What biomarkers should be tested in a patient with BRAF-negative, PD-L1 (Programmed Death-Ligand 1) inhibitor-resistant metastatic melanoma?

Biomarker Testing in Melanoma

All melanoma patients should undergo BRAF V600 mutation testing using an FDA-approved test or CLIA-approved facility before treatment selection, as this is the single most critical biomarker that determines eligibility for targeted therapy with BRAF/MEK inhibitor combinations. 1, 2

Essential Biomarkers for All Melanoma Cases

BRAF Mutation Testing (Mandatory)

• BRAF V600 mutation status must be tested in all patients with unresectable stage III or metastatic melanoma to identify candidates for BRAF/MEK inhibitor combinations (dabrafenib/trametinib, encorafenib/binimetinib, or vemurafenib/cobimetinib). 1, 2
• Testing should identify specific BRAF V600 variants including V600E, V600K, V600R, V600D, and other V600 mutations. 2
• BRAF testing is required regardless of whether immunotherapy or targeted therapy is planned first-line, as it determines second-line options. 1

Additional Mutation Testing for BRAF Wild-Type Disease

• NRAS mutation testing should be performed in BRAF wild-type melanoma, as NRAS-mutated disease has limited benefit from MEK inhibitor monotherapy (median PFS only 2.8 months) and should be treated with immunotherapy identical to wild-type melanoma. 1
• c-KIT mutation testing is recommended for acral, mucosal, and chronically sun-damaged melanomas, as specific c-KIT mutations may respond to imatinib or nilotinib in second-line settings, though activity is limited. 1
• NF1 mutation status may be identified through expanded molecular profiling, as NF1-mutated melanomas should receive immunotherapy rather than targeted therapy. 1, 3

Clinical and Laboratory Prognostic Biomarkers

Standard Clinical Parameters (Routinely Used)

• Serum lactate dehydrogenase (LDH) level is a negative prognostic and predictive marker for both targeted and immunotherapies, with elevated LDH (particularly >2× upper limit of normal) associated with worse outcomes. 1
• ECOG performance status serves as a negative prognostic marker, with higher scores predicting worse response to all systemic therapies. 1
• Number of metastatic sites correlates inversely with treatment outcomes for both immunotherapy and targeted therapy. 1
• Presence of brain metastases affects treatment selection, with asymptomatic brain metastases showing >50% response rate to nivolumab/ipilimumab combination versus 21% for nivolumab monotherapy. 1

PD-L1 Expression: Limited Clinical Utility

Current Status of PD-L1 Testing

• PD-L1 expression testing is NOT routinely recommended for treatment selection in melanoma, as the NCCN panel considers its use "an emerging research issue with nonuniform application" (category 2B). 1
• PD-L1 expression assays are "not in current form sufficiently reproducible, widely available, nor discriminative for screening patients with melanoma." 1
• The predictive value of PD-L1 is limited, with ROC curve analysis showing an area under the curve of only 0.56, indicating marginal improvement over random assignment. 1

When PD-L1 May Inform Treatment Decisions

• Descriptive analyses suggest patients with low/absent PD-L1 expression may benefit more from nivolumab/ipilimumab combination therapy relative to nivolumab monotherapy, while high PD-L1 expression patients may do equally well on nivolumab monotherapy without the increased toxicity of combination therapy. 1
• Both PD-L1-positive and PD-L1-negative subgroups derive clinical benefit from anti-PD-1 therapy compared to ipilimumab or chemotherapy. 1

PD-L1 in BRAF-Inhibitor Treated Patients

• PD-L1 expression does NOT predict outcome of BRAF inhibitor therapy in two independent cohorts totaling 141 patients, and therefore cannot be recommended as a predictive biomarker for BRAFi-based treatment. 4
• However, PD-L1 expression at baseline in BRAF-mutated patients correlates with resistance and poor prognosis, with PD-L1 positivity associated with shorter progression-free survival (HR 4.3) and melanoma-specific survival (HR 6.2). 5

Emerging and Investigational Biomarkers

Tumor Microenvironment Markers

• Tumor-infiltrating lymphocytes (TILs) or mononuclear cells (TIMC) show prognostic value, with absence of TIMC associated with shorter PFS (HR 2.5) and worse melanoma-specific survival (HR 3.1) in BRAF inhibitor-treated patients. 5
• Presence of TIMC correlates with better response to BRAF inhibitor treatment (OR 6.5). 5

Genomic Biomarkers Under Investigation

• Tumor mutational burden (TMB) correlates with response to immune checkpoint inhibitors across cancer types, with high mutational load facilitating immune response. 6
• Microsatellite instability (MSI-high/dMMR) status should be considered, as MSI-high tumors have higher response rates to immunotherapy, though this is rare in melanoma. 3
• Rare actionable alterations such as NTRK fusions may be identified through expanded molecular profiling. 3

Critical Pitfalls to Avoid

• Never rely on PD-L1 expression alone to exclude patients from anti-PD-1 therapy, as PD-L1-negative patients still derive significant clinical benefit. 1
• Do not use PD-L1 status to select between BRAF inhibitor combinations, as it does not predict response to targeted therapy. 4
• Consider retesting for BRAF mutations if disease progresses on immunotherapy, as false-negative results can rarely occur, though this is uncommon. 3
• BRAF inhibitor therapy should not be used too late in disease course, as clinical parameters like elevated LDH, multiple metastatic sites, and poor ECOG PS represent strong negative predictive biomarkers. 1

Practical Testing Algorithm

1. All patients: BRAF V600 mutation testing (mandatory)
2. If BRAF wild-type: Consider NRAS mutation testing
3. If acral/mucosal/chronically sun-damaged melanoma: c-KIT mutation testing
4. Optional expanded profiling: NF1, NTRK fusions, TMB, MSI status
5. Routine clinical markers: LDH, ECOG PS, number of metastatic sites, brain metastases status
6. PD-L1 testing: Optional and not required for treatment decisions, may inform choice between anti-PD-1 monotherapy versus combination therapy in select cases