Proteomic Profile and <i>In Silico</i> Analysis in Metastatic Melanoma with and without BRAF Mutation

<div><p>Introduction</p><p>Selective inhibitors of BRAF, vemurafenib and dabrafenib are the standard of care for metastatic melanoma patients with BRAF V600, while chemotherapy continued to be widely used in BRAF wild type patients.</p><p>Materials and Methods</p><p>In order to discover novel candidate biomarkers predictive to treatment, serum of 39 metastatic melanoma vemurafenib (n = 19) or chemotherapy (n = 20) treated patients at baseline, at disease control and at progression, were analyzed using SELDI-TOF technology. In silico analysis was used to identify more significant peaks.</p><p>Results</p><p>In patients with different BRAF status, we found 5 peptides significantly deregulated, with the down-regulation of the m/z 9176 peak strongly associated with BRAF mutation. At baseline as predictive biomarkers we identified 2 peptides - m/z 6411, 4075 – as significantly up-regulated in responders to chemotherapy and 4 peaks - m/z 5900, 12544, 49124 and 11724 - significantly up-regulated in longer vs shorter responders to vemurafenib. After response, 3 peptides (m/z 4658, 18639, and 9307) resulted significantly down regulated while 3 peptides m/z 9292, 7765 and 9176 appeared up-regulated respectively in chemotherapy and vemurafenib responder patients. In vemurafenib treated patients, 16 peaks appeared deregulated at progression compared to baseline time. In silico analysis identified proteins involved in invasiveness (SLAIN1) and resistance (ABCC12) as well as in the pathway of detoxification (NQO1) and apoptosis (RBM10, TOX3, MTEFD1, TSPO2). Proteins associated with the modulation of neuronal plasticity (RIN1) and regulatory activity factors of gene transcription (KLF17, ZBTB44) were also highlighted.</p><p>Conclusion</p><p>Our exploratory study highlighted some factors that deserve to be further investigated in order to provide a framework for improving melanoma treatment management through the development of biomarkers which could act as the strongest surrogates of the key biological events in stage IV melanoma.</p></div

Progression free survival in TMZ/FM treated patients with respect to m/z 6411 and 4075 overexpression.

<p>The median value of expression has been considered as cutoff to discriminate peak overexpression. p = 0.024. <b>—</b>: patients with basal level of the 2 peaks; ---: patients with overexpression of both peaks.</p

Differential expression among vemurafenib treated patients; in all patients who achieved a response 3 peptides resulted significantly up-regulated at response evaluation: m/z 9292, 7765 and 9176.

<p>Only m/z 9292 resulted significantly up-regulated in short responders patients, while m/z 9176 and 7765 were significantly up-regulated in the long responder group.</p><p>Differential expression among vemurafenib treated patients; in all patients who achieved a response 3 peptides resulted significantly up-regulated at response evaluation: m/z 9292, 7765 and 9176.</p

Mascot search result for BRAF mutated patients.

<p>Mascot search result for BRAF mutated patients.</p

Representative example of fractionated serum protein profiles of two temozolomide/fotemustine treated patients (from responder and a non-responder patients).

<p>Representative example of fractionated serum protein profiles of two temozolomide/fotemustine treated patients (from responder and a non-responder patients).</p

The 4 discriminating m/z peaks among BRAF V600E/K mutated and BRAFwt MM patients. m/z: mass-to-charge ratio; P was generated by peak comparison between BRAF mutated and wild type patients.

<p>The 4 discriminating m/z peaks among BRAF V600E/K mutated and BRAFwt MM patients. m/z: mass-to-charge ratio; P was generated by peak comparison between BRAF mutated and wild type patients.</p

Mascot search result for BRAF wild-type patients.

<p>Mascot search result for BRAF wild-type patients.</p

Patient characteristics.

<p>All samples were tested only for genetic evaluation of BRAF status.</p><p>Patient characteristics.</p

Differential expression among vemurafenib treated patients; in all patients who progressed 3 peptides resulted significantly up-regulated compared to basal time.

<p>Differential expression among vemurafenib treated patients; in all patients who progressed 3 peptides resulted significantly up-regulated compared to basal time.</p

Additional file 1: of The search for a melanoma-tailored chemotherapy in the new era of personalized therapy: a phase II study of chemo-modulating temozolomide followed by fotemustine and a cooperative study of GOIM (Gruppo Oncologico Italia Meridionale)

The methods of quantitative Real-Time PCR evaluation of the genes of BER and MGMT promoter methylation assessment are described in the additional file. (DOCX 13 kb