Dataset Information

Comparative Evaluation of Immunohistochemical Expression of p16 with p16 Microsatellite Marker by PCR in Surgical Margins of Oral Squamous Cell Carcinoma.

ABSTRACT: 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.

SUBMITTER: Babji D

PROVIDER: S-EPMC6848693 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Comparative Evaluation of Immunohistochemical Expression of p16 with p16 Microsatellite Marker by PCR in Surgical Margins of Oral Squamous Cell Carcinoma.

Babji Deepa D Nayak Ramakant R Bhat Kishore K Kotrashetti Vijayalakshmi V Hosmani Jagadish J Dindawar Santosh S Pattanshetty Smita S

Indian journal of otolaryngology and head and neck surgery : official publication of the Association of Otolaryngologists of India 20181031 Suppl 1

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 ...[more]

PMID: 31742049

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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.

Dataset Information

Comparative Evaluation of Immunohistochemical Expression of p16 with p16 Microsatellite Marker by PCR in Surgical Margins of Oral Squamous Cell Carcinoma.

Publications

Comparative Evaluation of Immunohistochemical Expression of p16 with p16 Microsatellite Marker by PCR in Surgical Margins of Oral Squamous Cell Carcinoma.

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