Year : 2010 | Volume
: 21 | Issue : 1 | Page : 10--15
Evaluation of circulating immune complexes and serum immunoglobulins in oral cancer patients - A follow up study
Sameena Parveen1, Neeraj Taneja2, Renuka J Bathi3, AC Deka4,
1 Department of Oral Medicine and Radiology, SB Patil Dental College and Hospital, Devi Colony, BIDAR - 585 401, India
2 Reader, Seema Dental College and Hospital, Virbhadra Road, RISHIKESH - 249 203 (UA), India
3 School of Dental sciences, Sharda University, Knowledge Park III, Greater Noida (UP), India
4 Karnataka Cancer Treatment and Reasearch Institute, Navanagar, Hubli, Karnataka, India
Reader, Seema Dental College and Hospital, Virbhadra Road, RISHIKESH - 249 203 (UA)
Background: High serum immunoglobulins and circulating immune complexes (IgG, IgM, IgA and CIC) values in patients with cancer have been used as tumor markers. Hence, the aim of the study was to estimate these immunological markers in pre- and post-treatment phases with a follow-up of 3-24 months and to understand the prognostic significance of the same in patients with oral cancer.
Materials and Methods: The malignancy group consisted of 56 patients with different stages (AJCC TNM) of oral cancer and 20 healthy control group. Samples were selected at random and subjected for sequential analysis of serum biochemical markers (IgG, IgA, IgM and CIC-circulating immune complexes levels) in the pre- and post-treatment period. Statistical method employed was the paired t test.
Results: We observed significant elevated levels of all the immunological markers ( P < 0.01) when compared with the control group. Sequential analysis of these markers revealed significant reduction in immunological markers in stage I and II patients. On the contrary, stage III and IV patients showed remarkably elevated levels of IgA and CIC one year after initial treatment.
Conclusions : All these immunological markers are indicative of tumor burden and Serum levels of CIC and IgA might be employed as prognostic indicators in oral cancer.
|How to cite this article:|
Parveen S, Taneja N, Bathi RJ, Deka A C. Evaluation of circulating immune complexes and serum immunoglobulins in oral cancer patients - A follow up study.Indian J Dent Res 2010;21:10-15
|How to cite this URL:|
Parveen S, Taneja N, Bathi RJ, Deka A C. Evaluation of circulating immune complexes and serum immunoglobulins in oral cancer patients - A follow up study. Indian J Dent Res [serial online] 2010 [cited 2021 May 16 ];21:10-15
Available from: https://www.ijdr.in/text.asp?2010/21/1/10/62800
The term "tumor marker" is applied to indicate the risk, presence, status or future behavior of cancer. The range of tumor markers in human cancers include oncofetal proteins, enzymes, hormones, polyamines, tumor associated antigens, circulating immune complexes (CIC), lipids, viral markers, immunoglobulins and glycoproteins. 
Immunoglobulin serves as a specific link between epitopes on tumor cell antigens and the host-effectors cells. Many authors have studied specific immunoglobulin response caused by malignancy.  Previous Investigations of serum immunoglobulin levels showed an increase in IgM, IgA, IgE and IgG in oral cancer patients when compared with normal individuals. ,, It has been reported that the levels of serum immunoglobulins increase with the progression of the disease. ,
Circulating immune complexes play a specific role as initiators of mechanism of tissue injury in many infections, autoimmune diseases and neoplastic diseases. A strong correlation exists between CIC levels and progress to cancer and to a slightly lesser extent in oral premalignancy. Studies have shown increased level of circulating immune complex levels in oral cancer , and in laryngeal cancer. 
Though the levels of CICs and serum immunoglobulins, which are already studied in oral cancer patients, very few studies are available on their pattern after completion of therapy. A follow-up study may fill the voids in this aspect. Hence, the present study was undertaken, which deals with the estimation of CIC and immunoglobulin levels of IgG, IgA, IgM in both pre-treatment and post-treatment phases, with follow-up period of 3 to 24 months in patients with oral cancer, to understand the prognostic significance of these parameters.
Materials and Methods
The present study included two groups viz. (i) Control group (C) of 20 normal healthy individuals without any illness (12 males and 8 females) with an age range of 40- 70 years. (ii) Study group of 56 patients with different stages of oral cancer [OC] (34 male and 22 female) with an age range of 40-70 years). The subjects of this study were selected at random from the patients who consulted at the department of oral medicine and radiology and Craniofacial unit, SDM College of Dental Sciences, Dharwad, and cancer patients who consulted and were admitted as in-patients at Karnataka Cancer Therapy and Research Institute, Navanagar, Hubli. Patient history and clinical findings were recorded in a specially prepared standard proforma. The initial clinical diagnosis of malignancy was later confirmed histopathologically and grouped according to AJCC TNM staging. Stage I patients: 04 (well differentiated), stage II patients: 09 "(04 well differentiated and 04 moderately differentiated), stage III patients: 16 (03 well differentiated, 07 moderately differentiated and 6 poorly differentiated) and stage IV patients: 27 (12 moderately differentiated and 15 poorly differentiated). Stage I and II patients were combined, as the number of cases in stages I and II were too small.
After obtaining permission from the ethical committee and informed consent from the patients, blood samples were taken from 56 patients during pre-treatment or before treatment (BT), immediately after treatment from 40 patients (AT), from 33 patients 6 months after treatment (AT6), from 37 patients 12 months after treatment (AT12) and from 31 patients from all four phases (BT, AT, AT6, AT12). After collection of the last blood sample at AT12 another 12 months of clinical follow-up of the patients was made.
Treatment administered in stage I and II patients was surgery and radiation. In stage III and IV patients, combined therapy was given (surgery, radiation and chemotherapy). Chemotherapy inclusive of Cisplatin injection 30 mg/m 2 IV infusion weekly for 6 weeks and 5-flurouracil injection 500 mg/m 2 IV injection weekly for 6 weeks. In radiotherapy, tumor dose was given as 2 Gy/day, 5 fractions per week for 6 weeks. Hence, a total dose was 60 Gy in 30 fractions. Total treatment period was 2-3 months.
The quantitative estimation of immunoglobulin IgG, IgA and IgM was done by single radial immunodiffusion technique. In principle, the method consists of radial diffusion of protein-antigen from a point application into an antibody containing gel forming a circular precipitate at the point of equivalence.  For the assay of immune complexes, polyethylene glycol (PEG) precipitation method was used. The test sample was prepared by diluting 0.1 ml of the serum with 0.2 ml of borate-buffered saline (BBS, PH 7.4) to which 2.7 ml of 4.16% polyethylene glycol (molecular weight 6000) in BBS was added. The mixture was incubated at 4°C overnight and the absorbance was measured in a spectro-calorimeter at 470 nm to assess its turbidity caused by precipitation of circulating immune complexes (CIC). The increase in turbidity caused by CIC was calculated by subtracting the optical density (OD) at 470 nm of the buffered-treated sample from the optical density (OD) at 470 nm of the PEG-treated sample. The CIC-PEG values are recorded as optical density (OD 470 x l000). 
Of 56 patients, 7 refused to complete the treatment and 6 patients died during the subsequent course of the treatment. Blood samples could not be collected from 12 patients due to unwillingness. Two subjects from stage IV reported for recurrence at six months after the treatment.
Data were analyzed using the SPSS statistical software. The values were expressed as meanąSEM. The levels of significance were determined by using the paired t test to evaluate in the marker levels during and/or after anticancer treatment in oral cancer patients. P 0.05, [Figure 2]. Pretreatment (BT) mean values of IgG were compared with post-treatment values (AT, AT6, AT12). The mean values of IgG decrease postoperatively irrespective of all stages, which was statistically significant (P 0.05) [Figure 3]. An immediate reduction was noted in IgM values immediately after the treatment from all the oral cancer cases included in the study when compared with pretreatment level [Figure 4]. During the follow-up period (AT6, AT12), the mean values of IgM reduced gradually but the reduced values were not significant in stage III and IV patients (P > 0.05). However, in stage I and II patients, maximum mean loss between BT and AT12 was 7.0%, which was statistically significant with P  prostate cancer by Ghankari et al. (1993)  and nasopharyngeal carcinoma by De-en et al. (1988). 
The CIC are formed by the interaction of antibodies with immunogenic antigens. Immune complexes with only a slight excess of antibody or antigen are soluble and activate complement. CIC formation may be viewed as a host defence against foreign antigens. Under normal circumstances CIC are cleared by phagocytosis. If CIC escape phagocytic clearance they may be deposited in endothelial or vascular structures provoking an inflammatory damage by tissue response.
The levels of CIC are often increased in patients with cancer when compared with controls. This has been reported by previous authors such as Balaram et al. (1987),  Abraham et al. (1987)  and Balan et al. (1991).  Our study is in full agreement with previous studies showing higher values of CIC in patients with oral cancer compared with controls. In contrast, Ristow et al. (1979)  observed no significant difference between the levels of CIC in controls and patient with colon cancer.
It has been reported that CICs generally increase with the progression of clinical stages in oral cancer patients. Vijay kumar et al. (1986)  and Yamanaka et al. (1988)  found that the serum levels of these patients were elevated according to the degree of advancement of the disease stage, showing good clinical correlations. Similarly, in our study we noted an increased CIC level with the progression of stages of malignancy. In contrast, Abraham et al. (1987),  Daniel et al. (1993)  and Balan et al. (1991)  found no correlations between immune complexes levels, stage of disease or nodal status.
The important aspect of our study focuses on the follow-up of 31 cases with oral cancer at an interval of 6 months (AT6) and 1 year (AT12) after completion of treatment. On post-therapy evaluation, we observed a decline in mean values of CIC (AT) compared with pretreatment (BT) in stages I and II. During follow-up period (AT6) and (AT12), again a gradual decline in the mean values of CIC, which was statistically significant (P 0.05). The high values in stages III and IV may be due to outburst of antigen into circulation. A probable cause may be necrosis of the tumor cells caused by either pressure from rapidly multiplying cells or decreased blood supply, resulting in precipitation of high titre values indicating poor prognosis.
Important point to be noted is that the two cases in which recurrence of the disease was seen were in stage IV. Both the cases showed very high values of CIC in pretreatment (BT). During post-therapy evaluation, the levels are gradually augmented during AT, AT6 and AT12 phase, indicating the increasing tumor burden. Our study is in accordance with previous study by Sarnath et al. (1985),  Krajjina et al. (1991)  and Abraham (1987)  in which the values remain elevated in patients showing recurrence.
In serum, the predominant immunoglobulin belongs to IgG class. It is the only maternal immunoglobulin that is normally transported across the placenta and provides natural immunity in the newborn. In our study, IgG was significantly elevated among patients with cancer compared to controls. The values were correlated with the stages of malignancy, which showed gradual increase in the values from stage I to stage IV. Studies by Kohli et al. (1987),  De-en et al. (1988)  and Balan et al. (1991)  have shown significant elevation of IgG in pretreatment cancer stages. The results of these previous studies with respect to IgG and corresponding increase in their levels with stages of malignancy were in accordance with our study. In contrast, Neuchrist et al. (1994)  found no significant difference between tumor and controls. The elevation of IgG levels in oral cancer patients may be due to dependencies of IgG on the intensity of the antigenic stimulation and functional capacity of the antibody producing mechanism.
Post-treatment (AT) values of IgG in our study showed a significant fall in their values among the oral cancer patients. There was a decline in IgG levels during post-therapy period, but the levels of IgG did not reach the normal baseline values of our study group, contrary to the study reported by Robert et al. (1986)  in which the levels reaches the baseline group. The values of IgG kept on decreasing gradually through AT6 to AT12 in all stages, which was statistically significant with P 0.05. De-en et al. (1987)  found that levels of IgG decreased markedly 6-8 months after completion of radiotherapy. In contrast, Khanna (1982)  found that the treatment modality (surgery and chemotherapy) does not seem to affect serum immunoglobulin levels.
Serum immunoglobulin (IgM) is the most common immunoglobulin expressed on the surface of B cells and first antibodies to appear in the serum after primary exposure to an antigen. The serum IgM levels are often increased in patients with cancer. This has been reported by Kohli et al. (1987),  De-en et al. (1988)  and Balan et al. (1991).  Our study also shows higher values of this parameter in patients with oral cancer when compared with controls, which is in agreement with previous studies. At the same time, the increased levels of IgM in cancer patients correlated with the clinical stages as observed by Khanna et al.  On the contrary, Rajendra et al.  found no significant increase in IgM levels with the progression of disease.
Post-treatment (AT) mean values of IgM from all cases irrespective of the stages reduced immediately after treatment when compared to pretreatment levels. During the follow-up period (AT6, AT12), the mean values of IgM reduced gradually but were not statistically significant in stages III and IV (P > 0.05). Kohli et al. (1987)  found a decrease in IgM level after radiotherapy; similarly, De-en et al. (1988)  in their study showed marginal decline immediately after completion of radiotherapy and significant decrease after 6-8 months of therapy. In contrast, Vasudevan (1971)  found no significant changes in values of IgM in the carcinoma of cervix.
In this study, increased levels of mean IgA values were found among patients with cancer when compared to control groups. Similar observations are made by Schantz et al. (1988),  Scully (1982)  and Vijay Kumar et al. (1986).  Increased IgA and IgG level were observed in oral cancer by Brown et al. (1975),  whereas Khanna et al. (1982)  reported a significant increase in IgA as well as IgM levels.
We noticed increased IgA levels with the progression of disease. These observations are consistent with the findings of Frank et al. (1988)  who reported the highest values of IgA in patients with advanced disease. Higher levels were associated with advanced disease rather than early primary disease. Serum concentrations of IgA was increased in patients with oral cancer as IgA is the predominant immunoglobulin secreted into mucosal surface of the nose, mouth, stomach, intestine, lungs, tears and colostrum. The increased serum IgA values in oral cancer cases may due the local immune response to the antigenic stimulation for the tumor.
In the post-treatment period, IgA levels reduced immediately after treatment during the follow-up period AT, AT6 and AT12 phase from stage I and II and stage III. However, the reduced levels were significant in stages I and II and not significant in stage III. In contrast, the post-treatment IgA mean values in stage IV did not show any reduction immediately after treatment; instead, there was marginal increase in values in AT period. The values kept increasing even in the follow-up period and reached maximum in AT12, which was statistically significant (P  found higher IgA anti-Fab activity in stage IV compared with stage I and II, demonstrating highest values in patients who died within 6 months after final diagnosis, suggesting an association between autoimmunity and final disintegration of physiologic body functions. Similarly, Schantz et al. (1988)  found that elevated IgA blood levels reflect the autoimmune nature of cancer. Elevated IgA blood levels results in altered immunological stage characterized by its tumor-promoting capacity, poor prognosis and higher probability of recurrent disease than those with normal IgA values. In two recurrent cases, the pretherapy levels were very high. After treatment (AT) there was elevation in the values of IgA, which increased again in the AT6 and AT12 phase, indicating increasing tumor burden and poor prognosis.
As mentioned earlier, we have noticed a significant increase in serum immunoglobulins and CIC in patients with untreated oral cancer when compared to those in the control group. The elevated levels correlated well with the extent of the malignant disease. The post-treatment values of IgG and IgM kept on decreasing from AT6 to AT12 in all stages, whereas IgA levels and CIC in stages III and IV started increasing from AT6 to AT12 phases.
Among the two recurrent cases, in one case the recurrence was in the primary site and in the other, the contralateral groups of lymph nodes were positive for the malignant disease. In these two cases, the pretreatment (BT) levels of all the parameters remained higher, which increased during AT, AT6 and AT12 phases, maintaining higher values than the other cases in stage IV. Increased serum immunoglobulins may reflect local infiltration of plasma cells adjacent to the neoplastic growth and superadded infection may further increase immunoglobulin levels. The persistent elevated levels of CIC could be due to the inability of the host to clear immune complexes because of immunosuppressive state or even after therapy the tumor antigen still persisted. Increased levels of immunoglobulins and CIC with advanced stage of cancer are indicative of an adverse prognosis. In the present study, the higher levels of IgA in stages III and IV in AT, AT6 and AT12, unlike IgG and IgM, may represent poor prognosis and possibly be related to the dormant state of disease. It is an established fact that cases from stages III and IV have poor prognosis compared with stages I and II. In the current study, during the clinical follow-up of cases after the collection of blood samples at 12-month period after the treatment (AT12), we noticed the poor prognosis among cases that showed higher values of CIC and IgA. Since all the parameters were increased in oral cancer patients, particularly IgA and CIC can be considered as tumor markers. Increase in these tumor markers could be due to residual tumor, metastasis, recurrence or subclinical disease. As seen in the present series of cases, the local recurrence was noticed only in two cases and poor prognosis observed in cases without clinically visible local recurrence. As Lorenz et al.  suggested the association between autoimmunity and final disintegration of physiologic body function in cases who showed higher values of IgA-anti-Fab activity. Similar explanation holds well in this study that showed poor prognosis. Although IgM and IgG showed higher levels in AT12 phases, IgA showed a remarkable difference in stage IV patients. Hence, IgA would be a better prognostic indicator compared with IgG and IgM. Therefore, serial monitoring of CIC and IgA could be used to know the prognosis in patients with oral cancer. However, there is scope for further studies to investigate the causes of death in the absence of visible local disease in treated patients with the larger sample size.
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