Most melanocytic tumors can be characterized as a benign nevus or a melanoma by a trained pathologist using traditional histopathological methods. However, a minority demonstrates ambiguous features and continues to be a diagnostic challenge. Genetic expression profiling (GEP) assays have been developed in an effort to resolve this dilemma. These assays measure mRNA levels of specified genes using reverse transcription quantitative polymerase chain reaction technology. The development of GEP assays, methodology, challenges associated with GEP validation and testing, and the suitability of a currently available GEP test for clinical use are reviewed.
Key points
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Genetic expression profiling (GEP) is an emerging diagnostic tool used to assist in discrimination between benign nevi and malignant melanomas.
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GEP evaluates a panel of genes with reverse transcription quantitative polymerase chain reaction to determine if mRNA expression is more consistent with a benign or malignant pattern.
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GEP tests face challenges with respect to demonstration of improvement upon the current pathologic “gold standard” for diagnosis of melanoma.
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National Comprehensive Cancer Network Guidelines do not recommend use of GEP diagnostic assays for routine clinical care.
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Like fluorescent in situ hybridization and array comparative genomic hybridization technologies, GEP may supply additional, independent information regarding challenging or ambiguous lesions; results should be considered in the context of the histopathologic findings, on a case-by-case basis.
Introduction
Accurate diagnosis of melanoma is critical. Thin melanomas have a better prognosis than thick melanomas, presumably because they represent earlier lesions that do not have the biological capacity to metastasize. Thus, if appropriately treated, most thin melanomas are curable. In contrast, most advanced melanomas are lethal, despite recent advances in targeted immunotherapies. Most melanocytic tumors can be readily categorized as benign or malignant with traditional histopathology. However, there is a subset of ambiguous/equivocal melanocytic tumors with the potential to metastasize that defy classification with conventional histologic criteria. The interobserver reproducibility among pathologists in the evaluation of challenging melanocytic neoplasms is also poor. Thus, the “gold-standard” histologic evaluation has the potential to miss a subset of lethal melanomas.
The consequences of missing these ambiguous, yet lethal melanomas are dire and frequently result in more aggressive, morbid, costly procedures and treatments, and death in some cases. In addition, the medicolegal risk has reinforced the need for providers to err on the side of caution. For these reasons, there has been a drift in the diagnostic criteria that may increase sensitivity of melanoma detection, at the expense of specificity. This shift toward cautious behavior may protect the patient and providers from misdiagnosis, but may also lead to overtreatment and a disproportionate increase in melanoma incidence rates relative to mortalities. Ideally, a diagnostic test that exceeds the current gold standard would be more objective and increase reproducibility and specificity without compromising sensitivity.
Introduction
Accurate diagnosis of melanoma is critical. Thin melanomas have a better prognosis than thick melanomas, presumably because they represent earlier lesions that do not have the biological capacity to metastasize. Thus, if appropriately treated, most thin melanomas are curable. In contrast, most advanced melanomas are lethal, despite recent advances in targeted immunotherapies. Most melanocytic tumors can be readily categorized as benign or malignant with traditional histopathology. However, there is a subset of ambiguous/equivocal melanocytic tumors with the potential to metastasize that defy classification with conventional histologic criteria. The interobserver reproducibility among pathologists in the evaluation of challenging melanocytic neoplasms is also poor. Thus, the “gold-standard” histologic evaluation has the potential to miss a subset of lethal melanomas.
The consequences of missing these ambiguous, yet lethal melanomas are dire and frequently result in more aggressive, morbid, costly procedures and treatments, and death in some cases. In addition, the medicolegal risk has reinforced the need for providers to err on the side of caution. For these reasons, there has been a drift in the diagnostic criteria that may increase sensitivity of melanoma detection, at the expense of specificity. This shift toward cautious behavior may protect the patient and providers from misdiagnosis, but may also lead to overtreatment and a disproportionate increase in melanoma incidence rates relative to mortalities. Ideally, a diagnostic test that exceeds the current gold standard would be more objective and increase reproducibility and specificity without compromising sensitivity.
Molecular diagnostic tests for melanoma
One candidate diagnostic tool is genetic expression profiling (GEP; Table 1 ). GEP is a relatively recent addition to the diagnostic armamentarium for melanoma and differs from DNA-based testing such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) (tumor cytogenetics). Over the past decade, ambiguous melanocytic tumors have been increasingly evaluated using FISH and/or CGH (see Table 1 ). FISH has the advantage of evaluating for large chromosomal deletions, duplications, and translocations in a subset of chromosomal loci that have a well-established association with melanoma. However, only a limited number of chromosomal loci are evaluated; the interpretation remains subjective, and specialized expertise is required. CGH overcomes some, but not all, of the subjectivity of FISH and allows evaluation of large chromosomal aberrations across the entire genome. However, CGH requires relatively large quantities of tissue and is limited by tumor heterogeneity, making it less reliable in thinner neoplasms. Although FISH and CGH are the most commonly used molecular diagnostic tests to date, GEP is an alternative molecular technology that could improve diagnostic accuracy, by increasing objectivity, reducing variability, and taking advantage of changes in the tumor’s biology. Table 1 summarizes the most salient features of the various diagnostic tests for melanocytic lesions.
| Test | Analytical Validation (Reproducibility) | Clinical Validation | Interpretation | Clinical Utility (Utilization of Results) | Performance vs Outcomes | |
|---|---|---|---|---|---|---|
| Performance | Reference Standard | |||||
| Histopathology | 38% discordance rate among experts reviewing “classic” examples of melanocytic neoplasms | No general systematic validation against outcomes performed; discordance rate for melanocytic tumors varies from 15% to 50% | Subjective | No systematic study of pathology report recommendations vs actual treatment performed | Favorable outcome: Accurately predicted in 47.1% of cases Unfavorable outcome: Accurately predicted in 73% of cases | |
| Favorable outcome accurately predicted in 47.1% of cases, unfavorable outcome accurately predicted in 73% of cases | Clinical follow-up ≥5 y | |||||
| IHC | Variability of IHC has been widely documented | Sensitivity: 69%–100%, Spec: uncertain | Histopathology | Subjective | Changed diagnosis in 11% of all lesions; confirmed or excluded differential diagnosis in 77% | N/A |
| FISH | Clinical validation not published, but required to be on file for CLIA-certified laboratories | Sensitivity range: 43%–100%, specificity range: 29%–80% | Pathologists within the same institutions that developed the test | Subjective | 88% of cases received a more definitive diagnosis | Sensitivity: 100% Specificity: 71% for 21 Outcome-proven cases |
| aCGH | Clinical validation not published, but required to be on file for CLIA-certified laboratories | Sensitivity range: 92%–96%, specificity range: 87%–100% | Institution’s original pathology report | Subjective | Not published to date | 9/10 cases had outcomes that matched aCGH result, 1/10 results was uncertain |
| GEP | 2.5% SD | Sensitivity: 90%, 91.5%, Specificity: 91%, 92.5% | Concordant diagnoses by 2–3 independent pathologists | Objective | 49.1% change in treatment recommendations | TBD |
Genetic expression profiling methodology
Tissue Collection
GEP is a relatively straightforward method used to detect abnormal expression of messenger RNA (mRNA). Ideally, the assay will follow Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines. GEP uses reverse transcription in combination with quantitative polymerase chain reaction (RT-qPCR) technology ( Fig. 1 ). The whole tumor (including the surrounding stromal microenvironment) or a dissected or a laser-captured component can be tested, using fresh, frozen, or formalin-fixed and paraffin-embedded tissue. Microdissection allows an enriched collection of malignant cells and improves the sensitivity by reducing contamination with benign cells. Conversely, use of the undissected tumor has the advantage of capturing expression changes induced in the tumor microenvironment, for example, normal stromal and inflammatory cells.
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