Metabolomic profiling of fresh and formalin-fixed paraffin-embedded samples reveals metabolome alterations in penile cancer

Penile squamous cell carcinoma represents a significant source of morbidity and mortality in Puerto Rico, with HPV-associated cases comprising 56% of cases. The link between clinical variables and metabolomic alterations has not been studied in penile cancer. This study aimed to characterize the non-targeted metabolomic profiles of fresh and formalin-fixed paraffin-embedded (FFPE) penile cancer samples to identify correlations with patient clinical variables: HPV infection status, tumor stage and tumor grade. In this study, 14 fresh and 37 FFPE penile cancer samples were processed for metabolomic analysis. HPV infection status was determined using INNO-Lipa HPV Genotyping and RHA kit HPV SPF10-LiPA25 kits. Sample aliquots were analyzed using GCMS/MS-TQ8050 equipment. Clinical variables were provided by the Departments of Pathology and Surgery. Filtering of raw data was conducted using AMDIS 32 and the NIST MS Spectrum database. Statistical analysis (T-test and Mann-Whitney test) of metabolomic profiles was done using MetaboAnalyst 6.0. Our study identified 52 metabolites in the fresh penile cancer sample cohort and 18 metabolites in the FFPE penile cancer sample cohort. The fresh sample cohort yielded eight metabolite classes: amino acids, sugar alcohols, fatty acids, carboxylic acids, nucleic acids, sterols, cofactor and vitamins, and others. The FFPE cohort yielded five metabolite classes: amino acids, sugar alcohols, fatty acids, carboxylic acids, and others. Statistical findings suggest that HPV infection status and tumor stage are significantly correlated with alterations in expression of several metabolites in both sample cohorts: isoleucine (p value = 0.011), homoserine (p-value = 0.0001), cysteine (p-value = 0.0385), glycolic acid (p-value = 0.0215), methylmalonic acid (p-value = 0.0475), malic acid (p-value = 0.0483) and heptanoic acid (p-value = 0.0461). Statistical findings found no significant correlation between tumor grade and metabolomic alterations. Our findings suggest that HPV infection status and tumor stage are associated with altered metabolomic pathways in penile cancer. Further understanding is needed to establish the clinical relevance of these altered metabolites and their potential role as novel metabolomic biomarkers or therapeutic targets to improve treatment outcomes for this disease.