Multiple predictive biomarker testing in melanoma: Another challenge in identifying the optimal approach on cytological samples

The management of cutaneous melanoma has changed dramatically in recent years thanks to the development of tyrosine kinase and immune‐checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important for the identification of patients who are potentially eligible for these treatments. One reliable approach to the molecular evaluation of metastatic melanoma is fine needle cytology (FNC). To examine the utility of this approach for assessing PD‐L1 expression levels, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas that were previously tested for BRAF and NRAS mutations.


| INTRODUC TI ON
Cutaneous melanoma is an aggressive neoplasm, accounting for about 1.7% of all novel cancer diagnoses and 0.6% of all cancerrelated deaths worldwide. 1,2 However, although its incidence continues to increase, mortality rates have significantly declined with the advent of immune checkpoint inhibitors (ICIs) and targeted therapies. [3][4][5][6] Accordingly, pathologists are now strongly encouraged to identify and report the presence of specific gene mutations in metastatic melanoma patients for whom targeted therapies are available. 7 Generally, the most common gene alteration in melanoma is V-Raf Murine Sarcoma Viral Oncogene Homologue B (BRAF), which is present in about 40%-60% of all cases. 8 Almost all BRAF mutations (97%) occur within codon 600 of exon 15. 9 In most cases (90%), this mutation results in the substitution of a valine with a glutamic acid (p.V600E). 10 Melanoma patients harbouring BRAF exon 15 p.V600 mutations benefit from targeted treatments with BRAF tyrosine kinase inhibitors (TKIs) and/or mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors. [11][12][13][14][15][16][17][18][19][20][21] The second most common mutated gene (15%-30%) in melanoma patients is Neuroblastoma RAS Viral Oncogene Homologue (NRAS). 9 Approximately 25% of NRAS mutations are represented by exon 3 p.Q61R and p.Q61K. 22 Like BRAF exon 15 p.V600 mutations, NRAS alterations seem to respond to MEK inhibitors. 23,24 Less prevalent is the KIT Proto-Oncogene, Receptor Tyrosine Kinase (KIT) mutation, which is reported in about 1%-7% of melanoma patients 25,26 ; this mutation shows high sensitivity to imatinib and nilotinib. 27,28 Another major biomarker that can be used to ascertain whether single or combination therapy is the most appropriate treatment is PD-L1. Indeed, the phase III CheckMate 067 study showed that patients with low PD-L1 expression (< 1% tumour proportion score [TPS]) benefit more from nivolumab/ipilimumab combination therapy than nivolumab monotherapy, whereas patients with high PD-L1 expression are best treated with nivolumab monotherapy. 29 Clearly, multiple biomarker testing is becoming ever more important in the identification of patients who would benefit from personalised treatments. Although tissue biopsies remain the gold standard material for molecular analysis, cytological preparations have also emerged as a useful source for predictive analysis. In particular, in the metastatic melanoma setting, fine needle cytology (FNC) is widely used for diagnostic and molecular evaluations. 30 However, since the cytomorphology of melanoma is heterogeneous-often requiring immunocytochemical (ICC) confirmation of melanocytic differentiation-and predictive biomarkers include both DNA-based analysis and ICC PD-L1 staining, proper management of cytological material is pivotal to guarantee a comprehensive diagnosis.
Therefore, the aim of the present study is to evaluate whether residual cell block (CB) material from metastatic melanomas that had previously been tested for BRAF and NRAS mutations provides adequate cellularity for PD-L1 assessment.

| MATERIAL S AND ME THODS
Cytological material that was obtained from metastatic melanomas diagnosed by FNC and subjected to molecular testing between January 2016 and July 2022 was retrieved from the archives of the Cytopathology and Predictive Molecular Pathology Service of "Federico II" University of Naples. Records regarding age, sex, anatomic location, and ancillary studies were collected.
In all cases, FNCs were performed by an experienced cytopathologist under ultrasound guidance; the diagnostic procedure and associated risks were discussed with all patients, and informed consent was obtained. In brief, cytology specimens were collected using a 23-gauge needle. A first pass was used to prepare direct smears, which were stained with Diff-Quik for rapid on-site evaluation and microscopically evaluated for cellular adequacy. Additional passes were performed in cases with low cellularity. Moreover, residual material from the needle was suspended in 5 mL of 10% neutral buffered formalin for CB preparation. For a subset of patients, molecular analysis was performed on CB material. For the remaining patients, a dedicated pass was performed for the molecular analysis to avoid sacrificing precious diagnostic material for the molecular testing. 31,32 Briefly, during the FNC procedure, an aliquot of the aspirated material was collected in a vial of nuclease-free water (Invitrogen, Ambion; Thermo Fisher Scientific). Vials were then immediately stored at −20°C until cytological evaluation. When metastatic melanoma was confirmed, DNA was extracted using the All For all cases, CBs were retrieved to evaluate their adequacy for PD-L1 evaluation. PD-L1 ICC was carried out using the companion diagnostic kit SP263 assay (Ventana) according to the manufacturer's instructions. 33 Briefly, a minimum of 100 viable tumour cells were required to consider a sample eligible for TPS evaluation.
Thirteen of these patients had a previous history of melanoma. ICC staining was performed in 18 of the 19 cases. In particular, in three cases, a single marker was used (S100 [two cases] and MART-1 [one case]), in seven cases, S100 and HMB-45 were used; in two cases S100 and a pan melanoma cocktail (HMB 45, MART-1 and tyrosinase) were used; in four cases S100, HMB-45, and MART-1 were used; and in two cases more than four ICC markers were used, due to their undifferentiated morphology.
Molecular testing was performed at the request of oncologists.
DNA was extracted from needle rinses collected in vials in nine cases and from CB sections in ten cases. However, three cases revealed just enough cells to meet the adequacy criteria. Finally, one case (10%) showed fewer than 100 cells.
In the sample group of cases in which DNA for molecular testing was extracted from the needle rinses collected in vials, 7/9 (77.8%) CBs were found to be adequate for PD-L1 evaluation. However, one of these contained just enough cells to meet the adequacy criteria.

| DISCUSS ION
Cytological material has been widely used to predict targeted treatment responses, especially in advanced-stage cancer patients.
Moreover, an increased understanding of the molecular events involved in malignancy and the consequent availability of multiple treatment options have led to increased demand for multiple, rather than single, predictive biomarker testing. In addition, with the development of immune checkpoint inhibitors, the assessment of PD-L1 expression has recently become standard for a wide range of cancer types, and several antibody clones and platforms have been developed to assess its expression levels. However, because PD-L1 is not a specific tumour marker, different scoring systems evaluating neoplastic and/or immunological cells have been developed to correctly quantify PD-L1 expression in different tumour types. 35  Although the application of CPS to cytological samples is feasible, [39][40][41] this system, as well as the IC score, is rather more com- Maximum 84

Mean 63
Clinical history

ACK N OWLED G EM ENT
We thank Paola Merolla for English language editing. Open Access Funding provided by Universita degli Studi di Napoli Federico II within the CRUI-CARE Agreement.

FU N D I N G I N FO R M ATI O N
The authors have not declared a specific grant for this review from any funding agency in the public, commercial or not-for-profit sectors. from Novartis for work unrelated to the current study. The other authors declare no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.