Preliminary reportElevated serum levels of M30 and M65 in patients with locally advanced head and neck tumors
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.
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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
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Usefulness of serum M30 and M65 levels to predict response to neoadjuvant chemotherapy in patients with breast cancer
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2014, Biomedicine and PharmacotherapyCitation Excerpt :M30 and M65 are relatively new markers that detect different circulating forms of the epithelial cell structural protein CK18. With these purposes, during more 10 years period, both M30-Apoptosense and M65 ELISAs were studied in either serum or plasma patients with many types of cancer, such as lung [15,16], colorectal [12–14], gastric [8–10], testicular [3], and head & neck carcinomas [17]. In most of the these trials, serum M30 and M65 levels were found to be elevated in cancer patients compared to healthy people and were significant prognostic and predictive values.
Serum m65 as a biomarker for metastatic renal cell carcinoma
2013, Clinical Genitourinary CancerCitation Excerpt :The biomarkers are increasingly being used in trials of new anticancer therapies. The prognostic significance of both the M30 and M65 assays has been evaluated, and it was found that the M30 and M65 assays could be important prognostic and predictive markers in several malignancies.12-17 However, the serum levels of M30 and M65 have not been evaluated in RCC in previous studies.
Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and survivin
2013, Cancer LettersCitation Excerpt :In contrast, the caspase-dependent Asp396-cleaved cytokeratin-18 fragment detected by the M30 antibody (M30) is reported to be an apoptosis specific marker [19,20]. M30 has been found to be elevated in diverse cancers [21–23] and was used in combination with the uncleaved cytokeratin-18 fragment (M65) to distinguish between apoptotic and necrotic cell death phenomenon [19,24–26]. In concordance with these studies, we observed elevated M30-levels in metastatic breast cancer.
Prognostic importance of markers for inflammation, angiogenesis and apoptosis in high grade glial tumors during temozolomide and radiotherapy
2012, International ImmunopharmacologyCitation Excerpt :Overexpression of caspases increases the apoptosis of M30 and M65 that are fragments of cytokeratin (CK) 18, released from normal and necrotic/apoptotic malignant cell deaths. These apoptotic fragments are biomarkers of tumor cell death in patients with different cancers and associated with poor prognosis [15,16]. The literature has controversial data about cytokine production in the glial tumors.