Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients

Eduardo Netto; Hugo Santos; Luís Carvalho; José Luis Capelo-Martínez; Miguel Rito; José Cabeçadas; Margarida Roldão

doi:10.1016/j.rpor.2020.05.007

Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients

Rep Pract Oncol Radiother. 2020 Sep-Oct;25(5):746-753. doi: 10.1016/j.rpor.2020.05.007. Epub 2020 Jul 11.

Authors

Eduardo Netto^{1

2}, Hugo Santos^{3

4}, Luís Carvalho^{3

4}, José Luis Capelo-Martínez^{3

4}, Miguel Rito⁵, José Cabeçadas⁵, Margarida Roldão^{1

2}

Affiliations

¹ Serviço de Radioterapia, Instituto Português de Oncologia Francisco Gentil, Lisboa, Portugal.
² NOVA Medical School, Universidade NOVA de Lisboa, Lisboa, Portugal.
³ BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Campus de Caparica, Portugal.
⁴ PROTEOMASS Scientific Society, Madan Parque, Rua dos Inventores, 2825-182 Caparica, Portugal.
⁵ Serviço de Anatomia-Patológica, Instituto Português de Oncologia Francisco Gentil, Lisboa, Portugal.

Abstract

Aim: Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis.

Background: Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis.

Methods: FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients' received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan-Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated.

Results: 28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein-Barr (EBV) and Herpes simplex (HSV) viruses' related proteins significantly present in early-stage primary NPC (T1 and T2, p < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p < 0.01). Median survival from this M1 group was <1 year (p < 0.001).

Conclusions: FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.

Keywords: Biomarkers; Metastases prediction; Nasopharyngeal carcinoma; Proteomic analysis.