Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors.

BACKGROUND: Around 50% of cutaneous melanomas harbor the BRAF(V600E) mutation and can be treated with BRAF inhibitors. DNA carrying this mutation can be released into circulation as cell-free BRAF(V600E) (cfBRAF(V600E)). Droplet digital PCR (ddPCR) is an analytically sensitive technique for quantifying small concentrations of DNA. We studied the plasma concentrations of cfBRAF(V600E) by ddPCR in patients with melanoma during therapy with BRAF inhibitors. METHODS: Plasma concentrations of cfBRAF(V600E) were measured in 8 controls and 20 patients with advanced melanoma having the BRAF(V600E) mutation during treatment with BRAF inhibitors at baseline, first month, best response, and progression. RESULTS: The BRAF(V600E) mutation was detected by ddPCR even at a fractional abundance of 0.005% in the wild-type gene. Agreement between tumor tissue BRAF(V600E) and plasma cfBRAF(V600E) was 84.3%. Baseline cfBRAF(V600E) correlated with tumor burden (r = 0.742, P < 0.001). cfBRAF(V600E) concentrations decreased significantly at the first month of therapy (basal median, 216 copies/mL; Q1-Q3, 27-647 copies/mL; first response median, 0 copies/mL; Q1-Q3, 0-49 copies/mL; P < 0.01) and at the moment of best response (median, 0 copies/mL; Q1-Q3, 0-33 copies/mL; P < 0.01). At progression, there was a significant increase in the concentration of cfBRAF(V600E) compared with best response (median, 115 copies/mL; Q1-Q3, 3-707 copies/mL; P = 0.013). Lower concentrations of basal cfBRAF(V600E) were significantly associated with longer overall survival and progression-free survival (27.7 months and 9 months, respectively) than higher basal concentrations (8.6 months and 3 months, P < 0.001 and P = 0.024, respectively). CONCLUSIONS: cfBRAF(V600E) quantification in plasma by ddPCR is useful as a follow-up to treatment response in patients with advanced melanoma.

Hypoglycemic Syndrome without Hyperinsulinemia. A Diagnostic Challenge.

The most common cause of organic fasting hypoglycemia in adults is the presence of an insulin-producing pancreatic adenoma, but when high insulin levels are not found, the differential diagnosis is challenging. Misdiagnosis can lead to an unnecessary pancreatectomy. Insulin concentrations may be low in some cases despite a clinical history suggestive of insulinoma. In these cases, a proinsulinoma should be suspected, although the rarity of this condition requires an extensive workup before reaching a final diagnosis. We describe an unusual case of a 38-year-old man with a severe hypoglycemic syndrome due to a proinsulin-secreting pancreatic adenoma. Insulin was measured by the specific assay and suppressed under the lower detection limit during fasting hypoglycemia. Serum proinsulin and C-peptide levels were abnormally elevated, and further tests revealed an islet cell tumor. The tumor was surgically removed, relieving the fasting hypoglycemia. Histopathological study showed a conventional well-differentiated neuroendocrine tumor with high immunoreactivity against proinsulin and with lesser intensity against insulin. Interestingly, GS-9A8 antibody clone used for immunostaining proinsulin did not cross-react with human insulin or C-peptide, providing an unbiased picture of proinsulin secretion. The resolution of symptoms, the fall of proinsulin concentrations after tumor removal and the histopathology study confirmed the diagnosis of proinsulinoma.

Circulating biomarkers in malignant melanoma.

Melanoma is an aggressive tumor with increasing incidence worldwide. Biomarkers are valuable tools to minimize the cost and improve efficacy of treatment of this deadly disease. Serological markers have not widely been introduced in routine clinical practice due to their insufficient diagnostic sensitivity and specificity. It is likely that the lack of objective responses with traditional treatment hinder biomarker research and development in melanoma. Recently, new drugs and therapies have, however, emerged in advanced melanoma with noticeable objective response ratio and survival. In this new scenario, serological tumor markers should be revisited. In addition, other potential circulating biomarkers such as cell-free DNA, exosomes, microRNA, and circulating tumor cells have also been identified. In this review, we summarize classical and emerging tumor markers and discuss their possible roles in emerging therapeutics.

Some basic aspects of HLA-G biology.

Human leukocyte antigen-G (HLA-G) is a low polymorphic nonclassical HLA-I molecule restrictively expressed and with suppressive functions. HLA-G gene products are quite complex, with seven HLA-G isoforms, four membrane bound, and other three soluble isoforms that can suffer different posttranslational modifications or even complex formations. In addition, HLA-G has been described included in exosomes. In this review we will focus on HLA-G biochemistry with special emphasis to the mechanisms that regulate its expression and how the protein modifications affect the quantification in biological fluids.

Relevance of MIA and S100 serum tumor markers to monitor BRAF inhibitor therapy in metastatic melanoma patients.

BRAF V600 mutation has been reported in more than 50% of melanoma cases and its presence predicts clinical activity of BRAF inhibitors (iBRAF). We evaluated the role of MIA, S100 and LDH to monitor iBRAF efficiency in advanced melanoma patients presenting BRAF V600 mutations. This was a prospective study of melanoma patients harboring the BRAF V600 mutation and treated with iBRAF within a clinical trial (dabrafenib) or as part of an expanded access program (vemurafenib). MIA, S100 and LDH were analyzed in serum at baseline, and every 4-6 weeks during treatment. Eighteen patients with melanoma stages IIIc-IV were enrolled with 88.8% of response rate to iBRAF. Baseline concentrations of all the tumor markers correlated with tumor burden. MIA and S100 concentrations decreased significantly one month after the beginning of treatment and, upon progression, their concentrations increased significantly above the minimum levels previously achieved. MIA levels lower than 9 µg/L one month after the beginning of treatment and S100 concentrations lower than 0.1 µg/L at the moment of best response were associated with improved progression-free survival. In conclusion, MIA and S100 are useful to monitor response in melanoma patients treated with iBRAF.