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Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

ABSTRACT: BACKGROUND:Histological assessment of resected margins has some drawbacks. We therefore aimed to identify a panel of metabolic markers for evaluating the surgical margins of oral squamous cell carcinoma during surgery. METHODS:A total of 28 case of OSCC samples were enrolled in the study. Gas chromatography-mass spectrometry based untargeted metabolic analysis was employed to acquire the metabolic perturbation of the distance-related surgical margins in the development group. The acquired MS data were then subjected to univariate and multivariate analysis by MetaboAnalyst. Ultra-high performance liquid chromatography-tandem mass spectrometerbased targeted metabolomics for quantitative analysis of the validation group was performed to verify the results of the development group. Another 60 OSCC patients with dysplastic surgical margins were used to further validate the results of the development group by immunohistochemical examination of key enzyme expression, and correlate them with clinicopathological parameters and clinical outcomes. FINDINGS:We finally identified 4 amino acids as negative margin markers, and 6 amino acids as dysplastic margin markers. IHC analysis showed that asparagine synthetase positive expression in dysplastic surgical margins and its higher expression was correlated with tumor recurrence and local relapse-free survival. INTERPRETATIONS:We developed a panel of metabolic molecular markers to supplement the evaluation of negative and dysplastic margins. FUND: This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2012); Center of Nanjing Clinical Medicine Tumor (Since 2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

SUBMITTER: Yang XH

PROVIDER: S-EPMC6838421 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

Yang Xi-Hu XH Zhang Xiao-Xin XX Jing Yue Y Ding Liang L Fu Yong Y Wang Shuai S Hu Shi-Qi SQ Zhang Lei L Huang Xiao-Feng XF Ni Yan-Hong YH Hu Qin-Gang QG

EBioMedicine 20191017

<h4>Background</h4>Histological assessment of resected margins has some drawbacks. We therefore aimed to identify a panel of metabolic markers for evaluating the surgical margins of oral squamous cell carcinoma during surgery.<h4>Methods</h4>A total of 28 case of OSCC samples were enrolled in the study. Gas chromatography-mass spectrometry based untargeted metabolic analysis was employed to acquire the metabolic perturbation of the distance-related surgical margins in the development group. The ...[more]

PMID: 31631041

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Project description:The aim of this study is to provide an extended, retrospective validation of a previously described 4-gene signature (MMP1, COL4A1, P4HA2, and THBS2) predictive of oral carcinoma recurrence. We assessed an independent cohort including 245 histologically normal surgical margins from 62 patients with OSCC. Gene expression was determined using a color-coded probe assay for quantitative gene expression analysis. All margins and tumor blocks were cut (4-5µm tick) and stained with Hematoxylin-Eosin and tissues were subjected to needle microdissection using the stereo microscope Leica EZ4 (Leica Microsystems, Wetzlar, Germany) before RNA extraction, in order to isolate pure tumor or normal cell populations. RNA from FFPE samples was isolated using the RecoverAll Total Nucleic Acid Isolation kit (Ambion/Life Technologies, Carlsbad, CA, USA), following our previously reported protocol with modifications to improve RNA yield [PMID:23835716]. RNA was stored at -80ºC until RNA extraction and gene expression validation⁠. In addition, tumor tissues from the same patients were collected for further analysis (not in scope of the presented study). Probes were designed for the 4-gene signature (MMP1, COL4A1, P4HA2 and THBS2) and contained one capture probe linked to biotin and one reporter probe attached to a color-coded molecular tag for each gene sequence, according to the nCounterTM code-set design. RNA samples were randomized using a numerical ID and were then subjected to NanoString nCounterTM analysis by the Princess Margaret Genomics Centre (https://www.pmgenomics.ca), Toronto, ON, Canada. Patient samples were analyzed in two batches consisting of 125 and 152 margins tissue samples. The detailed protocol for mRNA transcript quantification analysis, including sample preparation, hybridization, detection and scanning followed the manufacturer’s recommendations and was described in [PMID: 21549012]. We used 400 ng of total RNA isolated from FFPE tissues for detection of probe signals. Raw data were analyzed using the nCounterTM digital analyzer software (http://www.nanostring.com/support/ncounter/) and gene expression levels were subjected to further bioinformatic and statistical analyses.

Project description:Oral squamous cell carcinoma (OSCC) is most common oral cancer with multifactorial etiology. Surgical therapy is treatment of choice but known to have recurrence. The main reason for recurrence is associated with surgical margins which need to be tumor free. Changes at genetic level cannot be ascertained only through routine light microscopy in surgical margins, even though they are tumor free. Detection of early marker like p16 can help in predicting the risk of recurrence. Hence study aimed to detect p16 microsatellite marker (D9s1747) in surgical margins of oral squamous cell carcinoma and compare the same with p16 marker through immunohistochemistry. Total of 40 paraffin embedded tissue samples diagnosed and surgically treated cases of OSCC were included. From each sample one tumor proper and one surgical margin was obtained. From paraffin embedded tissue sample 2 sections of 4 µm thick was obtained from tumor proper and tumor margin. One section was stained with hematoxylin and eosin and other section was stained immunohistochemically using p16 antibody. DNA extraction was done for tumor proper and surgical margin tissue and PCR analysis was carried for p16 microsatellite marker (D91747). Out of 40 cases 37 cases showed positivity in tumor proper for p16 with IHC. Out of 37 cases 23 cases showed positivity for both tumor proper and surgical margin. There were 3 cases negative for tumor proper. Out of these 3 cases, 1 (33.3%) case was positive for surgical margin. Out of 40 cases 27 cases showed positivity for tumor proper with p16 microsatellite marker. Out of 27 cases 16 cases showed positivity for both tumor proper and surgical margin. There were 13 cases negative for tumor proper. However there were 8 (61.5%) cases negative which were in tumor proper but showed positivity for surgical margin. Other 5 cases were negative in both tumor proper and surgical margin. Our study reveals that surgical margins of OSCC exhibit alteration in p16 markers both by IHC and PCR techniques. p16 and p16 microsatellite marker detection in margins indicates field change. Further studies with larger sample size comparing expression with clinical and histological parameter and follow up has to be done to substantiate our findings.

Project description:BackgroundOral squamous cell carcinoma (OSCC) is the most common type of oral cancer, which remains a major cause of morbidity and mortality in patients with head and neck cancers. However, the critical immune-related signatures and their prognostic values have rarely been investigated.Materials and methodsGene differential analysis was used to measure the differences of gene expression between the groups. Correlation analysis was used to assess the association between the gene expression levels and immune-related risk score/DNA methylation levels. The gene set enrichment analysis (GSEA) was used to identify the pathways or cell types enriched by those identified differentially expressed genes (DEGs).ResultsIn this study, we identified four immune-related gene signatures, including CTSG, TNFRSF4, LCORL, and PLAU, that were significantly associated with the overall survival in OSCC patients from the Cancer Genome Atlas (TCGA) OSCC cohort. Moreover, these four immune-related signatures were differentially expressed between the OSCC and nontumor tissues. The two groups (high and low risk) stratified by the immune-related risk scores had significantly different OS and mortality rates. The gene expression patterns and prognostic values of these immune-related signatures were also verified in two independent validation cohorts. Furthermore, the downregulated genes in the high-risk group (which were also upregulated in the low-risk group) were significantly enriched in the cell type-specific signatures of type 2 T helper cell (Th2), plasmacytoid dendritic cell (pDC), and memory B cell. In contrast, the upregulated genes in the high-score group were enriched in growth factor receptor-related signaling pathways, such as the VEGFA-VEGFR2 signaling pathway, PI3K-Akt signaling pathway, focal adhesion-PI3K-Akt-mTOR signaling pathway, and PDGF pathway, suggesting that those pathways were inversely correlated with immune cell infiltration.ConclusionIn summary, the immune-related signatures had the potential for predicting the risk of OSCC patients. Moreover, the present study also improved our understanding of the association between the growth factor receptor pathways and immune cell infiltration in OSCC.

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Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

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Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

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