Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma

Abstract

BACKGROUND:The cytodiagnosis of melanoma in fine-needle aspiration (FNA) specimens can be challenging, often requiring the use of immunocytochemistry. As constitutively activating mutations in the BRAF oncogene are present in at least 40% of melanomas, the use of FNA material to interrogate the BRAF mutational status is likely to increase. Because cell blocks, traditionally used for these studies, can occasionally exhibit insufficient tumor cellularity, the authors investigated the utility of direct smears for immunocytochemistry and BRAF mutational analysis. METHODS: Immunocytochemistry for S-100, HMB-45, and Mart-1 was prospectively performed on direct smears in 17 FNAs of metastatic melanoma. Next, BRAF sequencing was performed using DNA isolated from archived Diff-Quik-stained direct smears for 15 cases.In parallel, sequencing was performed using DNA obtained from corresponding cell blocks. RESULTS: S-100 positivity in the tumor cells was observed in all 17 cases. HMB-45 and Mart-1 positivity was noted in 81% and 88% of cases, respectively. All 3 markers were positive in 76% of cases. Next, of the 15 archived melanoma FNAs tested, BRAF mutations were observed in 8 (53%); 5 and 3 melanomas harbored the V600E and V600K mutation, respectively. Corresponding cell blocks were also tested for all 15 cases, yielding concordant BRAF results in 14 (93%); 1 cell block yielded a false-negative result. CONCLUSIONS: Cytologic direct smears represent a robust and valuable source of cellular material for immunocytochemistry and molecular studies, especially in instances in which inadequate cell block cellularity is anticipated or encountered. Cancer (Cancer Cytopathol) 2012;120:52-61. V C 2011 American Cancer Society.KEY WORDS: melanoma, BRAF, immunocytochemistry, direct smear, cytology, S-100, HMB-45, Mart-1.In 2010, it is estimated that >68,000 men and women were diagnosed with melanoma and 8700 died of the disease.1 The morbidity and mortality associated with melanoma stems from a multitude of factors including challenges in establishing a diagnosis, limited understanding of the molecular pathogenesis underlying the disease, unpredictable presentation of metastatic foci, and limited treatment options for those with advanced disease. 2,3 Surgical therapy is more effective in patients with localized or early melanoma; however, melanoma that has spread to locoregional lymph nodes or distant sites is largely refractory Fine-needle aspiration (FNA) represents a minimally invasive technique and an accurate, safe, and affordable means to achieve a tissue diagnosis. FNA is therefore becoming an increasingly used modality to establish a diagnosis of metastatic melanoma.3-6 An early definitive diagnosis of metastatic melanoma can facilitate prompt, appropriate management including surgical removal or avoidance of unnecessary surgery and accurate staging of patients in clinical trials. 6Because melanoma can exhibit a variety of cytomorphologic features and mimic other neoplasms such as carcinomas and sarcomas, identifying melanoma...

High prevalence of somatic MAP2K1 mutations in BRAF V600E–negative Langerhans cell histiocytosis

“…Primers are listed in supplemental Table 1 on the Blood Web site. Sanger sequencing of exon 15 of the BRAF gene was performed for one case with a BRAF insertion mutation, as described by Hookim et al 7 Results and discussion Similar to previous reports, a BRAF V600E mutation was detected in 3 of the 8 cases initially screened. An E102_I103del mutation in MAP2K1 was identified in one BRAF WT case using the Ion Comprehensive Cancer panel and confirmed by Sanger sequencing.…”

Triage of Cytologic Direct Smears for Ancillary Studies: A Case-Based Illustration and Review

“…Alternatively, Diff-Quik-stained cytologic direct smears represent a rich, underused, and ''untapped'' source of cellular material for ancillary molecular studies. 2,10,[13][14][15] Furthermore, generation of unstained smears allows for the performance of immunocytochemistry, if necessary. 2,14,16,17 Insights gained from these studies provide opportunities to optimize cytologic specimen triage for ancillary immunocytochemical and molecular studies.…”

“…2,10,[13][14][15] Furthermore, generation of unstained smears allows for the performance of immunocytochemistry, if necessary. 2,14,16,17 Insights gained from these studies provide opportunities to optimize cytologic specimen triage for ancillary immunocytochemical and molecular studies. In our practice, when appropriate, we prepare multiple direct smears for a given FNA needle pass or needle passes (Figure 11) by distributing the cellular material over multiple slides.…”

Kba.62 and S100 protein expression in cytologic samples of metastatic malignant melanoma

“…S100 protein is one of the most sensitive markers for melanoma, being expressed in 96-100% of primary and metastatic melanomas on tissue samples, 5,7,9 and in 7-100% of fine-needle aspirates and effusion samples, including a series of 81 FNA cases of MMM. [10][11][12][13][14][15] The sensitivity of the highly specific melanoma markers HMB-45 and MART-1 on cytology samples of MMM is reported to be approximately 78-100% 14,16 and 88-100%, 12,15,17 respectively. However, the specificity of S100 protein for melanomas is limited (75-87%).…”