|Year : 2020 | Volume
| Issue : 2 | Page : 230-236
Assessment of cervical lymph node metastasis based on total RNA from saliva and tumor tissue in patients with oral squamous cell carcinoma: An observational study
Kiran B Jadhav1, Vandana Shah2, Ghansham Parmar3, Nirali Chauhan4, Naveen Shah5, Nidhi Gupta6
1 Department of Oral Pathology and Microbiology, K.M. Shah Dental College and Hospital, Sumandeep Vidyapeeth, Vadodara, Gujarat, India; Department of Oral Pathology and Microbiology, Vasantdada Patil Dental College and Hospital, Sangli, Maharashtra, India
2 Department of Oral Pathology and Microbiology, K.M. Shah Dental College and Hospital, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
3 Department of Pharmacy, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
4 Department of ENT, Smt. B.K. Shah Medical College and Research Centre, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
5 Department of Oral and Maxillofacial Surgery, K.M. Shah Dental College and Hospital, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
6 Department of Oral Pathology and Microbiology, Vasantdada Patil Dental College and Hospital, Sangli, Maharashtra, India
|Date of Submission||08-Feb-2020|
|Date of Decision||28-Apr-2020|
|Date of Acceptance||04-May-2020|
|Date of Web Publication||09-Sep-2020|
Kiran B Jadhav
PhD Scholar, Department of Oral Pathology and Microbiology, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, Piparia, Vadodara, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In case of oral squamous cell carcinoma (OSCC) most patients die within first 2 years due to metastasis. To overcome the limitations and drawbacks of the present available methods of assessment of lymph nodes metastasis, the search for alternative method is needed.
Aim: The aim of the study is to evaluate the sensitivity, specificity and diagnostic accuracy of salivary and tumor tissue RNA for assessment of lymph node metastasis in patients with OSCC.
Methodology: Patients histologically diagnosed with OSCC were included as participants. The unstimulated saliva and tumor tissue were collected and stored at deep freeze before surgical therapy. The pretreatment lymph node metastasis assessment was done by radioimaging investigation. The posttreatment histopathological status of cervical lymph nodes was noted. The RNA was isolated and quantified from stored saliva sample and tumor tissue. The collected data were statistically analyzed for specificity and sensitivity and significance.
Results: The area under curve for salivary RNA level is 0.647 and for tumor tissue RNA level is 0.628 with moderate predictability at 95% confidence interval. It was observed that the sensitivity was 63.50% and 71.40% and specificity was 62.70% and 58.80% for saliva and tumor tissue respectively with diagnostic accuracy of 63%–65%. The Kappa statistics showed moderate degree of agreement with high statistical significance (P ≤ 0.05).
Conclusion: Saliva and tumor tissue RNA can be a good marker for pretreatment assessment of lymph node metastasis in patients with OSCC. Although the diagnostic accuracy which range from 63% to 65%, further characterization and study of specific mRNA, siRNA and miRNA may come out with high diagnostic accuracy.
Keywords: Lymph node metastasis, oral squamous cell carcinoma, RNA, saliva, tumor
|How to cite this article:|
Jadhav KB, Shah V, Parmar G, Chauhan N, Shah N, Gupta N. Assessment of cervical lymph node metastasis based on total RNA from saliva and tumor tissue in patients with oral squamous cell carcinoma: An observational study. J Oral Maxillofac Pathol 2020;24:230-6
|How to cite this URL:|
Jadhav KB, Shah V, Parmar G, Chauhan N, Shah N, Gupta N. Assessment of cervical lymph node metastasis based on total RNA from saliva and tumor tissue in patients with oral squamous cell carcinoma: An observational study. J Oral Maxillofac Pathol [serial online] 2020 [cited 2020 Sep 30];24:230-6. Available from: http://www.jomfp.in/text.asp?2020/24/2/230/294645
| Introduction|| |
Oral cancer is the most frequent type of cancer of the head and neck area, with oral squamous cell carcinoma (OSCC) being the most common single entity. OSCC has a significant recurrence rate and about 40% of patients show metastasis to cervical lymph nodes. The detection of nodal metastasis at the time of diagnosis is moreover associated with 50% reduction in survival in 5 years., Routine clinical examination and palpation have demonstrated only 68% accuracy in pretreatment detecting metastasis. The use of CT scan may increase the accuracy but occult metastasis may still remain for 20%–45% of patients. Although sentinel node biopsy holds great promise, widespread application is limited by the lack of rapid and accurate, intraoperative detection of metastatic disease in the sentinel node(s). Furthermore, it is expensive technique. The present available methods for pretreatment assessment of lymph node metastasis has their own drawbacks and limitations because of false-positive and false-negative results.
Patients with lymph node metastasis have a markedly worse prognosis than patients without metastasis. Only 25%–40% of patients with lymph node metastasis at presentation will achieve 5-year survival, compared to approximately 90% of patients without metastasis., Even after surgical removal of draining lymph nodes and radiation the node negative patients estimated to have a 20% or greater risk of metastasis. The ability to better predict lymph node metastasis could allow therapy better tailored to each patient.
A variety of nucleic acid-based biomarkers has been demonstrated as novel and powerful tools for the detection of cancers.,, However, most of these markers have been identified either in cancer cell lines or in biopsy specimens from late invasive and metastatic cancers. We are still lacking in establishing the biomarkers assessment in oral cancer patients using easily available, noninvasive sample of saliva and small bit of tumor tissue of same patients before any surgical therapeutic intervention. The present study was undertaken to assess whether salivary and tumor tissue total RNA level can be used to assess the cervical lymph node metastasis in patients with OSCC.
| Methodology|| |
Source of subjects
The study was conducted at an Institution with hospital-based level setup, wherein patients reporting from different levels of strata. One hundred and fourteen patients histopathologically diagnosed with OSCC were included as participants. The written informed consent was obtained from each participant before obtaining any samples. This study was approved by institutional ethical review board. The following exclusion criteria were imposed before selecting the participants.
- Participants who are undergoing or have already taken either surgical, radiotherapy and/or chemotherapy
- Participants with recurrence of OSCC
- Participants with any salivary gland lesion and or medication which can alter saliva properties were excluded from the study.
The plan of study is as follows
Clinical records: Demographic details, a thorough clinical history, were recorded for each participant. The pretreatment records of radio imaging assessment of neck lymph nodes were also documented.
Whole unstimulated saliva was collected before the incisonal biopsy procedure, using standard techniques, as described by Navazesh. The samples of saliva were stored at −20°C until further analysis.
The tumor tissue was obtained during incisional biopsy for histological diagnosis. The tissue bit was immediately immersed in “RNA Later” RNA Stabilization Solution (Qiagen) in 1.5 ml eppendorf tube and stored at −20°C until further analysis.
The confirmation of cervical lymph node metastasis was done through histological examination of dissected lymph nodes from surgically resected specimen after the routine surgical therapy. The grading of OSCC was done based on Broader's grading system. All histological examinations were carried out by two independent oral pathologists who were blinded for further molecular assessments.
Isolation and purification of total RNA from stored saliva and tumor tissue sample were done using Qiazol Reagent [Figure 1]a and RNeasy® Mini Kit (Qiagen, Germany) and protocol given by Rio DC (Cold Spring Harb Protocol) with modification as per our optimization [Figure 1]b. The isolated purified RNA was quantified using QIAxpert (Qiagen, Germany) which works on UV spectrophotometer [Figure 1]c and [Figure 1]d.
|Figure 1: (a) Addition of Quiazol and chloroform will lead to clear separation (arrow) of nucleic acids from other cellular contents. The buffy coat indicates the cellular debris. (b) The eppendorf tube shows RNA pellet at the bottom (arrow). (c and d) Quantification of RNA in saliva and tumour tissue by using QIAxpert|
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All the collected data which include the demographc details, total RNA (ng/dl) of saliva and tumor tissue of each participants and cervical lymph node metastasis status at pretreatment level (through Radioimaging) and post treatment level (through histopathology of dissected lymph nodes), was compiled and statistically analyzed for significance.
| Results|| |
A total of 114 patients with histologically confirmed diagnosis of OSCC and who have undergone surgical therapy for the same were included as participants. The average age of participant included was 47.8 years old. The male predominance is seen as 81 male participants were included against 33 female participants. Out of 114 participants included in study 76 participants (67%) were showing well-differentiated squamous cell carcinoma, whereas 35 (31%) participants fall under moderately differentiated squamous cell carcinoma, and only 3 participants (2%) showed poorly differentiated squamous cell carcinoma. Based on the postsurgical histological examination of lymph nodes, 55% (63) of participants showed the presence of metastasis whereas 45% (51) showed the absence of any metastasis.
Receiver operating characteristics curve was plotted to find the cut off value of total RNA from saliva and tumor tissue of all participants [Graph 1] which will help to assess the specificity and sensitivity of predictivity of lymph node metastasis. The area under the curve is 0.647 and 0.628 for the saliva and tumor tissue, respectively. Indicating moderate predictivity and both are significant [Table 1]. The cut off values are determined based on highest combination of sensitivity and specificity [SD- [Table 1]. For saliva, the cut off value was 66.95 ng/dl and for tumor tissue it is 197.1 ng/dl.
On comparison of salivary RNA level (cutoff of 66.95) with lymph node metastasis based on histopathology, it shows sensitivity of 63.5 % and specificity of 62.7%. The test has a positive predictive value of 67.8% and negative predictive value of 58.2%. The test and the gold standard agree on 72 out of 114 having a diagnostic accuracy of 63.16%. The κ = 0.26 indicates moderate agreement with a P = 0.008 [Table 2].
|Table 2: Sensitivity, specificity, negative predictive value, positive predictive value and diagnostic accuracy|
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On comparison of tumor tissue RNA level (cutoff of 197.1) with lymph node metastasis based on histopathology, it shows sensitivity of 71.4 % and specificity of 58.8%. The test has a positive predictive value of 68.2% and negative predictive value of 62.5%. The test and the gold standard agree on 75 out of 114 having a diagnostic accuracy of 65.78%. The κ = 0.304 indicates moderate agreement with a P = 0.001 [Table 2].
Based on above results, the inference can be drawn that when total RNA level is above the 66.95 ng/dl in saliva of patients suspected with OSCC, the chances of presence of positive lymph node metastasis is 63.16% [Graph 2]. Similarly for tumor tissue when total RNA level goes above 197.1 ng/dl, the chances of the presence of positive lymph node metastasis are 65.79% [Graph 3].
| Discussion|| |
Saliva contains clinically discriminatory protein and RNA biomarkers of oral cancer, Recently, human RNA obtained from cell-free saliva was shown to be a biomarker for oral cancer., Park et al. have shown for the first time cell-free saliva from healthy individuals contains more than 3000 species of mRNA, out of which 17 mRNA that were present in higher amounts in patients with oral cancer than in healthy persons. Based on the salivary mRNA concentration, they have developed prediction model for 4 genes which has shown 91% sensitivity and specificity for oral cancer detection. In the present study, we could isolate the pure form of RNA and we could quantify it in saliva sample collected before any intervention. Although the volume of RNA in saliva is less as compared to what we observed the volume of total RNA in small bit of tumor tissue [Table 1].
There are three major sources of RNA in whole saliva, major salivary glands, gingival crevicular fluid and oral mucosal cells. One more explanation of how RNAs enter the saliva is cell death. Cell lysis at the salivary ducts, gingival pockets or the oral epithelium can lead to the release of RNA into the saliva. It is also possible that RNAs are actively secreted. When RNA level in saliva and tumor tissue was compared with lymph node metastasis, the higher value was observed in metastasis positive group as compared to metastasis negative group though it is statistically not significant [SD –[Table 2]. These RNAs could originate from secreting cells or they could be produced elsewhere in the body, travel through the circulatory system, and be secreted into the saliva., The ribose nucleic acids (RNAs) in the saliva are produced either locally or from serum.,, Serum-derived RNAs are transported through acinar cells and gingival crevicular fluid and also by transcellular (active transport or passive diffusion) and paracellular routes (ultrafiltration). Cellular necrosis and apoptosis are believed to be the principal mechanisms of release of ribose nucleic acids in saliva.,
Park et al. have shown that saliva contains both full length and partially degraded mRNA. RNA entered the saliva through different sources and association with macromolecules may protect the salivary RNA from degradation. As per their study, the half-life of endogenous salivary mRNA is 12.2 min which is far more than 4.4 min of exogenous salivary mRNA. Since salivary RNA can be preserved for long period, it is good candidate for molecular-based assessment of oral diseases.
The presence of elevated RNAse activity in saliva of oral cancer patients making RNA more susceptible to degradation as reported by Kharchenko and Shpakov. However, we could consistently detect the RNA in saliva of all participants. In this present study, we have neither used the RNAse inhibitor nor we stored the saliva sample at −80°C as like protocol followed by Li et al.
We have done modification in Cold Spring Harbour Protocol given by Rio DC for purification of RNA. We have used Beta-mercaptoethanol (ß-ME) as a reducing agent that will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality. In combination with the strong, but temporary denaturing effects of guanidinium isothiocyanate contained in buffer RLT of the RNeasy Kits, any RNases present in the material to be extracted from will be completely inactivated.
Furthermore, we used lithium chloride (LiCl) to precipitate RNA, although precipitation with alcohol and a monovalent cation such as sodium or ammonium ion is much more widely used. Barlow et al. have shown through rabbit reticulocyte ribosome precipitation technique, LiCl precipitation offers major advantages over other RNA precipitation methods in that it does not efficiently precipitate DNA, protein or carbohydrate. It is the method of choice for removing inhibitors of translation or cDNA synthesis from RNA preparations. It also provides a simple rapid method for recovering RNA fromin vitro transcription reactions.
We compared our method with technique followed by Pandit et al., for RNA extraction from saliva. In their study, RNA was extracted from both the cell pellet and the cell-free supernatant of saliva. In the present study, we have used only supernatant from saliva to avoid debris, exfoliated cells, mixture of tumor cells and microbes if any. The RNA yield observed in the present study was ranging from 15.8 ng/μl to 636 ng/μl as compared to the study by Pandit et al. which showed RNA yield was ranging from 75 to 356 ng/μl. The reason for wide range of RNA yield in our study may be due to more number of patients and modification in techniques.
It is conceivable that disease-associated RNA can find its way into the oral cavity through the salivary gland or circulation through the gingival crevicular fluid. For OSCC, the local tumor is the source of elevated level of RNA in saliva., The present study has shown the elevated level of RNA in saliva as well as tumor tissue in OSCC patients with metastasis [SD –[Table 2]. Li et al. have found in a series of experiments that various mRNAs are upregulated in the saliva of patients suffering from OSSC. In present study though the levels of RNA in saliva and tumor tissue were found to be elevated in metastatic group as compared to non metastatic group with diagnostic accuracy ranging from 63-65%, the radioimaging technique shows high diagnostic accuracy of 75.44% [Table 2]. Low diagnostic accuracy of salivary and tumor tissue RNA as compared to radioimaging techniques may be due to its non specificity. Further study of RNA with specific focus on miRNA, siRNA, or mRNA may show high diagnostic accuracy with high sensitivity and specificity Zhang et al. had conducted systematic review and meta-analysis regarding long noncoding RNA (ncRNA) LINC00152 as a novel predictor of lymph node metastasis. Fang et al. have shown that increased expression of long ncRNA UCA1 in tongue squamous cell carcinoma correlates well with lymph node metastasis. Thus, enhanced expression of UCA1 lncRNA might promote cancer metastasis in TSCCs. The key feature of all ncRNAs is that they are not translated into proteins, but rather function directly at the RNA level.,
The present study was restricted to isolation, purification and quantification of total RNA in saliva and tumor tissue and we did not carry out further analysis of characterization of RNA as LncRNA, snRNA, miRNA and gene expression. There is lot of future scope for such further analysis.
Salivary RNA and tumor tissue RNA can reflect certain clinical and pathological features of OSCC. Salivary total RNA and tumor tissue total RNA can be indicator for cervical lymph node metastasis in patients with OSCC. Further specification of this total RNA and segregation and study of mRNA, siRNAs, miRNAs with targeted pathways may come out with new tools for the assessment of OSCC patients at presurgical stage. Thus, these tools in future may help to overcome the limitations faced at present for the assessment of lymph node metastasis in OSCC patients.
Authors would like to acknowledge the support for molecular biology lab by Principal, Department of Pharmacy, Sumandeep Vidyapeeth. We would like to thank to the Department of Pathology and Department of ENT, Smt. BK Shah Medical College and Research Centre, Sumandeep Vidyapeeth, Piparia, Vadodara for all their support and help. Also, we thank all the postgraduate students from Department of Oral Pathology and Microbiology, Department of Oral Surgery, K M Shah Dental College and Hospital and Department of ENT, Smt. BK Shah Medical College and Research Centre, Sumandeep Vidyapeeth, Piparia, Vadodara for their help in terms of patients
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]