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Original article

Clinical significance of serum M30 and M65 levels in patients with breast cancer Faruk Tas *, Senem Karabulut, Ibrahim Yildiz, Derya Duranyildiz Institute of Oncology, University of Istanbul, Istanbul, Turkey

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 September 2014 Accepted 18 October 2014

M30 and M65 are relatively new assays that detect different circulating forms of the epithelial cell structural protein cytokeratin18. The objective of this study was to determine the clinical significance of the serum levels of M30 and M65 in patients with breast cancer. A total of 80 patients with a pathologically confirmed diagnosis of breast cancer were enrolled into the study. Serum M30 and M65 concentrations were determined by the solid-phase sandwich ELISA method. Serum samples were obtained on first admission before any type of treatment. The median age at diagnosis was 52 years, range 30 to 81 years. The baseline serum M30 and M65 levels in patients with metastatic disease were significantly higher than those in the non-metastatic patients (P = 0.017 and P = 0.003, respectively). Moreover, serum M65 level was also elevated in patients with large tumor size (P = 0.02). No correlation was found between these serum assay levels and response to chemotherapy (P > 0.05). However, the significant relationship was found between the serum levels of M30 and M65 (rs = 0.96, P < 0.001). Neither serum M30 nor serum M65 had significantly effect on survival (P = 0.50, and P = 0.52, respectively). In conclusion, although both serum M30 and M65 levels are elevated in metastatic disease, no predictive and prognostic roles on survival were found in patients with breast cancer. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Breast cancer M30 M65 Serum

1. Introduction Cytokeratins (CK) are characteristically expressed in various types of epithelial cells and released into the blood in either intact or caspase-cleaved forms during apoptotic and necrotic cell death [1–3]. They remain relatively stable in the circulation of cancer patients [3]. Circulating CKs, including cytokeratin18 (CK18), have been recognized for years as tumor markers in the diagnosis of cancer [2,4–6]. Although traditionally, detection of a CK antigen in circulation, such as tissue polypeptide antigen, CYFRA21-1 was an indicator of tumor burden, they are being proposed to be markers of apoptosis rather than tumor volume nowadays [2]. M30 and M65 are relatively novel described sandwich ELISA assays that determine different circulating forms of the protein CK18 in either plasma or serum and are proposed to be surrogate endpoints of different forms of cell death [2,3]. M30 detects a neoepitope mapped to positions 387 to 396 of CK18 that is only revealed after caspase cleavage of the protein and is postulated as a selective biomarker of apoptotic cell death [2,3]. However, M65 recognizes a common epitope present in the full-length protein as

well as the 21-kDa caspase cleaved fragment and therefore, it measures not only apoptosis, but also intact CK18 that is released from cells undergoing necrosis [2,3]. Both assays have now been validated as ‘‘fit for purpose’’ in the analysis of serum and plasma collected from subjects entered into clinical trials [2,3,7]. Both assays have been extensively applied as pharmacodynamic biomarkers of chemotherapy-induced cell death in a range of different cancer types treated with different chemotherapeutic agents. Additionally, these assays have been reported as having prognostic markers in several malignancies, such as testicular, gastric, pancreatic, colorectal, lung cancers, and others [3,8– 17]. Although these available findings existed in the other tumors, there are a few clinical studies to investigate the clinical significance of these assays in breast cancer [18–23]. The diagnostic, predictive and prognostic importance of measuring soluble products of CK18 is underlined by the previous studies demonstrating conflicting results. The present study was designed to assess the clinical significance of serum M30 and M65 levels in breast cancer patients. 2. Material and methods 2.1. Patients

* Corresponding author. Institute of Oncology, Istanbul University, Capa, 34390 Istanbul, Turkey. Tel.: +00 90 212 534 80 78; fax: +00 90 212 534 80 78. E-mail address: [email protected] (F. Tas).

A total of 80 female breast cancer patients admitted to Istanbul University, Institute of Oncology were enrolled into the study.

http://dx.doi.org/10.1016/j.biopha.2014.10.021 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

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2

Diagnosis of breast cancer was histologically proved either with tru-cut biopsy of the primary lesion or metastatic site and staged according to seventh edition of AJCC. All the patients were treatment-naive for at least 3 months before accrual. For histological evaluation, tissue sections (2 mm) were deparaffinized and stained using hematoxylin and eosin. Grading of tumors was established according to modified Bloom-Richardson grading system. Estrogen receptor (ER), progesterone receptor (PR) and HER2 status were evaluated in the sample sections using appropriate antibodies. The immunohistochemical staining was assessed upon visual inspection with optical microscope and considered as positive if the percentage of cells staining positive were more than 1%. In case of 2(+) staining by IHC, HER2 gene amplification was analyzed by fluorescent in situ hybridization (FISH). All patients were treated with multidisciplinary approach. Overall 66 (83%) patients received an anthracycline-based regimens and 14 (17%) received only hormone therapy either aromatase inhibitor or tamoxifen  LHRH analogues. Forty patients received adjuvant treatment after mastectomy or lumpectomy. Other 40 patients with locally-advanced and metastatic disease were treated with anthracycline-based chemotherapy (n = 37) and only hormonal therapy (n = 3). In these patients, response to treatment was evaluated according to revised RECIST criteria version 1.1. Informed consent was obtained from all patients and the study was reviewed and approved by our local ethical committee. 2.2. Measurement of serum M30 and M65 levels

Table 1 Patient characteristics. Variables

n

No. of patients Age (years) Median (range): 52 (30–81) < 50/  50 Grade of histology 1/II/III/unknown Estrogen receptor (ER) –/+/unknown Progesteron receptor (PR) –/+/unknown HER2 –/+/unknown Tumor status (T)a 1/2/3/4 Lymph node status (N)a 0/1/2/3 Metastasis status (M) 0/1 Serum hemoglobin level (12g/dL) Low/Normal Serum white blood cell (WBC) count (10 000) Normal/High Serum platelet (PLT) level Normal/Elevated Erythrocyte sedimentation rate (ESR) (50/h) Normal/Elevated Serum LDH level (450 U/L) Normal/Elevated Serum CA15-3 level (35 U/mL) Normal/Elevated Response to chemotherapy Yes/No Last status Alive/Dead

80

n :58 non-metastatic diseases.

Table 2 Results (median and range) of comparisons between the assays and various clinical/ laboratory variables. Variables

Blood samples were obtained from patients with breast cancer by venopuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at – 20 8C until analysis.

a

The M30 Apoptosense1 ELISA is a solid-phase sandwich enzyme immunoassay. Standards, controls and samples react with a solid phase capture antibody M5 directed against K18 and the HRP-(horseradish peroxidase) conjugated M30 antibody directed against the K18Asp396 neo-epitope. The M651 ELISA is a solid-phase sandwich enzyme immunoassay. Standards, controls and samples react with a solid phase capture antibody M6 directed against K18 and the HRP (horseradish peroxidase) conjugated M5 antibody directed against a different epitope of K18. Unbound conjugate was removed by a washing step. TMB substrate was added. The color development was stopped and the absorbance was read. The resulting color was directly proportional to the concentration of the analyte. The M30 Apoptosense1 ELISA and M651 ELISA were performed at room temperature. All reagents were allowed to reach room temperature before performing the assay. All reagents were vortexed prior to use. The wash solution was diluted with fresh deionised water. The M30 HRP conjugate was diluted with M30 conjugate dilution buffer and mixed. Twenty-five microlitres of M30 standard (duplicated), M30 control low, M30 control high and samples were pipetted per well. Seventy-five microlitres of the

36/44 6/36/31/7 16/57/7 28/45/7 53/19/8 19/32/1/6 22/20/19/19 58/22 31/49 68/12 75/5 10/43 59/13 36/40 18/12 53/27

Age, years < 50  50 Grade I–II III ER Negative Positive PR Negative Positive HER2 Negative Positive T status 1 2–4 N status Negative Positive M status Negative Positive Hemoglobin Low Normal WBC Normal High PLT Normal High ESR Normal High Serum LDH level Normal High Serum CA 15-3 level Normal High Response to chemotherapy Yes No

Assays M30 (U/I)

M65 (U/I)

0.23 60 (36–1169) 67 (27–1178) 0.97 63 (27–1178) 64 (35–1010) 0.71 59 (35–1178) 64 (27–1178) 0.76 64 (35–1178) 62 (27–1169) 0.85 64 (27.4–1169) 60 (35–1178) 0.96 56 (27–529) 62 (35–94) 0.86 58 (36–529) 62 (27–113) 0.017 61 (27–529) 69 (36–1178) 0.57 64 (40–1010) 62 (27–1178) 0.41 62 (35–1178) 69 (27–1169) 0.42 62 (27–1178) 65 (56–94) 0.22 65 (27–1178) 57 (36–817) 0.08 62 (27–1010) 82 (44–1178) 0.62 60 (35–1169) 63 (27–1178) 0.43 68 (37–1178) 63 (36–1169)

0.13 211 (94–2800) 240 (106–2895) 0.56 221 (133–2895) 209 (94–2252) 0.52 308 (94–2776) 221 (133–2895) 0.91 228 (94–2895) 225 (133–2800) 0.46 221 (194–2800) 229 (1161–2895) 0.02 170 (94–1389) 222 (129–609) 0.31 198 (94–1389) 215 (129–609) 0.003 211 (94–1389) 441 (156–2895) 0.09 246 (159–2392) 205 (94–2895) 0.56 225 (94–2895) 201 (142–2799) 0.87 221 (94–2895) 230 (168–284) 0.16 229 (133–2800) 190 (94–2392) 0.19 218 (94–2252) 335 (142–2800) 0.19 200 (94–2800) 224 (129–2895) 0.26 439 (129–2895) 224 (156–2780)

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

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diluted M30 HRP conjugate solution was added to each well. The wells were covered with sealing tape. The microplate was incubated on shaker for 4 hours at 600 rpm. The M65 HRP conjugate was diluted with M65 conjugate dilution buffer and mixed.25 mL of M65 standard (duplicated), M65 control low, M65 control high and samples were pipetted per well. Seventy-five microlitres of the diluted M65 HRP conjugate solution was added to each well. The wells were covered with sealing tape. The microplate was incubated on shaker for 2 hours at 600 rpm. The plate was washed in a plate washer 5 times with 250 mL/well with diluted wash solution. 200 mL of TMB substrate was added to each well. The microplate was incubated in darkness at room temperature for 20 minutes. Fifty microlitres of stop solution was added to each well. The absorbance at 450 nm was determined in a microplate reader and the results were recorded. 2.3. Statistical analysis Continuous variables were categorized using median values as cut-off point. Assessment of relationships, comparisons between various clinical/laboratory parameters and serum levels of M30/ M65 assays were accomplished using Mann-Whitney U test. Spearman’s rank order correlation was used for correlation analysis. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. Kaplan-Meier method was used for estimation of survival of patient and differences in survivals were assessed by the log-rank statistic. A P value < 0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.

3. Results Between February 2010 and June 2011, 80 female breast cancer patients were enrolled into the study. Median age of diagnosis was 52 years old (range: 30–81) (Table 1). Twenty-two (28%) patients had metastatic disease where most frequent site of metastasis was bone (n = 15, 68%). Majority of the patients with non-metastatic disease had node-positive (n = 58, 69%).

3

Table 2 shows the correlation between the serum levels of both M30/M65 and clinicopathological factors. A few of the prognostic parameters analyzed were correlated significantly with the serum assay concentrations. Both serum M30 and M65 levels were significantly higher in the metastatic patients than non-metastatic patients (P = 0.017 and P = 0.003, respectively). Additionally, the serum M65 levels were also elevated in patients with larger tumor size (> 2 cm) compared to ones with small tumors (P = 0.02). No correlation was found between these serum levels and response to chemotherapy (P = 0.43 for serum M30 and P = 0.26 for serum M65). The significant relationship was found between the serum M30 and M65 levels (rs = 0.96, n = 80, P < 0.001), Spearman’s correlation) (Fig. 1). The median follow-up time was 25 months (range: 2.7– 38 months). At the end of the observation period, 27 (34%) patients were dead. The median survival for all patients was 35.6  3.8 months (95% CI = 28.05–43.14) and 1-, 2-, 3-year overall survival rates were 87.5% (95% CI = 80–95), 72.4% (95% CI = 63–82), and 66.3% (95% CI = 53–80), respectively. Negativity of ER (P = 0.01), and unresponsiveness to chemotherapy (P = 0.04) had statistically significant and presence of metastasis tended to be worse survival (P = 0.07) (Table 3). However, neither serum M30 nor serum M65 had significantly effect on survival (P = 0.50, and P = 0.52, respectively) (Figs. 2 and 3).

4. Discussion Assessment of soluble CK18 fragments has been shown to facilitate discrimination between normal and cancer patients and to be of value in prognosis assessment and monitoring of response to therapy [2,3,7,14,19–23]. 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

Fig. 1. Correlation between serum M30 and M65 levels in patients with breast cancer (rs = 0.96, n = 80, P < 0.001), Spearman’s correlation.

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

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4 Table 3 Univariate analyses of survival. Variables

Median survival month ( SE)

All patients Age, years < 50  50 Grade I–II III ER Negative Positive PR Negative Positive HER2 Negative Positive T status 1 2–4 N status Negative Positive M status Negative Positive Hemoglobin Low Normal WBC Normal High PLT Normal High ESR Normal High Serum LDH level Normal Higher Serum CA 15-3 level Normal Higher Response to chemotherapy Yes No Serum M30 level High (> median) Low (< median) Serum M65 level High (> median) Low (< median)

35.6 (3.8)

P

30.2 (2.1) 27.7 (1.6)

0.51

29.6 (1.8) 26.2 (1.7)

0.67

20.7 (2.6) 30.1 (1.5)

0.01

27.2 (2.1) 30.1 (1.8)

0.22

28.1 (1.6) 25.5 (2.0)

0.56

30.3 (1.9) 27.0 (1.9)

0.56

33.6 (2.3) 26.1 (1.6)

0.20

31.0 (1.5) 25.5 (2.1)

0.07

29.0 (2.0) 28.8 (1.6)

0.85

29.6 (3.2) 29.2 (1.5)

0.30

28.4 (1.4) 24.6 (3.5)

0.62

28.8 (1.8) 22.1 (3.2)

0.19

29.6 (1.5) 21.1 (3.1)

0.08

30.0 (1.7) 24.0 (1.4)

0.13

26.2 (1.5) 22.5 (3.8)

0.04

27.1 (1.9) 30.1 (1.9)

0.50

28.2 (2.0) 29.1 (1.7)

0.52

compared to healthy people and were significant prognostic and predictive values. These assays were also studied in serum or plasma of patients with breast cancer [18–23]. In literature, only a limited number of studies have investigated serum M30 and M65 levels in human breast cancer [18–23]. In pioneering study, Biven et al. evaluated the relationship between serum M30 level and tumor response to chemotherapy in 32 recurrent breast cancer patients [18]. They found that the increases in serum M30 level were higher in patients with response than in those with no response (P = 0.0001). In another study performed by same investigators, the levels of circulating M30 are increased in 201 patients with breast cancer (152 patients with primary breast cancer and 49 patients with recurrence) (P = 0.001) [19]. Estrogen receptor (ER)-negative patients showed a higher level of M30 than ER-positive patients (P = 0.04). Other studied clinical parameters such as, tumor size, number of involved lymph nodes, menopausal status, progesterone receptor (PR) and histological type were not found as a

prognostic factor. Patients with recurrent disease showed higher M30 levels than both healthy control (P < 0.0001) and patients with primary cancer (P = 0.008). Additionally, in these relapsed patients, M30 levels were found to be correlated with the number of involved organs (P = 0.041) and performance status (P = 0.014). Patients with a larger number of involved organs and patients with impaired performance status showed a higher concentration of M30 levels in their sera. A difference in M30 levels was observed among patients with different major relapsed sites (P = 0.02). Patients with skin metastasis had higher M30 levels than those without skin metastasis (P = 0.016) and patients with local recurrence showed lower M30 levels compared with those with other relapsed sites (P = 0.024). However, they found that no association between serum M30 levels and patient prognosis. In order to evaluate the relationship between M30 level and chemotherapy response in neoadjuvant treatment of breast cancer, Demiray et al. studied in 42 patients with breast cancer received 4 cycles of anthracycline-based neoadjuvant chemotherapy [20]. Serum M30 levels were significantly increase postchemotherapy at 24 and 48 hours (P < 0.001 and P = 0.003, respectively). Significantly positive correlation was observed between tumor size and baseline M30 level (P = 0.041). Additionally, baseline serum M30 level in grade 3 tumors was significantly higher than grade 1– 2 tumors (P < 0.001). However, ER, PR, clinical stages and axillary node status were not correlated with baseline M30 level. This trial revealed a significant relationship between increases of M30 serum levels and overall response to therapy. While M30 level of responders increased and reached statistically significant level at 24 and 48 hours compared with baseline levels (P < 0.001 and P = 0.004, respectively), non-responders also showed such increase without statistical significance (P = 0.055 and P = 0.28, respectively). In evaluating the use of cytokeratin 18 as a serum biomarker for monitoring chemotherapy induced cell death in breast cancer, M30 and M65 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/ 5-fluorouracil (CEF) therapy [21]. Blood samples were collected before each cycle of treatment and 1 and 3 days after treatment. Baseline both serum M30 and M65 level in large-size tumors were significantly higher than small tumors (P = 0.005 and P = 0.0044, respectively). However, no significant correlation was observed between tumor receptors (ER and PR) and baseline M30/M65 level. The patients with either lymph node involvement or bone metastasis had only higher M65 levels than those without involvement or metastasis (P = 0.0046 and P = 0.028) contrary to serum M30 levels. The results show that increases in serum CK18 levels are associated with clinical response to CEF chemotherapy. In conclusion, CK18 biomarkers were found as useful for early prediction of the response to CEF therapy in breast cancer and might be useful biomarkers for clinical trials. In another study, Ulukaya et al. studied M30 serum levels in 37 breast cancer patients for a useful tool to predict the effectiveness of cancer treatment than conventional methods [22]. Patients received neoadjuvant chemotherapy consisting of either CEF or epirubicin and docetaxel (ED). Similar to other studies mentioned earlier, serum M30 levels were detected before chemotherapy, 24 and 48 hours after chemotherapy. The M30 level of the metastatic group was significantly higher than that in the non-metastatic group (P < 0.05). Additionally, they found that M30 levels differ depending on the receptor status. ER-negative or PR-negative patients had higher M30 levels compared with those in ER-positive or PR-positive ones (P < 0.05 for each other). However, no significant correlation was found between baseline serum M30 level and age of patients. Serum M30 levels significantly elevated at 24 hours (P < 0.05) and increased about three times in patients with tumor regression after chemotherapy.

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

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Fig. 2. Overall survival curves in patients with breast cancer according to M30 levels (P = 0.50).

These findings indicate that serum M30 is increased following chemotherapy and this measurement might be a useful tool to predict the effectiveness of chemotherapy.

Because biomarkers predicting response to neoadjuvant chemotherapy in locally confined breast cancer are highly needed, Stoetzer et al. prospectively assessed serial blood levels of

Fig. 3. Overall survival curves in patients with breast cancer according to M65 levels (P = 0.52).

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

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apoptotic biomarkers including M30 in 51 local and 28 metastatic breast cancer patients [23]. Levels of M30 was only elevated in patients with metastatic disease as compared with healthy persons, not local breast cancer patients (P = 0.014 and P = 0.169, respectively). However, there was no difference in M30 levels in the response groups before, during and after neoadjuvant chemotherapy contrary to the other trials. Therefore, this is the study in which these markers in breast cancer were evaluated. Eighty patients with a pathologically confirmed diagnosis of breast cancer were enrolled in this study. Both the serum M30 and M65 levels were significantly higher in the metastatic patients (P = 0.017 and P = 0.003, respectively). Additionally, the serum M65 levels were also elevated in larger tumor size (> 2 cm) compared to small tumors (P = 0.02). No correlation was found between these serum levels and response to chemotherapy (P = 0.43 for M30 and P = 0.26 for M65). The significant relationship was found between the serum levels of M30 and M65 (P < 0.001). Neither serum M30 nor serum M65 had significantly adverse effect on survival (P = 0.50, and P = 0.52, respectively). Therefore, the results from our study suggest that measuring baseline serum levels of CK18 in patients with breast cancer may help to differentiate metastatic disease from early stages. However, these markers are no potential role of tumor response and prognostic value on survival in patients with breast cancer. In conclusion, it seems that there are conflicting results and findings in the a few studies performed earlier. The small sample size of our study could be considered as significant limitation and might have influenced these results. However, our study contributes to the literature. Further clinical biomarker studies utilizing serial CK18 levels in a larger patient population are necessary to determine the potential clinical significance of these assays in breast cancer. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Source of funding: none declared. References [1] Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathol 2000;40:403–39. [2] Cummings J, Hodgkinson C, Odedra R, Sini P, Heaton SP, Mundt KE, et al. Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity. Mol Cancer Ther 2008;7:455–63. [3] De Haas EC, di Pietro A, Simpson KL, Meijer C, Suurmeijer AJH, Lancashire LJ, et al. Clinical evaluation of M30 and M65 ELISA cell death assays as circulating biomarkers in a drug-sensitive tumor, testicular cancer. Neoplasia 2008;10: 1041–8.

[4] Weber K, Osborn M, Moll R, Wiklund B, Luning B. Tissue polypeptide antigene (TPA) is related to the non-epidermal keratins 8, 18 and 19 typical of simple and non-squamous epithelia: re-evaluation of a human tumor marker. EMBO J 1984;3:2707–14. [5] Silen A, Wiklund B, Andersson EL, Nilsson S. A novel IRMA and ELISA for quantifying cytokeratin 8 and 18 fragments in the sera of healthy individuals and cancer patients. Scand J Clin Lab Invest 1995;55:153–61. [6] Barak V, Goike H, Panaretakis KW, Einarsson R. Clinical utility of cytokeratins as tumor markers. Clin Biochem 2004;37:529–40. [7] Greystoke A, Cummings J, Ward T, Simpson K, Renehan A, Butt F, et al. Optimisation of circulating biomarkers of cell death for routine clinical use. Ann Oncol 2008;19:990–5. [8] Bilici A, Ustaalioglu BB, Ercan S, Seker M, Yilmaz BE, Orcun A, et al. The prognostic significance of the increase in the serum M30 and M65 values after chemotherapy and relationship between these values and clinicopathological factors in patients with advanced gastric cancer. Tumour Biol 2012;33:2201–8. [9] Bilici A, Ustaalioglu BBO, Ercan S, Orcun A, Seker M, Salepci T, et al. Is there any impact of plasma M30 and M65 levels on progression-free survival of patients with advanced gastric cancer? Cancer Chemother Pharmacol 2011;68:309–16. [10] Yaman E, Coskun U, Sancak B, Buyukberber S, Ozturk B, Benekli M. Serum M30 levels are associated with survival in advanced gastric carcinoma patients. Int Immunopharmacol 2010;10:719–22. [11] Dive C, Smith RA, Garner E, Ward T, George-Smith SS, Campbell F, et al. Considerations for the use of plasma cytokeratin 18 as a biomarker in pancreatic cancer. Br J Cancer 2010;102:577–82. [12] Ausch C, Buxhofer-Ausch V, Olszewski U, Hinterberger W, Ogris E, Schiessel R, et al. Caspase-cleaved cytokeratin 18 fragment (m30) as marker of postoperative residual tumor load in colon cancer patients. Eur J Surg Oncol 2009;35:1164–8. [13] Ausch C, Buxhofer-Ausch V, Olszewski U, Schiessel R, Ogris E, Hinterberger W, et al. Circulating cytokeratin 18 fragment M65- a potential marker of malignancy in colorectal cancer patients. J Gastrointest Surg 2009;13:2020–6. [14] Koelink PJ, Lamers CB, Hommes DW, Verspaget HW. Circulating cell death products predict clinical outcome of colorectal cancer patients. BMC Cancer 2009;9:88. [15] Ulukaya E, Yilmaztepe A, Akgoz S, Linder S, Karadag M. The levels of caspasecleaved cytokine 18 are elevated in serum from patients with lung cancer, helpful to predict the survival. Lung Cancer 2007;56:399–404. [16] Ustaalioglu BO, Bilici A, Ercan S, Orcun A, Seker M, Ozkan A, et al. Serum M30 and M65 values in patients with advanced stage non-small-cell lung cancer compared with controls. Clin Transl Oncol 2012;14:356–61. [17] Ozturk B, Coskun U, Sancak B, Yaman E, Buyukberber S, Benekli M. Elevated serum levels of M30, M65 in patients with locally advanced head, neck tumors. Int Immunopharmacol 2009;9:645–8. [18] Biven K, Erdal H, Hagg M, Ueno T, Zhou R, Lynch M, et al. A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera. Apoptosis 2003;8:263–8. [19] Ueno T, Toi M, Biven K, Bando H, Ogawa T, Linder S. Measurement of an apoptotic product in the sera of breast cancer patients. Eur J Cancer 2003;39:769–74. [20] Demiray M, Ulukaya EE, Arslan M, Gokgoz S, Saraydaroglu O, Ercan I, et al. Response to neoadjuvant chemotherapy in breast cancer could be predictable by measuring a novel serum apoptosis product, caspase-cleaved cytokeratin 18: a prospective pilot study. Cancer Invest 2006;24:669–76. [21] Olofsson MH, Ueno T, Pan Y, Xu R, Cai F, van der Kuip H, et al. Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy. Clin Cancer Res 2007;13:3198–206. [22] Ulukaya E, Karaagac E, Ari F, Oral AY, Adim SB, Tokullugil AH, et al. Chemotherapy increases caspase-cleaved cytokeratin 18 in the serum of breast cancer patients. Radiol Oncol 2011;45:116–22. [23] Stoetzer OJ, Fersching DMI, Salat C, Steinkohl O, Gabka CJ, Hamann U, et al. Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and surviving. Cancer Lett 2013;336:140–8.

Please cite this article in press as: Tas F, et al. Clinical significance of serum M30 and M65 levels in patients with breast cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.10.021

Clinical significance of serum M30 and M65 levels in patients with breast cancer.

M30 and M65 are relatively new assays that detect different circulating forms of the epithelial cell structural protein cytokeratin18. The objective o...
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