Research Article

Bevel: Original Research

C-Reactive Protein: Precise, Penurious and Evidential Diagnostic and Prognostic Biomarker for Oral Squamous Cell Carcinoma: An In vitro study

Deepak Chandrakant Kelgandre, Jigna Rakesh Pathak, Shilpa Nilesh Patel, LS Poonja, Niharika Swain

Abstract

Background: The number of patients with oral carcinomas is increasing gradually, although the diagnostic modalities and therapeutic management of oral cancer is improving, the treatment outcome and prognosis of oral cancer have improved little. The absence of definite early warning signs for most head and neck cancers suggests that sensitive and specific biomarkers are likely to be important in screening high-risk patients. Aims: To analyse Serum C- Reactive protein levels in Oral Squamous Cell Carcinoma.Settings and Design: A prospective study was performed in Oral squamous cell carcinoma patients who reported our institute. Serum C Reactive protein levels were analysed. Methods and Material: Ninety histopathologically proven cases of Oral squamous cell carcinoma (study group) and 90 normal healthy individuals (control group) selected for study. Statistical analysis used Independent Sample t-test, One Sample t-test and One-way Analysis of Variance followed by TuckeyŐs POST HOC test. Results: Statistically significant increase in serum C- Reactive protein level was observed in Oral Squamous Cell Carcinoma as histopathological grade increased. Conclusion: Serum C- Reactive protein levels in Oral squamous cell carcinoma may be useful diagnostic and prognostic biomarkers in clinical practice and our data warrant a large-scale study to establish and confirm the clinical utility of the same as a prognostic & diagnostic biomarker.

 

Keywords: Oral Squamous Cell Carcinoma; C reactive protein; CRP

 

Deepak Chandrakant Kelgandre, Jigna Rakesh Pathak, Shilpa Nilesh Patel, LS Poonja, Niharika Swain. C-Reactive Protein: Precise, Penurious and Evidential Diagnostic and Prognostic Biomarker for Oral Squamous Cell Carcinoma: An In vitro study. International Journal of Oral & Maxillofacial Pathology; 2015:6(3):07-13. ©International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta Software Private Limited. All Rights Reserved.

 


Introduction

The number of patients with oral carcinomas is increasing gradually.1 Failure or delay in the early diagnosis of oral squamous cell carcinoma (OSCC) is one of the primary causes of high mortality and morbidity in cancer patients.2 Further insights into the mechanisms leading to malignancy are prerequisite for identifying new biomarkers for OSCC from the serum. The idea of screening and following patients with malignancy by serum analysis is appealing from several points of view including its ease, economical advantage, non-invasiveness and possibility of repeated sampling3. Serum C reactive protein (CRP) levels are increased in cancers of colon4, bladder5, breast4, esophagus6 and liver.5 Therefore, aim of the present study was to analyze Serum CRP levels in OSCC and objectives of the study were to compare serum CRP levels of OSCC patients with normal healthy individuals, to correlate serumCRP levels of OSCC patients with histopathological grades of OSCC and to determine the diagnostic and prognostic implications of serum CRP levels in OSCC patients.

 

Materials and Methods

In the present prospective study 90 OSCC patients who reported to our institute were selected as a study group. Similarly, 90 normal healthy age matched individuals were selected as a control group. Ethical clearance was taken from ethical committee before starting the study. A written informed consent has been taken from all the individuals. Detailed customized case history of all the patients was taken. Inclusion criteria were histopathologically diagnosed OSCC patients, having no history of any other malignancies. Individuals having history of prior treatment for OSCC or other malignancies, suffering from granulomatous diseases like tuberculosis or inflammatory or infectious diseases were excluded.

 

Routine blood investigations like complete blood count, random blood sugar level, bleeding and clotting time was done. Incisional biopsy was performed in clinically suspected cases of oral cancer (Figure 1) under local anesthesia by using 2% Lignocaine. Tissue samples were processed by routine method and paraffin embedded blocks was prepared. Paraffin embedded specimen was cut into sections of 5µm thickness by using the soft tissue microtome and were stained by using hematoxylin and eosin stain. (Figure 2).


Figure 1: Various clinical forms of Oral Squamous Cell Carcinoma such as homogeneous whitish patch involving labial and buccal mucosa (a), Non homogeneous red-white patch (b), ulceroproliferative growth into the labial vestibule (c) and in the  buccal vestibule (d) with involvement of submandibular lymph nodes.

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Figure 2: Histopathological grading of Oral Squamous Cell Carcinoma A & B: Well differentiated, C: Moderatelly differentiated, D: Poorly differentiaed grade.

 


According to descriptive criteria for OSCC grading provided by Royal College of Pathologists and WHO 20057, patients were categorized into three grades i.e. Well Differentiated, Moderately Differentiated and Poorly Differentiated with 30 cases each. Under the normal aseptic conditions 5ml venous blood was collected from both the groups. Using centrifugation machine at 3000rpm for ten minutesŐ serum was separated. Serum analysis for CRP was done in both the groups.

 

Principle of RHELAX-CRP slide test (Beacon Diagnostic Pvt Ltd., Goa) for detection of CRP is based on the principle of agglutination. The test specimen (serum) is mixed with RHELAX-CRP latex reagent and allowed to react. If CRP concentration is greater than 0.6mg/dl a visible agglutination is observed. If CRP concentration is less than 0.6mg/dl, then no agglutination is observed. During the estimation of serum CRP first we used the qualitative method in which Reagent and sample were brought to room temperature before use then One drop of test specimen (serum) was pipette on the slide using the disposable pipette, then One drop of RHELAX-CRP latex reagent was added to the drop of test specimen on the slide, test specimen and the latex reagent were mixed uniformly over the entire circle by using the mixing stick, then slide was rocked gently, back and forth, observing for agglutination macroscopically at two minutes. After qualitative method semi quantitative method was used. In this method we used the isotonic saline serial dilutions (D) of the test specimen positive in the qualitative method which was prepared as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and so on. Each dilution of the test specimen was Pipette onto separate reaction circles, then one drop of RHELAX-CRP latex reagent was added to the drop of test specimen on the slide following which test specimen and the latex reagent were mixed uniformly over the entire circle by using the mixing stick. Slide was then rocked gently, back and forth, observing for agglutination macroscopically at two minutes. (Figure 3)

 

Result of qualitative method was interpreted as positive if agglutination is positive and indicated presence of detectable levels of CRP in the test specimen. If agglutination was absent then it is considered as a negative test result and indicated absence of detectable levels of CRP in the test specimen. Results of semi quantitative method was interpreted as Agglutination in the highest serum dilution corresponds to the approximate amount of CRP in mg/dl present in the specimen. Concentration of CRP was calculated by using the formula CRP (mg/dl) = S _ D Where, S= Sensitivity of reagent i.e. 0.6 mg/dl and D= Highest dilution of serum showing agglutination.

 

All collected data was entered into a SPSS 17.0 Analysis was done using statistical tests such as independent Sample t-test, one Sample t-test and one-way ANOVA followed by TuckeyŐs POST HOC test wherever applicable. The level of significance was set at 5%. All p-values less than 0.05 were treated as a significant.

 

Results

Serum CRP was not detected in any of the control group individuals. Mean serum CRP level in OSCC group was 19.20mg/l. A significant difference of serum CRP level was observed between OSCC and control group with p-value being <0.001. As serum CRP was not detected in healthy individuals (controls) for statistical analysis value 1 was considered for comparison.

 

When serum CRP level of control group was compared with well, moderate and poorly differentiated OSCC, the p-value was 0.01 which was statistically highly significant. When serum CRP level of well-differentiated group was compared with moderately and poorly differentiated OSCC group, p-value was 0.01 and hence was statistically highly significant. When serum CRP level of moderately differentiated group was compared with poorly differentiated OSCC group, the p-value was 0.319 which was statistically insignificant. (Table 1)

 

The serum CRP levels in control group and OSCC groups are graphical represented as star dots imply the mean serum CRP levels in each group and dotted line signifies levels <6mg/l which are not detected in the serum using slide test method. (Graph 1) There is an increase in serum CRP levels from control to poorly differentiated OSCC. Table 2 describes the serum CRP level and inflammatory infiltration in well, moderate and poorly differentiated OSCC groups. Out of total n=30 cases of well differentiated OSCC, n=27 cases showed mild inflammatory response, and n=3 case showed moderate inflammatory response. Out of total n=30 cases of moderately differentiated OSCC cases, n=15 cases each showed mild and moderate inflammatory response.  Out of total n=30 cases of poorly differentiated OSCC cases, n=3 cases showed Mild inflammatory response, n=24 cases showed moderate inflammatory response, while n=3 cases showed marked inflammatory response.       

 

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Figure 3: Rhelax Reagent Kit used for Serum CRP estimation (Beacon Diagnostic Pvt Ltd.)

 

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Graph 1: Graphical presentation of serum CRP levels in control group and OSCC groups

 

Discussion

Oral and pharyngeal cancer, grouped together is the sixth leading cancer in the world and ranks in the top three in high incidence areas.8 It traditionally caries a poor prognosis with more than 50% of the cases diagnosed at an advanced stage, with the five-year survival rate remaining low at ~30 to 50%.8 Lack of noninvasive and reliable screening test has been quoted as the main reason.

 

The linkage of inflammation and cancer was first reported by Rudolf Virchow in 1863, when he identified leukocyte infiltration in neoplastic tissues, and suggested that the sites of chronic inflammation were the origin of cancer.9 Since then, approximately 25% of all cancer patients are reported to have an association with chronic inflammation of either infectious or non-infectious causes. Indeed, it has recently been suggested that cancer- related inflammation may represent the seventh hallmark of cancer, in addition to the six hallmarks identified by Hanahan and Weinberg.10 CRP is an acute-phase protein and a marker for inflammation. It is synthesized in hepatocytes as a part of the inflammatory response to tissue damage induced by infection, trauma, and malignant diseases.11 The synthesis of CRP in the hepatocytes is regulated by pro-inflammatory cytokines like interleukin-1, interleukin-6, and tumor-necrosis factor, which are also reported for different malignancies. Therefore, these pro-inflammatory cytokines are currently the subject of intense studies as influencing factors in various types of tumors.11 Increased levels of IL-6 have been shown in head and neck cancers including oral SCC with several mechanisms proposed for such a rise. It is increasingly recognized that in addition to tumor stage, the disease progression depends on a complex interaction between the tumor and the hostŐs inflammatory response.12

 

Group

Study Groups

p value

Control Group

Well Differentiated

0.001

Moderately Differentiated

0.001

Poorly Differentiated

0.001

Well Differentiated

Control Group

0.001

Moderately Differentiated

0.001

Poorly Differentiated

0.001

Moderately Differentiated

Control Group

0.001

Well Differentiated

0.001

Poorly Differentiated

0.319

Poorly Differentiated

Control Group

0.001

Well Differentiated

0.001

Moderately Differentiated

0.319

Table 1: Multiple comparisons of serum CRP level of control group with different histopathological grades of OSCC.

 

The normal serum CRP level in healthy individuals range from 5 to 6mg/l.13 We measured CRP level by slide test method, using RHELAX-CRP reagent. Sensitivity of RHELAX- CRP latex reagent is 6 mg/l, so when the CRP level was more than 6 mg/l, only then it was considered positive. In our study, serum CRP was not detected in any control group individuals. Mean serum CRP level in study group was 19.2 mg/l. Our results showed the statistically significant increased serum CRP levels in study group as compared to control group which was in agreement with study done by Ewa Jablonska et al (1997)14, Khandavilli et al (2009)13, Kim CH, Hwang SY (2010)15, Kruse et al (2010)16, Faraz Ahmed Tariq et al (2011)17, Chen HH et al (2011)18, Huang et al (2012)19, and Gallo O et al (2012)20. Ewa Jablonska et al in 199714 showed high serum concentrations of IL-1_, IL-6, TNF-_ & CRP levels in OSCC group as compared to control group. Kim CH, Hwang SY (2010)15 evaluated the serum CRP level before and after the oral cancer resection and reconstructive surgery at immediate postoperative day and after 1 & 5days. Results showed that CRP level was statistically higher in oral cancer patients before surgery than normal levels. Gallo et al20 examined levels of IL-6 and of several acute-phase proteins, including CRP in head and neck cancer patients and found that serum IL-6 and CRP level significantly increased as compared to control group. Result of our study was partly in accordance with study done by Kruse et al (2010)16, they found only 69% patients of oral cancer had the increased preoperative serum CRP level while 31% had within the normal range.


Histopathological grade of OSCC

Inflammatory Infiltration

Mean CRP level (mg/l)

Mild

Moderate

Marked

Well differentiated

27

03

00

13

Moderately differentiated

15

15

00

21

Poorly differentiated

03

24

03

23.4

Table 2: The serum CRP level and inflammatory infiltration in well, moderate and poorly differentiated OSCC groups.

 


We found that serum CRP level gradually increased from well, moderate to poorly differentiated OSCC grade. Statistically significant difference was present between well & moderate grade, and between well & poor grade, but insignificant difference between moderate & poor grade. In the literature various studies mentioned the correlation of serum CRP level with different clinical stages of oral cancer, but there was no study, which correlated the serum CRP levels with different histopathological grades. Ewa Jablonska et al (1997)14 showed that serum CRP level increases according to higher clinical stage & stated that their finding support the role of acute phase response during malignant disease, including oral carcinoma. Khandavilli et al (2009)13 demonstrated that patients with T3 and T4 tumors of OSCC had significantly higher levels of preoperative serum CRP (p = 0.046) than those with T1 and T2 tumors, and stated that this may be due to large tumors secreting higher amounts of IL-6 leading to increased hepatic synthesis of CRP. Their results also showed that survival of patients with preoperative elevation of serum CRP was significantly less favorable than that of patients without serum CRP elevation, thus they inferred that a raised preoperative CRP was associated with worse overall survival. In 2012, Huang et al19 evaluated the pre operative serum CRP level in OSCC cases and found that increased CRP level was significantly associated with pathologic tumor status, pathologic nodal metastasis, tumor depth, disease-free survival and overall survival, thus they concluded that concurrent high levels of CRP act as a predictor for lymph node metastasis, advanced tumor stage and tumor recurrence and it has a significant potential as a biomarker for risk stratification in OSCC.

 

Results of our study showed that inflammatory response increases from well to moderate to poorly grade which is in accordance with the study done by Vieira et al in 2008.21 CRP is an acute-phase protein and a marker for inflammation. The synthesis of CRP in the hepatocytes is regulated by pro-inflammatory cytokines like interleukin-1, interleukin-6, and tumor-necrosis factor, which are also reported for different malignancies like colon4, bladder5, breast4, esophagus6 and liver5. Increased levels of IL-6 have been shown in head and neck cancers including oral SCC with several mechanisms proposed for such a rise. These include secretion by, peripheral blood mononuclear cells, T-lymphocytes, tumor cells and by intra- and peri-tumour resident normal cells.13 The exact mechanism whereby IL-6 influences tumor cells is not known, with some reports suggesting that IL-6 has a dual effect by stimulating growth of some cells while inhibiting that of others.11 It is believed that IL-6 acts through the membrane bound receptor complex consisting of IL-6 binding protein and glycoprotein 130 to promote tumor cell proliferation with increased levels of IL-6 having a direct influence on tumor cell growth and progression. Increased levels of IL-6 have been shown to correlate with increased levels of serum CRP in patients with head and neck cancers.14

 

Two hypotheses could be associated with increased CRP levels as a sign of chronic inflammation. First, the induction hypothesis: Chronic inflammation results in excessive cell proliferation and activation of a cascade of cellular actions, leading to induction of irreversible DNA damage.9 Second, the response hypothesis: The immune response of the host as a consequence of tumor growth itself could be the reason for the elevation in CRP levels.22

 

There are some other biologic underlying mechanisms that explain the association between levels of CRP and risk of cancer. Several findings support the hypothesis that elevated levels of CRP is a marker of occult cancer. First, tumor growth can cause tissue inflammation around the tumor and thus increase plasma levels of CRP.9 Second, tumor cells are known to produce various cytokines and chemokineŐs that attract leukocytes, and some cancerous cells have been shown to express CRP and secrete interleukin-6 and interleukin-8, which stimulate CRP production in the liver.9 Finally, it is possible that CRP is a part of a host immune response to the tumor. It is evident that inflammatory cells act as tumor promoters, producing an attractive environment for tumor growth, inducing DNA damage, promoting angiogenesis, and favoring neoplastic spread and metastasis.

 

Results of our study showed that serum CRP level increases significantly in OSCC as compared to control group. It shows that inflammation increases markedly in OSCC, which may play an important role in carcinogenesis. In response to increased inflammation, hepatocytes synthesize more amount of CRP. The inflammation increases from well, moderate to poorly differentiated grade, which may cause the increases in CRP levels. In the literature it is mentioned that the prognosis in OSCC get worse as grade increases from well to poorly differentiated, also it is proven that elevated preoperative serum CRP is associated with worse overall survival. In our study serum CRP level increases from well, moderate to poorly differentiated grade. Therefore, we suggest that serum CRP can be used as a diagnostic and also prognostic biomarker for OSCC. Also the simplicity of measuring CRP activity combined with its cost effectiveness gives an added advantage to consider CRP as a tumor marker in oral cancer. However, during evaluation of serum CRP level one has to keep in mind that the level of these markers altered in different systemic conditions such as various cancers, infections etc., and so these factors should be ruled out.

 

Acknowledgement

We would like to acknowledge the staff members of the Dept. of Oral Pathology for their support and guidance.

 

Author Affiliations:

1.Dr.Deepak Chandrakant Kelgandre, Senior lecturer,           Oral Pathology and Microbiology, YCMM & RDFS Dental College and Hospital, Ahmednagar, 2.Dr.Jigna Rakesh Pathak, Professor, 3.Dr.Shilpa Nilesh Patel, Professor and Head, 4.Dr.LS Poonja, Professor, 5. Dr.Niharika Swain, Senior lecturer, Oral Pathology and Microbiology, MGM Dental College and Hospital, Navi Mumbai, India.    

 

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22.  Siemes C, Visser LE, Coebergh JW, Splinter TA, Witteman JC, Uitterlinden AG, et al. C-reactive protein levels, variation in the C-reactive protein gene, and cancer risk: The Rotterdam Study. J Clin Oncol. 2006;24:5216-22.

 

Corresponding Author

Dr. Deepak chandrakant kelgandre

Dept of Oral Pathology and Microbiology,

YCMM & RDFS Dental College and Hospital,

Ahmednagar, India.

E-mail: drdeepak144@gmail.com

Ph: +91 8108929898


 

 

 

 

Source of Support: Nil, Conflict of Interest: None Declared.


 

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