Expert Opinions in Ancillary Testing and Second Opinions

Navigating Ambiguous Melanocytic Neoplasms: A Practical Roundtable on Ancillary Testing

J Clin Aesthet Dermatol. 2026;19(6 Suppl 3):S4–S5.

by Etan Marks, MD, and Drazen Jukic, MD

Dr. Marks is with Advanced Dermatology and Cosmetic Surgery, Delray Beach, Florida. Dr. Jukic is with Georgia Dermatology and Skin Cancer Center in Savannah, Georgia, and Mercer University School of Medicine, Macon, Georgia.

FUNDING: Funding for this article was provided by Castle Biosciences.

DISCLOSURES: Dr. Marks is a consultant for Castle Biosciences. Dr. Jukic is a speaker for Castle Biosciences and Roche.

Introduction

Ambiguous melanocytic lesions remain one of the most persistent diagnostic challenges in dermatopathology.¹ While most cases can be confidently classified on routine histology, a small but critical subset exists in diagnostic gray zone—particularly within the Spitz and Clark’s (dysplastic) nevus spectra. In these scenarios, ancillary testing can be invaluable, but only when applied thoughtfully and interpreted in context.²

In this roundtable discussion, Dr. Etan Marks and Dr. Drazen Jukic explore how experienced dermatopathologists integrate immunohistochemistry (IHC), gene expression profiling (GEP), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) into real-world diagnostic workflows—while keeping patient care, cost, and downstream clinical impact front and center.

First Principles: Most Cases Are Straightforward, BUT Some Are Not

Dr. Jukic begins by emphasizing an often-overlooked truth: the majority of melanocytic lesions are diagnosed with confidence. “In about 90–95% of cases, you know exactly what you’re dealing with and how to prove you’re right. The challenge is the remaining 5–10%.” Those difficult cases demand balance—avoiding overtreatment of benign lesions while ensuring malignant lesions are not undertreated. The goal is not perfection, but responsible risk stratification.

The diagnostic process begins the same way every time: careful assessment of hematoxylin and eosin (H&E) staining. If the diagnosis is clear, it should be rendered without unnecessary testing. Ancillary studies enter the equation only when reasonable doubt persists or when a second observer could plausibly reach a different conclusion.³

Two Major Problem Categories Exist: Spitz and Clark’s Nevi

Spitz lesions, as Dr. Jukic notes, are inherently atypical.

“There’s no worse atypia than what you see in a Spitz nevus—but it’s the wrong kind of atypia.” In his practice, suspected Spitz lesions are routinely worked up before being shown to colleagues. A typical panel includes:

• • PReferentially expressed Antigen in MElanoma (PRAME), with caution
  • p16
  • Cyclin D1
  • SOX10 (distribution)
  • Proliferation markers (Ki-67 or pHH3), often in dual antibodies with Melan-A or HMB45

 

Most classic Spitz nevi demonstrate retained p16, relatively controlled proliferation, and expected maturation patterns. However, when discordant immunophenotypes or dual melanocytic populations emerge, further molecular testing becomes appropriate.

At that point, Dr. Jukic commonly turns to:

• • MyPath Melanoma,⁴ and/or
  • FISH,⁵ when sufficient dermal cells are present.

 

Each result is treated as a “checkbox,” contributing to an integrated interpretation rather than dictating the diagnosis outright.

 

Clark’s (Dysplastic) Nevi: A Different Set of Questions

Clark’s nevi present distinct challenges.⁶ Most lack a substantial dermal component, making the presence of atypical dermal melanocytes more concerning. Here, the key diagnostic question often becomes:

• • Is this melanoma in situ arising in a nevus, or
  • Is it early invasive melanoma?

 

In these cases, Dr. Jukic finds PRAME, HMB45, and proliferation markers—interpreted together—more informative than p16 or cyclin D1. Deeper levels can be critical, as additional melanocytic populations may only become apparent with further sectioning.

For dysplastic lesions, Dr. Jukic rarely uses FISH, citing limited tissue, sampling uncertainty, and lower added value. Instead, when ambiguity persists, MyPath Melanoma is his preferred adjunct.

MyPath Melanoma: A Tool, Not a Verdict

Both discussants are clear: no ancillary test replaces pathology judgment. “MyPath isn’t making the diagnosis—it’s another tool, just like immunohistochemistry,” Dr. Marks summarizes. Dr. Jukic agrees, stressing that gene expression results must always be reconciled with morphology and clinical context. Importantly, MyPath results frequently prompt a second, more informed look at the slides:

“Sometimes the study doesn’t change the case—it changes me.”

FISH vs MyPath: Strengths and Limitations

Dr. Jukic characterizes FISH as having high specificity but limited sensitivity.⁷ A positive result is valuable; a negative one may not be. Sampling concerns, particularly when melanoma arises in a nevus, further complicate interpretation. By contrast, MyPath analyzes gene expression across the submitted tissue, reducing uncertainty about which cells are being assessed.⁸ “If the MyPath result is malignant, that tells me there’s enough malignant signal there to matter.”

PRAME: Helpful, but Not Definitive

PRAME receives special attention, and caution.⁹ Dr. Jukic likens its trajectory to that of HMB45: useful, but often overinterpreted. He has seen:

• • PRAME-positive nevi (focal and diffuse)
  • PRAME-negative melanomas, particularly lentigo maligna

 

“PRAME is just another antibody. You have to know how—and when—to trust it.”

Cases with PRAME discordance often benefit from broader molecular context, where MyPath may clarify what immunohistochemistry alone cannot.

 

Why NGS Isn’t Ready for Prime Time (Yet)

Next-generation sequencing offers enormous data—but limited clinical clarity in routine melanocytic diagnostics. Dr. Jukic points to 3 major issues:

• 1. Cost
  2. Data overload
  3. Lack of actionable interpretation without molecular pathology expertise

 

“If the oncologist calls me and asks what to do with the results, and I don’t have an answer—that’s a problem.” While NGS may ultimately prove valuable in prognostication or therapy selection, Dr. Jukic believes current workflows relying on morphology, IHC, and validated GEP remain more reliable for diagnosis.

Cost, Tissue, and Turnaround Time

From a practical standpoint:

• • • Tissue usage for IHC, FISH, and MyPath is modest when handled correctly
    • MyPath turnaround: typically 3–5 business days once received
    • FISH: often longer and more variable
    • NGS: may take weeks—often too slow for clinical decision-making

   

Dr. Jukic stresses the broader economic context:

“If a $1,000 test prevents unnecessary sentinel node biopsies, imaging, and lifelong surveillance, that’s a net healthcare savings.”

 

Final Takeaways

This discussion reinforces several core principles:

• • Ancillary tests support—but never replace—expert histopathology
  • Spitz and Clark’s nevi require distinct diagnostic strategies
  • MyPath Melanoma is most valuable when used in the scenarios expressed above and interpreted thoughtfully¹⁰
  • PRAME is helpful, but not definitive
  • NGS is currently impractical for routine diagnostic use

 

As Dr. Jukic concludes:

• “If you can use and interpret immunohistochemistry properly, you can use MyPath. The key is knowing what questions you’re trying to answer—and explaining those answers clearly to clinicians.”

 

 

References

1. Barnhill RL, Elder DE, Piepkorn MW, et al. Revision of the melanocytic pathology assessment tool and hierarchy for diagnosis classification schema for melanocytic lesions: a consensus statement. JAMA Netw Open. 2023;6(1):e2250613.
2. AUC Committee Members, Fung MA, Vidal CI, et al. Appropriate use criteria for ancillary diagnostic testing in dermatopathology: new recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology Appropriate Use Criteria Committee. J Cutan Pathol. 2022;49(3):231–245.
3. Hosler GA, Murphy KM. Ancillary testing for melanoma: current trends and practical considerations. Hum Pathol. 2023;140:5–21.
4. Goldberg MS, Cockerell CJ, Rogers JH, et al. Appropriate statistical methods to assess cross-study diagnostic 23-gene expression profile test performance for cutaneous melanocytic neoplasms. Am J Dermatopathol. 2024;46(12):833–838.
5. North JP, Garrido MC, Kolaitis NA, et al. Fluorescence in situ hybridization as an ancillary tool in the diagnosis of ambiguous melanocytic neoplasms: a review of 804 cases. Am J Surg Pathol. 2014;38(6):824–831.
6. Drozdowski R, Spaccarelli N, Peters MS, Grant-Kels JM. Dysplastic nevus part I: historical perspective, classification, and epidemiology. J Am Acad Dermatol. 2023;88(1):1–10.
7. Kerl K, Palmedo G, Wiesner T, et al. A proposal for improving multicolor FISH sensitivity in the diagnosis of malignant melanoma using new combined criteria. Am J Dermatopathol. 2012;34(6):580–585.
8. Clarke LE, Warf MB, Flake DD, et al. Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma. J Cutan Pathol. 2015;42(4):244–252.
9. Kunc M, Żemierowska N, Skowronek F, Biernat W. Diagnostic test accuracy meta-analysis of PRAME in distinguishing primary cutaneous melanomas from benign melanocytic lesions. Histopathology. 2023;83(1):3–14.
10. Marks E, Jarell A, Ludzik J, et al. A physician’s guide to the use of gene expression profile ancillary diagnostic testing for cutaneous melanocytic neoplasms. J Clin Aesthetic Dermatol. 2023;16(4):12–20.

 

 

Second Opinions in Dermatopathology: Balancing Expertise, Objectivity, and Emerging Molecular Tools

J Clin Aesthet Dermatol. 2026;19(6 Suppl 3):S6–S7.

by Etan Marks, MD, and Clay J. Cockerell, MD, MBA, JD

Dr. Marks is with Advanced Dermatology and Cosmetic Surgery, Delray Beach, Florida. Dr. Cockerell is with Cockerell Dermatopathology, Dallas, Texas.

FUNDING: Funding for this article was provided by Castle Biosciences.

DISCLOSURES: Dr. Marks is a consultant for Castle Biosciences. Dr. Cockerell is a speaker for Castle Biosciences.

Introduction

Second opinions have long played a central role in dermatopathology, particularly for melanocytic lesions, where diagnostic uncertainty can carry profound clinical and medicolegal consequences.¹ Yet as ancillary testing and molecular diagnostics continue to evolve, many practitioners are asking an important question: When is a second opinion truly necessary, and when can objective testing provide equivalent—or even superior—support?

In this roundtable discussion, Dr. Etan Marks speaks with Dr. Clay J. Cockerell, a dermatopathologist and Juris Doctor, about why dermatopathologists seek second opinions, the benefits and limitations of consultative diagnoses, medicolegal considerations, and how tools such as immunohistochemistry (IHC) and gene expression profiling (GEP) fit into modern practice.

Why Do Dermatopathologists Seek Second Opinions?

According to Dr. Cockerell, motivations vary widely. “Sometimes it’s simply reassurance. Other times it’s lack of confidence, lack of experience, or uncertainty about how to integrate clinical findings.” While melanocytic lesions constitute the highest stakes consults, Dr. Cockerell notes that a significant proportion (30–40%) of second opinions involve nonmelanocytic diagnoses, including keratinocytic and inflammatory conditions.

The most critical consults, however, are those involving potentially invasive melanocytic lesions, where misclassification could lead either to overtreatment or to dangerous undertreatment.^2,3

How Much Workup Is Done Before a Case Is Sent?

Practices vary considerably. When cases are submitted by board certified dermatopathologists, they are often already worked up with:

• • Deeper sectioning
  • Immunohistochemical stains
  • PReferentially expressed Antigen in MElanoma (PRAME), p16, and other melanocytic markers

“Usually they’ve done the stains and they’re still not sure—that’s why they send it.”

In contrast, some cases arrive as hematoxylin and eosin (H&E)-only consultations, particularly from solo or resource limited practices or non dermatopathologists seeking foundational guidance.

 

The Limitations of Second Opinions

While second opinions are valuable, Dr. Cockerell cautions against viewing them as infallible. “The farther away you send the case, the more dogmatic, and sometimes more incorrect the opinion.” Several challenges exist:

• • Subjectivity in morphologic interpretation
  • Low interobserver concordance, sometimes as low as 40–50% for ambiguous lesions
  • Overinterpretation of immunostains, particularly highly sensitive markers like Melanoma Antigen Recognized by T cells 1 (MART-1) and PRAME

 

Dr. Cockerell also criticizes overly verbose reports:

• • “I almost never go to page 2. A second opinion should be a communication between colleagues, not an exercise in obfuscation.”

 

Medicolegal Protection: Myth vs Reality

A common belief is that a second opinion functions as a legal “safety net.”⁴ Dr. Cockerell is clear: there are no magic bullets. Instead, medicolegal defensibility hinges on whether the pathologist met the standard of care, defined as what a reasonable dermatopathologist with similar training would do under comparable circumstances.^5,6

Second opinions can help establish reasonableness, but they do not eliminate liability, particularly if:

• • • The consultant is wrong, or
    • The original pathologist uncritically defers judgment.

   

“You can do everything right and still get sued.”

 

Can Ancillary Testing Replace Second Opinions?

Increasingly, dermatopathologists are turning to objective diagnostic tools like IHC, fluorescence in situ hybridization (FISH), and GEP as alternatives or complements to consultative review.⁷ Dr. Cockerell supports this approach:

• • “If you’re board certified and you work the case up appropriately, you don’t have to get a second opinion.” When these tools are used responsibly and interpreted in context, they can support a defensible diagnosis without external consultation.

 

GEP–MyPath Melanoma

Dr. Cockerell reports using MyPath Melanoma routinely in his laboratory, particularly for:

• • Ambiguous melanocytic lesions
  • Equivocal or conflicting immunostain results

 

However, he emphasizes that no test should be followed blindly. “You’re still the captain of the ship.” There are cases where molecular results conflict with morphology or clinical impression and in those situations, judgment prevails. GEP adds data, but it does not absolve the pathologist of interpretive responsibility.^8,9

Second Opinions vs Molecular Testing: Not Either/Or

Dr. Marks raises a practical concern: some consultants hesitate to contradict molecular results. Dr. Cockerell counters this, noting that all information, whether it is morphology, immunostains, molecular studies, and/or clinical context, must be integrated. In some cases, uncertainty remains despite exhaustive workup.¹⁰ This diagnostic gray zone has contributed to greater adoption of terms like melanocytoma. Acknowledging uncertain biologic behavior, providing transparent communication, and offering medicolegal protection through accuracy rather than overconfidence is the key. “You didn’t call it benign. You didn’t call it malignant. You did your best and rendered a reasonable opinion.”

Practical Takeaways for the Practicing Dermatopathologist

This discussion highlights several key principles:

• • Second opinions are valuable but not inherently objective
  • Overreliance on either consultants or stains can mislead
  • Molecular tests offer additional clarity, not definitive answers
  • Following a reasonable, documented workup is what matters most legally

 

Looking ahead, Dr. Cockerell does not foresee molecular tools or artificial intelligence replacing second opinions entirely, but he does expect them to reduce unnecessary external consultations and empower more dermatopathologists to manage complex cases independently. “If you want to practice dermatopathology and read pigmented lesions, these tools can give you confidence. You don’t have to send everything out.”

References

1. Geller BM, Frederick PD, Knezevich SR, et al. Pathologists’ use of second opinions in interpretation of melanocytic cutaneous lesions: policies, practices, and perceptions. Dermatol Surg. 2018;44(2):177–185.
2. Elmore JG, Eguchi MM, Barnhill RL, et al. Effect of prior diagnoses on dermatopathologists’ interpretations of melanocytic lesions: a randomized controlled trial. JAMA Dermatol. 2022;158(9):1040–1047.
3. Elmore JG, Barnhill RL, Elder DE, et al. Pathologists’ diagnosis of invasive melanoma and melanocytic proliferations: observer accuracy and reproducibility study. BMJ. 2017;357:j2813.
4. Carney PA, Frederick PD, Reisch LM, et al. How concerns and experiences with medical malpractice affect dermatopathologists’ perceptions of their diagnostic practices when interpreting cutaneous melanocytic lesions. J Am Acad Dermatol. 2016;74(2):317–324.
5. High WA. Malpractice in dermatopathology: principles, risk mitigation, and opportunities for improved care for the histologic diagnosis of melanoma and pigmented lesions. Clin Lab Med. 2008;28(2):261–284.
6. Troxel DB. Medicolegal aspects of error in pathology. Arch Pathol Lab Med. 2006;130(5):617–619.
7. AUC Committee Members, Fung MA, Vidal CI, et al. Appropriate use criteria for ancillary diagnostic testing in dermatopathology: new recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology Appropriate Use Criteria Committee. J Cutan Pathol. 2022;49(3):231–245.
8. Goldberg MS, Cockerell CJ, Rogers JH, et al. Appropriate statistical methods to assess cross-study diagnostic 23-gene expression profile test performance for cutaneous melanocytic neoplasms. Am J Dermatopathol. 2024;46(12):833–838.
9. Marks E, Jarell A, Ludzik J, et al. A physician’s guide to the use of gene expression profile ancillary diagnostic testing for cutaneous melanocytic neoplasms. J Clin Aesthetic Dermatol. 2023;16(4):12–20.
10. Cockerell CJ. Commentary on atypical melanocytic proliferations. Dermatol Surg. 2018;44(2):175–176.