Preliminary report
Elevated serum levels of M30 and M65 in patients with locally advanced head and neck tumors

https://doi.org/10.1016/j.intimp.2009.02.004Get rights and content

Abstract

Background

M30-Apoptosense and M65 ELISAs can detect caspase cleaved and intact forms of cytokeratin 18. As biologic marker of cell death, both assays can be useful to evaluate prognosis and chemotherapy response in the patients with breast, lung, and endometrium cancer. In the present study, we measured serum M30 and M65 levels in the patients with locally advanced head and neck tumors, and compared with healthy controls.

Materials and methods

A total of 40 consecutive patients with locally advanced head and neck tumors were included in this study. The sera were collected from the patients and 32 healthy controls. Median age was 51 years (range: 19–80) and squamous cell carcinoma of head and neck was major histopathologic subtype. Primary tumors were localized in larynx, nasopharynx, hypopharynx and tongue. The mean serum M30 concentration was 112.7 ± 59 U/L in patients and this was significantly higher than healthy controls (mean 106.5 ± 17.6 U/L) (p < 0.05). Serum M65 levels were also higher in patients with head and neck tumors when compared to controls but not statistically significant (261.7 ± 175 U/L vs 200.2 ± 164.5 U/L, p = 0.077). There was no statistically significant correlation among age, stage and localization of tumor and serum M30 and M65 levels.

Conclusion

According to our knowledge, this is the first study which evaluated serum M30 and M65 levels in head and neck tumors. We found increased serum levels of M30 and M65 levels in head and neck tumors. Significantly higher levels of serum M30 may have prognostic importance in this type of tumor. Larger studies are needed to evaluate its' prognostic importance.

Introduction

Cytokeratin 18 (CK18) is major component of epithelial and endothelial cell intermediate filaments. It is cleaved by caspases during apoptosis and its' fragments are released into the serum [1], [2], [3]. M30 is a mouse monoclonal IgG2b antibody. M30-Apoptosense ELISA can recognize a neo-epitope mapped to positions 387–396 of 21 kD fragment of CK18. M30 antibody is a selective biomarker of apoptosis, because it detects only caspase-cleaved fragment of CK18, not native or intact form [4]. Another new ELISA, M65 ELISA uses two mouse monoclonal antibodies (clones M5 and M6, both IgG2b) specific for conventional epitopes on CK18. M65 ELISA measures both caspase cleaved and intact form of CK18 which are released during apoptotic and non-apoptotic cell death [5].

There is a large amount of soluble and insoluble cytokeratins in the epithelial cancers. During apoptotic and non-apoptotic cell death, intact or caspase-cleaved forms of CK18 are released to the circulation and remained stable in the circulation. M30 and M65 ELISAs, are increasingly used for the evaluation of responses to anti-cancer drugs in several tumor types [6], [7]. The stability of M30 and M65 antigens in the plasma of healthy volunteers was reported as 4–6 months after storage at − 80 °C [8]. In the cancer patients, if plasma samples were storage at − 80 °C over 2 years, M30 and M65 antigens were not degrading with the time [9].

The recent clinical trials have shown that serum M30 levels were elevated in the patient with breast and lung cancer and can be useful as a prognostic marker [10], [11]. Moreover, serum M30 and M65 levels were correlated with prognosis and reflected chemotherapy induced apoptosis in the patients with disseminated testicular germ cell tumor [12]. Serum M30 levels were found to be correlated with the tumor load, performance status and thereby prognosis in the patients with recurrent breast cancer [10]. Similarly, in the patients with disseminated testicular germ cell tumor, circulating M30 and M65 levels were found to be correlated with classic prognostic markers lactate dehydrogenase, α-fetoprotein, and β-human chorionic gonadotropin, probably reflecting tumor load [12].

In the present study, serum M30 and M65 levels were measured by ELISA in the patients with head and neck tumors and healthy volunteers. We aimed to evaluate the correlation of serum M30 and M65 levels with tumor localizations and stages.

Section snippets

Patients and methods

This is a case control study. Patients with previously untreated, aged between 18 and 80 years, histologically confirmed, locally advanced head and neck cancers were enrolled in the study. Blood samples were collected from 40 (male/female: 14/26) consecutive patients with head and neck cancer and age matched, randomly selected 32 healthy controls (male/female:10/22, aged between 18 and 78 years) who attended the blood bank at Gazi University Hospital, Ankara, Turkey. Blood samples from the

Assay methods

M30-Apoptosense® ELISA (PEVIVA, USA) is a one step in vitro immunoassay for the quantitative determination of the apoptosis-associated CK18Asp396 (M30) neo-epitope in serum and plasma. Our measuring range is 0–1000 U/L. The values of standards are 0, 75, 150, 250, 500, 750 and 1000 U/L respectively. Within assay (WA % CV) reproducibility is < 10% and between assay (BA % CV) reproducibility is < 10%. The minimal detectable concentration of CK18 Asp396 neo-epitope in M30-Apoptosense® ELISA is

Statistical analysis

Statistical analysis was performed using SPSS 11.5 version for Windows program. M30 and M65 levels have been presented on the basis of average (± standard deviation). Median values were compared using the Mann–Whitney unpaired test with two-tailed significance. Significance of ORs was estimated with the x2 test with two-tailed significance.

Results

A total of 40 (male/female: 14/26) patients were enrolled in this study. Median age was 51 years (range: 19–80 years). Most common areas of tumors were larynx (32%) and nasopharynx (25%). The majority of patients were diagnosed as having squamous cell carcinoma of head and neck except for 3 (7.5%) patients with undifferentiated tumor of nasopharynx. All of patients had locally advanced disease. The patients with distant metastasis were excluded from the study but 3 (7.5%) patients had

Discussion

Cytokeratin 18 (CK 18) is a major component of intermediate filaments of epithelial cells and tumors. During apoptotic cell death, CK 18 is cleaved into fragments by caspases which are proteolytic enzymes. These fragments of intact form of CK18 levels can be measured by using new enzyme linked immunosorbent assays (ELISAs). The M30 antibody detects only the caspase-cleaved fragments of CK18. M65 ELISA utilizes the M5 antibody, it can detect full length protein. M30 is postulated as a selective

References (17)

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