TBS automation wash buffer. The chromogenic reaction was initiated by Vector ImmPACT DAB and subsequently quenched and washed. The PRAME IHC slides were viewed using an Olympus microscope. PRAME IHC was scored where 0 indicates no visible staining and +4 indicates staining in ≥76% of cells.4 Lower grading of PRAME IHC (ie, from 0 to +3), was not performed; lesions were designated negative or inconclusive, meaning some patchy, dim, or focal staining, but not enough to meet the +4/≥76% criteria.

Case Selection

Initial case selection was based on 23-GEP use with PRAME-positive, -inconclusive, and -negative lesions. Final case selection was based on the review of H&E and PRAME IHC slides to select cases that were of the intent-to-use population. Owing to the high discrepancy of the reviews for Cases 6 and 7, the authors of this manuscript re-evaluated the histopathology and determined final malignant potential.

RESULTS

Eight histopathologically equivocal cases are presented (Table 1). All cases had various levels of diagnostic discordance as assessed by histopathological review, which did not include the results of PRAME IHC or 23-GEP. Five cases had 23-GEP benign results with corresponding PRAME IHC results of negative (n = 1), inconclusive (n = 2), and positive (n = 2). Three cases had 23-GEP malignant results and also had negative (n = 1) or inconclusive PRAME IHC results (n = 2).

TABLE 1.Case Series Diagnostic SummaryHistopathological reviews*#benign/#UMP/#malignantPRAME IHC23-GEPCase 11/2/1InconclusiveBenignCase 21/2/1NegativeBenignCase 33/2/0InconclusiveBenignCase 43/1/0PositiveBenignCase 53/1/0PositiveBenignCase 61/2/1InconclusiveMalignantCase 73/0/1InconclusiveMalignantCase 80/1/3NegativeMalignant*Histopathological reviews are expressed as #benign diagnoses/#UMP diagnoses/#malignant diagnoses.

Case Series Diagnostic Summary

Histopathological reviews are expressed as #benign diagnoses/#UMP diagnoses/#malignant diagnoses.

Utility of 23-GEP Resulted Benign Lesions

Case 1, Figure 1, presents an atypical intraepidermal junctional lesion on the upper back of a 61-year-old woman. Histopathological review resulted in diagnostic discordance with 1 benign diagnosis, 1 malignant diagnosis, and 2 UMP diagnoses. There were large, atypical nests and some pagetoid spread, which were worrisome features of this lesion. PRAME IHC was mostly negative, showing some patchy, positive staining areas. 23-GEP provided a benign result, mitigating the overall malignant potential of the lesion.

Atypical intraepidermal junctional lesion (A, B) located on the upper back of a 61-year-old female patient. This lesion received 1 diagnosis of benign, 2 diagnoses of unknown malignant potential (UMP), and 1 diagnosis of melanoma. Further analyses revealed that 23-GEP results were benign and PRAME IHC interpretation was negative (pigment is melanin) (C, D). All images at 2× and 10× magnification.

Case 2, Figure 2, presents an atypical compound Spitz nevus on the medial elbow of a 40-year-old woman. Histopathological review indicated a high level of discordance, underscoring the ambiguity of this lesion. Diagnostic results were 1 designation for benign, 1 for malignant, and 2 for UMP. The H&E showed Kamino bodies within an inflamed lesion. It was also heavily pigmented with marked atypia. This concerning lesion revealed PRAME IHC negative staining and a benign result for 23-GEP. Taken together, the PRAME IHC and 23-GEP helped quell the malignant potential of this lesion despite the concerning histopathological features.

Inflamed melanocytic lesion with Spitz features (A, B) located on the right elbow of a 40-year-old female patient. This lesion received 1 diagnosis of benign, 2 diagnoses of unknown malignant potential (UMP), and 1 diagnosis of melanoma. Subsequently, this lesion received a benign 23-GEP result and was also PRAME IHC-negative (pigment is melanin) (C, D). All images at 2× and 10× magnification.

Case 3, Figure 3, reveals a severely atypical melanocytic lesion on the mid back of a 30-year-old man. Nests were visualized with marked inflammation and severe atypia. The histopathological review of this lesion was split, with 3 reviewers indicating a benign lesion and 2 indicating a UMP lesion. PRAME IHC did not help to resolve the ambiguity and showed inconclusive results with focal and patchy staining. 23-GEP provided a benign result, supporting a benign diagnosis.

Atypical compound melanocytic lesion (A, B) on the back of a 30-year-old male patient. On multiple diagnostic reviews, this lesion received 3 benign diagnoses and 2 unknown malignant potential (UMP) diagnoses. Further analysis revealed focal and patchy positive PRAME IHC staining (brown, nuclear stain with background melanin) (C, D) and benign 23-GEP results. All images at 2× and 10× magnification.

Case 4, Figure 4, demonstrates a severely atypical melanocytic lesion on the posterior lateral distal thigh of a 34-year-old woman. The histopathological assessment of this lesion was 3 indications for benign and 1 indication for UMP. The lesion was small in size but with a marked amount of pagetoid spread. PRAME IHC showed areas of diffuse positivity with additional patchy areas of staining that could potentially influence the diagnosis of this lesion. 23-GEP was benign, which reinforced most histopathologic diagnoses.

Atypical melanocytic lesion located on the distal thigh of a 34-year-old female patient. This lesion received 3 diagnoses of benign and 1 diagnosis of unknown malignant potential (UMP). 23-GEP results were benign and PRAME IHC (brown, nuclear) interpretation was positive, which were not part of the histopathological review process. All images at 2× and 10× magnification.

Case 5, Figure 5, is an atypical junctional melanocytic proliferation in the toenail matrix of a 13-year-old boy. The age and lesion site argued against melanoma, but the lesion stained positive for PRAME IHC at the junction with possible overstaining of the keratinocytes. The histopathological review, which took place without knowing the PRAME result, supported most benign results, where 3 reviewers indicated a benign diagnosis and 1 indicated a UMP diagnosis. The 23-GEP result was benign. In this case demonstration, 23-GEP was able to provide information for challenging a potential overdiagnosis of melanoma in the context of PRAME positivity.

Junctional atypical melanocytic proliferation (A, B) located in the toenail matrix of a 13-year-old male patient. This lesion received 3 diagnoses of benign and 1 diagnosis of unknown malignant potential (UMP). Subsequently, this lesion received a benign 23-GEP result and positive PRAME IHC interpretation (brown, nuclear) (C, D). All images at 2× and 10× magnification.

Utility of 23-GEP Resulted Malignant Lesions

Case 6, Figure 6, reveals a malignant melanoma, superficial spreading subtype, on the upper back of a 72-year-old man. The histopathological review results indicated that this lesion was highly ambiguous, where 1 dermatopathologist designated benign, 1 designated malignant, and 2 designated UMP. The lesion showed marked fibrosis and potentially a melanoma arising in a background dysplastic nevus. PRAME expression was observed, but it was not strong/diffuse. 23-GEP was able to clarify the malignant potential of the lesion with a malignant result as determined by additional review by the authors.

Atypical melanocytic lesion (A, B) on the back of a 72-year-old male patient. On multiple diagnostic reviews, this lesion received 1 benign diagnosis, 2 unknown malignant potential (UMP) diagnoses, and 1 malignant diagnosis. Further analysis revealed positive PRAME IHC staining (brown, nuclear) (C, D) and malignant 23-GEP results. All images at 2× and 10× magnification.

Case 7, Figure 7, reveals a lesion clinically diagnosed as malignant melanoma on the mid back of a 72-year-old man. Initial histopathological assessment revealed an atypical melanocytic lesion with concerning features. There were some nests in the dermis where superficially invasive melanoma was considered. There was marked pagetoid spread as highlighted in the inset of Figure 7B. This lesion received 3 designations of benign and 1 designation of malignant during histopathological review. PRAME IHC was inconclusive and revealed patchy positive staining. 23-GEP defended the final diagnosis of melanoma with a malignant test result as determined by additional review by the authors.

Atypical melanocytic lesion (A, B) on the back of a 72-year-old male patient. This lesion received 3 benign diagnoses and 1 melanoma diagnosis. A malignant 23-GEP test result and inconclusive PRAME (brown, nuclear with partial positivity) IHC results (C, D) were received for this lesion after review. Images at 2× and 10× magnification. The inset in panel B highlights the marked pagetoid spread within this melanoma.

Case 8, Figure 8, presents a nodular malignant melanoma on the upper back of a 57-year-old woman. Most reviewing dermatopathologists (3) indicated a malignant designation and 1 indicated a UMP designation. PRAME IHC was primarily negative. The 23-GEP test result was malignant, supporting a malignant diagnosis of this lesion in the context of negative PRAME staining.

A concerning melanocytic lesion through histopathology (A, B) located on the back of a 57-year-old woman. This lesion received 1 diagnosis of UMP and 3 diagnoses for melanoma (nodular type). This lesion received a malignant 23-GEP result; however, it was PRAME IHC focal and weakly positive (nuclear, brown) (C, D). All images at 2× and 10× magnification.

DISCUSSION

Equivocal melanocytic lesions present a diagnostic quandary where additional histopathological and diagnostic tools may be used to obtain a definitive diagnosis.23,24 As the evidence regarding diagnostic discordance grows, objective diagnostic tools continue to play a prominent role in obtaining a definitive diagnosis for melanocytic lesions.25 This case series demonstrates that the 23-GEP adds independent and objectively quantifiable gene expression profile information that can augment interpretation of H&E and PRAME IHC information in 8 diagnostically challenging melanocytic lesions. Specifically, the 23-GEP was able to provide an objective result in 5 lesions with inconclusive PRAME IHC staining, which served to strengthen or abate the malignant potential in the context of conflicting histopathological features. In addition, 23-GEP was able to align with histopathological impressions in the context of conflicting PRAME IHC results in 3 cases.

Dermatopathologists commonly use PRAME IHC staining to provide diagnostic information in combination with a histopathological impression.26 PRAME has been shown to have accuracy metrics of 70%–100% sensitivity and 75%–100% specificity in lesions using histopathological consensus as the comparator.5 However, studies comparing PRAME IHC interpretation to diagnoses with known outcomes are rare.27 Moreover, a limitation of PRAME IHC testing is the subjective interpretation of the staining, where accuracy and thresholds for positive/negative likely differ between laboratories and between individuals, with the possibility of inadvertently altering those thresholds over time as the pathologist gains experience. Although, the most common threshold for PRAME positivity (≥76%) was used in this study, alternative cutoffs for PRAME IHC staining have been proposed.9 Further, PRAME IHC staining commonly provides results that contrast with the original histopathological impression or results in staining that is neither confirmatory nor contrary to a melanoma diagnosis. In addition, the subjective interpretation nature of PRAME IHC allows dermatopathologists to align the results with their pretest bias. In these circumstances, additional diagnostic information, ideally objective information that can be interpreted in combination with known clinical, histopathological, and IHC/molecular information can be sought to arrive at a confident diagnosis.

In contrast to the subjective nature of PRAME IHC, the 23-GEP offers objective information using GEP and a validated algorithm to provide diagnostic clarity. The 23-GEP has demonstrated accuracy metrics of 91.5% sensitivity and 92.7% specificity when compared with histopathology and 90.4%–96.3% sensitivity and 87.3%–96.2% specificity in studies with known patient outcomes.14–19 Notably, the 23-GEP test includes the PRAME gene in its qRT-PCR-based testing algorithm. As described in the original validation, each component of the test demonstrated a distinct expression pattern with minimal correlation to the others, indicating that each contributes independent information to the overall test result. Although studies correlating PRAME transcriptional data and protein detectible by IHC are not available, full concordance of PRAME IHC positivity with a 23-GEP malignant test result is not expected. In addition, in a recent study, cases with 23-GEP test results and PRAME IHC reported an overall kappa statistic (concordance) of 0.29, which is described as poor to fair agreement.28,29 Importantly, as demonstrated in the cases discussed in this study, 23-GEP added diagnostic information independent of the PRAME IHC result, indicating that 23-GEP uses additional components to provide a final objective test result.

In conclusion, synergistic utilization of diagnostic tools is often needed to arrive at a confident and correct diagnosis; however, diagnostic tools should always be used in the context of histopathological impressions and, therefore, are not a replacement for careful histopathological assessment. When PRAME IHC is used but does not contribute toward a confident diagnosis because of inconclusive staining or disagreement with histopathologic assessment, the 23-GEP can be used to increase the diagnostic accuracy.