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Evaluation of pleural fluid YKL-40 as a marker of malignant pleural effusion
Egyptian Journal of Chest Diseases and Tuberculosis (2015) 64, 489–495
H O S T E D BY
The Egyptian Society of Chest Diseases and Tuberculosis
Egyptian Journal of Chest Diseases and Tuberculosis www.elsevier.com/locate/ejcdt www.sciencedirect.com
ORIGINAL ARTICLE
Evaluation of pleural fluid YKL-40 as a marker of malignant pleural effusionq Adel Attia
a,*
, Ayman Rasmy b, Amal Amin c, Manal Alanazi
d
a
Chest Department, Faculty of Medicine, Zagazig University, Egypt Oncology Department, Faculty of Medicine, Zagazig University, Egypt c Microbiology Department, Faculty of Medicine, Fayoum University, Egypt d Internal Medicicne Department, King Fahad Hospital Dammam, Saudi Arabia b
Received 19 December 2014; accepted 8 January 2015 Available online 16 March 2015
KEYWORDS YKL-40; Pleural effusion; Malignant pleural effusion
Abstract The glycoprotein YKL-40 is synthesized both by cancer cells and by tumor-associated macrophages and plays a functional role in tumor progression. Consequently, high serum YKL40 levels have been associated with a poor prognosis in patients with several cancer types. The aim of this study is to assess pleural effusion and serum concentrations of YKL-40 in patients with different types of pleural effusions and to evaluate the diagnostic performance of YKL-40 in detecting malignant pleural effusion. Patients and methods: A prospective cohort study was carried out of 88 consecutive patients presenting with pleural effusions. The patients were divided into four groups: 22 patients with transudative effusions, 24 patients with parapneumonic pleural effusion and 8 patients with tuberculous pleural effusion and 34 patients with malignant pleural effusions. Blood and pleural fluid YKL-40 levels were measured using enzyme-linked immunosorbent assay. Results: Both serum HE4 levels and pleural effusion YKL-40 levels were significantly higher in patients with malignant effusions than in patients with transudative or non-malignant exudative effusions. A pleural fluid YKL-40 cut-off value of 256 ng/mL was found to predict malignant pleural effusions with a diagnostic sensitivity of 85.3% and specificity of 90.7%. Conclusion: The current study reports a finding of increased serum and pleural fluid YKL-40 levels in patients with malignant pleural effusions compared to non-malignant effusions. ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction q
All the authors contributed to research design. Participated in the writing of the manuscript and data analysis, performed research, and contributed with analytical tools. * Corresponding author. Tel.: +20 1224733108, +966502591539. E-mail address: [email protected] (A. Attia). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.
Pleural effusion is a commonly encountered presentation in clinical practice, with a large number of possible causes, including disease localized to the lung, primary or metastatic pleural disease or a manifestation of systemic disease. The differential diagnosis of a pleural effusion is wide, and pleural
http://dx.doi.org/10.1016/j.ejcdt.2015.01.003 0422-7638 ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
490 fluid analysis is usually the first step toward identifying the cause of a pleural effusion. It gives an indication about the underlying pathophysiologic process and the need for further investigations [1]. Light’s criteria is the most commonly used parameter for the differentiation between exudative and transudative effusions [2] as it is a very robust test with a diagnostic accuracy of 93–96% [3]. Malignant pleural effusions are a frequent cause of exudative effusions, and their diagnosis often poses a clinical challenge as pleural fluid cytology is only positive in around 60% of cases on average [4,5]. More invasive procedures such as thoracoscopy or thoracotomy may be required to reach a diagnosis, but at the expense of higher morbidity [6]. Numerous studies have been published analyzing the potential utility of different tumor markers in pleural effusions [7,8]. The chitinase-like protein YLK-40, also known as human cartilage glycoprotein 39, is a glycoprotein produced in association with various inflammatory and malignant processes [9,10]. It is mainly secreted by macrophages, synovial cells, chondrocytes, and neutrophils [11,10]. Although the exact physiological function of YKL-40 protein is unknown, the pattern of its expression suggests a role in inflammation and tissue remodeling. Elevated serum YKL-40 levels have been found in a wide range of inflammatory diseases including asthma, cirrhosis, sarcoidosis, osteoarthritis, cardiovascular disease, diabetes mellitus, rheumatoid arthritis, chronic obstructive pulmonary disease, and inflammatory bowel disease. High serum concentration of YKL-40 has also been found in various malignancies and seems to be a marker for decreased survival [12–14]. YKL-40 is not specific for SCLC. It is secreted by many different types of solid carcinomas and also by non-malignant cells in tissues characterized by inflammation, tissue degradation/remodeling or ongoing fibrogenesis. YKL-40 is produced by macrophages, neutrophils, malignant tumor cells, mesothelial cells and arthritic chondrocytes [15], and has been found in the fibrotic liver [16]. Elevated serum YKL-40 levels are found in patients with non-malignant diseases such as severe active rheumatoid arthritis, severe bacterial infections and liver fibrosis [17]. YKL-40 has been shown to be associated with lung injury pathogenesis contributing to inflammation, remodeling and cellular proliferation [18]. The aim of the current study was to assess pleural effusion and serum concentrations of YKL-40 in patients with pleural effusions of varying characteristics, and to evaluate the diagnostic performance of YKL-40 in detecting malignant pleural effusions. Patients and methods A prospective cohort study was carried out of 88 consecutive patients with pleural effusions seen in the pulmonary and Oncology Departments of King Fahad Hospital Dammam. The patients were subsequently divided into four groups according to the type of effusion: transudative effusion (TPE), parapneumonic pleural effusion (PPPE), tuberculous pleural effusion (TBPE), and malignant pleural effusion (MPE). Light’s criteria were calculated to differentiate exudative from transudative effusions. An effusion was classified as malignant if tumor cells were found in the pleural fluid or pleural biopsy, or in a patient with disseminated malignancy and no alternative explanation for an exudative effusion.
A. Attia et al. Parapneumonic effusion was defined as; the presence of pleural effusion in patients with community-acquired pneumonia. We used The American Thoracic Society criteria for defining the pneumonia [newly occurred respiratory symptoms, fever and abnormal breath sounds, together with a new pulmonary infiltration on chest X-ray, and consistent laboratory findings (leukocytosis count and high serum CRP levels) and neutrophilic pleural effusion] [19]. Pleural fluid samples were diagnosed as tuberculous effusion by pleural biopsy specimen showing granulomas with caseification necrosis, or pleural fluid positive on Ziehl-Nielsen stain or Lowenstein-Jensen culture, or pleural fluid consistent with clinical signs and symptoms of tuberculosis together with lymphocyte predominance in pleural effusion and good response to anti-tuberculosis treatment. The study thereafter comprised twenty-two patients with transudative effusions, twenty-four patients with parapneumonic effusions, eight patients with tuberculous effusion and thirty-four patients with malignant effusions. Patients with transudative effusions comprised 12 women and 10 men and were aged (Mean ± SD) 60.2 ± 6.5 years. Patients with parapneumonic effusions comprised 13 women and 11 men and were aged (Mean ± SD) 59.8 ± 5.4 years. Patients with tuberculous effusions comprised 3 women and 5 men and were aged (Mean ± SD) 58.9 ± 8.9 years. Patients with malignant effusions comprised of 18 males and 16 females and were aged; (Mean ± SD) 63.5 ± 8.3 years (Table 1). Twenty-three patients had lung cancer, 2 metastatic colon adenocarcinoma, 2 patients with non Hodgkin lymphoma, 2 patients with metastatic gastric cancer and 5 patients with metastatic breast cancer. Of the 23 patients with bronchial carcinoma, histopathologically the cases comprised of 11 adenocarcinoma (3 of them were well differentiated and 8 were moderately differentiated), 12 cases of squamous cell carcinoma (3 of them were moderately differentiated and the other 9 poorly differentiated). Five out of the 11 distant metastatic cases originated from breast cancer, and they were microscopically invasive ductal carcinoma not otherwise specified (NOS), ranging from grade II/III to grade III/IV. All patients included in this study had unilateral pleural effusion except 2 patients of the transudative group with congestive heart failure. As per institution guidelines, the study was approved by King Fahad Hospital Ethics Committee. Informed consent was obtained from each patient included in the study. Thoracocentesis was performed under the aseptic technique, and pleural effusion and serum samples were collected at the same time. The pleural fluid was collected in tubes containing EDTA for determination of the cellular content, in plain tubes for determination of LDH, protein, albumin and YKL-40 levels. Paired serum samples were analyzed for urea, creatinine, albumin, total protein, and LDH levels. These parameters were measured using Siemens Diagnostics (Marburg GmbH, Germany) (RxL Dimension Clinical Chemistry analyzer) [20]. Pleural fluid pH was measured using blood gas analyzer, GEM premier 3000 (instrumentation laboratory, USA). Sample for pleural fluid pH was collected anaerobically, kept on ice and analyzed immediately within five minutes of collection. Pleural adenosine deaminase (ADA) levels were also measured as UI/mL. For serum YKL-40 analysis, the blood samples were centrifuged at 3000g for 10 min at 4 C. After the centrifugation, the serum was removed and transferred into a clean test tube. All pleural and serum samples were stored in a refrigerator at 80 C until YKL-40 analysis and they were dissolved and stored for 24 h at
Evaluation of pleural fluid YKL-40 Table 1
491
Demographic data, clinical and biochemical criteria of the studied groups.
Parameters
TPE N = 22
PPPE N = 24
TBPE N=8
MPE N = 34
Age Sex BMI BUN (mmol) S. creatinine (lmol/L) S. protein (g/L) S. albumin (g/L) Blood WBCs · 103/mm3 Pl. WBCs Pl. lymphocytic count Pl. neutrophils count Pl. PH
60.2 ± 6.5 M,10/F,12 20.8 ± 1.6 6.9 ± 4.7 79 (42–45) 62 ± 0.6 30 ± 0.34 5.5 ± 1.3 45.50(1–314) 23.18 ± 10.95 23.2 ± 11.1 8 ± 0.26
59.8 ± 5.4 M,11/F,13 25.3 ± 3.4 4.9 ± 2.7 79.05(42–43) 71 ± 0.9 36 ± 0.45 12.7 ± 3.2 980(345–4610) 17.72 ± 9.36 72.5 ± 13.05 7.21 ± 0.03
58.9 ± 8.9 M,5/F,3 25.3 ± 3.4 6.9 ± 4.7 79(42–39) 71 ± 0.7 36 ± 0.58 11.2 ± 2.4 950(339–3245) 57.54 ± 8.6 25.76 ± 11.5 7.19 ± 0.04
63.5 ± 8.3 M,18/F,16 23.9 ± 3.4 6.19 ± 4.7 79(42–41) 71 ± 0.5 37 ± 0.98 5.7 ± 1.9 1025(475–5346) 64.3 ± 9.7 24.7 ± 9.4 7.22 ± 0.108
TPE: transudative pleural effusion, PPPE: parapneumonic pleural effusion, TBPE: tuberculous pleural effusion, MPE: malignant pleural effusion, BMI: body mass index, BUN: blood urea nitrogen.
2–8 C before the study. YKL-40 protein concentrations were determined in serum and pleural fluids using a specific kit (MICROVUE YKL-40 EIA Kit, Quidel, San Diego, USA) by an enzyme-binding immunosorbent test (ELISA). Standards, controls and the concentration of YKL-40 presented in the test specimens were measured by spectrophotometer (PerkinElmer LAMBDA UV/Vis Spectrophotometer, Massachusetts, USA) and the results are calculated from the generated standard curve using linear regression analysis according to the producer’s instructions [21]. Statistical analysis Comparison among the groups was conducted using analysis of variance (ANOVA) test for normally distributed variables. The non-parametric Mann–Whitney U test was used for variables with non-Gaussian distribution. For the choice of the optimal cut-off, Receiver Operating Characteristic (ROC) curves were constructed and the Youden index was calculated [22]. The Youden index is defined as follows: (sensitivity + specificity) 1. The best cut-off has the highest Youden index. The commercial statistical software package used was SPSS11.0 (SPSS, Inc., Chicago, IL, USA). Results Demographic data, clinical and biochemical criteria of the studied groups are illustrated in Table 1. Both serum and pleural fluid levels YKL-40 were significantly higher in the exudative pleural effusions group (i.e. parapneumonic effusion, tuberculous effusion and malignant effusion) than the transudative group (p < 0.01). Median (range) values of serum YKL-40 were 154(56–237.3) & 123(45–187) ng/mL and pleural fluid YKL-40 values were 194(85–265) & 141(78–187) ng/mL in exudative and transudative groups respectively: (Fig. 1a). In the exudative pleural effusion group pleural fluid YKL-40 positively correlated with serum YKL-40 (r: 0.49 & p < 0.001). Similarly serum YKL-40 positively correlated with serum LDH (r: 0.383 & p = 0.002). Fig. 1b and d illustrates serum and pleural effusion levels of YKL-40. Both serum YKL-40 levels and effusion YKL-40 levels were significantly
higher in malignant effusions than in transudative or parapneumonic effusion, and tuberculous effusion (p < 0.05) while YKL-40 pleural fluid/serum ratio did not show significant difference between the four groups. When malignant effusions were classified according to the histopathological diagnosis, there was no significant difference between pleural and serum YKL-40 levels in the different tumor types (p > 0.05). The pleural effusion/serum ratio of LDH and albumin, pleural effusion LDH level, YKL-40 serum level and YKL-40 pleural effusion levels were evaluated as indicators for malignant effusion using Receiver Operating Characteristic curve (ROC). Effusion YKL-40 outperformed the other markers followed by serum YKL-40, pleural fluid LDH, Pleural fluid/serum LDH ratio then finally pleural fluid/serum albumin ratio (Fig. 1c). Pleural YKL-40/serum YKL-40 ratio >1.6 yielded 71% sensitivity, 73% specificity and likelihood ratio 2.8 for diagnosing TBPE, with an area under the curve of 0.785 (95% CI: 0.711–0.890). The results of pleural ADA levels were found to be high in pleural tuberculosis compared to other pleural fluids (p < 0.001) although we did not find such a correlation between serum and pleural YKL-40 and ADA levels. The area under the curve (AUC) for fluid YKL-40 was 0.886 (95% confidence interval [CI] was 0.8–0.973). The AUC for the serum YKL-40 was 0.798 (95% CI was 0.689– 0.907), while the AUC for the pleural fluid LDH was 0.722 (95% CI was 0.617–0.827). The AUC for pleural fluid LDH/ serum ratio was 0.615 (95% CI was 0.499–0.731). The AUC for pleural fluid lymphocyte count was 0.69 (95% CI was 0.544–0.836). The AUC for pleural fluid neutrophil count was 0.22 (and 95% CI was 0.123–0.317). Using the Youden index, the best cut-off value for pleural fluid YKL-40 to predict malignant effusions was 256 ng/mL. This cut-off value had a diagnostic sensitivity of 85.3% and specificity of 90.7%. The best cut-off value for pleural fluid YKL-40 to differentiate between exudative and transudative effusions was 212 ng/mL. This cut-off value had a diagnostic sensitivity of 74.3% and specificity of 75.7% (see Tables 2 and 3). Discussion The diagnosis of malignancy in pleural effusion is a challenging clinical problem. Several studies have been published
492
A. Attia et al.
Figure 1 HE4 levels in the different groups and YKL-40 AUC under a ROC curve. Boxplots (a, b and d) illustrate YKL-40 values in serum, and pleural effusion in patients with transudative effusion (TPE), parapneumonic pleural effusion (PPPE), tuberculous pleural effusion (TBPE) and malignant pleural effusions (MPE). The boxplots represent the interquartile range from the 25th to the 75th percentiles. The whiskers below and above the boxes represent the minimum and maximum values. The line across each box represents the median value. (c) ROC curves were constructed by plotting the sensitivity versus 1-specificity at different cut-off values for each analyte. EPE: Exudative pleural effusion, P: Pleural, S: Serum.
Table 2 Pleural, serum YKL-40 and pleural/serum ratio values in the transudative and exudative effusion patients. Parameters
TPE (n = 22)
EPE (n = 66)
p value
Pleural YKL-40 ng/mL Serum YKL-40 ng/mL PL/S YKL-40 ratio
141(78–187) 123(45–187) 1.05(0.8–5.5)
194(85–265) 154(56.7–237.3) 2.16(1.01–6.02)
<0.001 <0.05 <0.001
TPE: transudative pleural effusion, EPE: exudative pleural effusion, PL/S: pleural/serum.
reporting the presence of elevated levels of markers in malignant pleural effusions [23], however, none of these markers gained consensus as a highly sensitive marker for neoplastic effusions due to varied sensitivity and specificity [24] or specificity for certain neoplasms (e.g. mesothelioma) [25]. The search therefore continues for a marker or combination of markers that will increase the ability to diagnose malignancy
in a pleural effusion, and to select those patients who would benefit from more extensive invasive investigation when the pleural fluid cytology is negative. Proposed cut-off values for pleural tumor markers have varied in several studies; however some authors have suggested that cut-off points for a tumor marker that allows 100% specificity are necessary to substantiate a diagnosis of a malignant pleural effusion. Adopting such high cut-off points has resulted in the sensitivity of single tumor markers to predict malignancy to be as low as 17–30% [8]. The current study found a significantly higher level of YKL-40 in serum of patients with exudative effusion than those with transudative ones. The best cut-off value for pleural fluid YKL-40 to differentiate between exudative and transudative effusions was 212 ng/mL. This cut-off value had a diagnostic sensitivity of 74.3% and specificity of 75.7%. Most importantly, a marked significant increase was found in serum of patients with neoplastic pleural effusions than those with non-malignant exudative effusions and transudative effusions. This finding was also mirrored by an increased
Evaluation of pleural fluid YKL-40 Table 3
493
Pleural, serum, and pleural/serum ratio values of some biochemical parameters evaluated in the study.
Parameters
TPE (n = 22)
PPPE (n = 24)
TBPE (n = 8)
MPE (n = 34)
Protein(g/L) Pleural Serum PL/S ratio
22.7 ± 2.7 62 ± 5.9 1.9 ± 0.21
39 ± 2.4 70.2 ± 5.7 1.8 ± 0.17
43 ± 1.9 70.3 ± 5.9 1.8 ± 0.21
41.6 ± 6.4 70.1 ± 5.2 1.7 ± 0.23
Albumin(g/L) Pleural Serum PL/S ratio
16 ± 4.1 30 ± 4.8 0.52
32.29 ± 1.5 36 ± 3.5 0.84 ± 0.09
28.11 ± 1.8 36.2 ± 1.4 0.81 ± 0.08
29 ± 4.9 37.4 ± 3.6 0.80 ± 0.09
LDH(U/L) Pleural Serum PL/S ratio
59.68 ± 19.9 151 ± 47.9 0.42 ± 0.14
755 ± 274.3 209.2 ± 86.1 4.16 ± 2.4
721 ± 187.46 301.8 ± 84 5.12 ± 3.5
843.1 ± 501.3 332.8 ± 214 3.03 ± 1.19
Pleural ADA(U/mL)
12(1–25)
16(1–39)
48(23–100)
14(1–37)
YKL-40 (ng/mL) Pleural Serum PL/S ratio
141(78–187) 123(45–187) 1.05(0.8–5.5)
154(55–224) 149(59–200) 1.09(0.81–5.44)
182(65–235) 109(52–200) 4.11(1.3–6.7)
255(135–335) 205(59–312) 1.28(0.91–5.92)
TPE: transudative pleural effusion, PPPE: parapneumonic pleural effusion, TBPE: tuberculous pleural effusion, MPE: malignant pleural effusion, PL/S: pleural/serum, ADA: adenosine deaminase, LDH: lactic dehydrogenase.
pleural fluid YKL-40 level in patients with malignant effusions compared to those with transudative or non-malignant exudative effusions. This finding may be explained by increased local secretion of YKL-40 that is produced by the neoplastic tissues with subsequent overload to the systemic circulation resulting in increased serum YKL-40. The current study did not find a significant difference in pleural fluid YKL-40 between primary pulmonary neoplasm and secondary metastatic pleural effusions. This increase in pleural fluid YKL-40 may be due to increased secretion by the malignant cells in cases of primary pulmonary neoplasm or local irritation of respiratory epithelial cells with subsequent increase in YKL-40 local production in secondary metastatic pleural effusions. Furthermore the observed lack of correlation between serum or pleural fluid YKL-40 and the histopathological type of the tumor may be explained by the fact that all patients with malignant pleural effusion were advance metastatic stage IV patients according to TNM staging system. The metastasis may have caused increased production of YKL-40 either through local irritation or cellular destruction irrespective of the histopathological tumor origin. The biologic functions of YKL-40 in cancer cells are unknown, and very few studies have evaluated the functional role of YKL-40 expression in cancer cells. It has been suggested that YKL-40 plays a role in the proliferation and differentiation of malignant cells, protects the cells from undergoing apoptosis, YKL-40 is a relatively new biomarker of inflammation and it has been further investigated in several pathologic conditions [26]. Funkhouser et al. [27] reported that the levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders correlating with disease severity. Kim et al. [28] studied YKL-40 levels in different lung diseases and pleural effusions, they found that the results of serum YKL-40 were the highest in tuberculosis compared with other pleural effusions including parapneumonic,
and cardiogenic effusions. Bouzas et al. [29] demonstrated a significant relation between chitotriosidase and ADA activities, although we did not find such a correlation between serum and pleural YKL-40 and ADA levels. Contrary to our results, Kim et al. [28] reported that serum YKL-40 level of the patients with pleural tuberculous was higher than that of the patients with effusions due to malignant or cardiac causes. The current study showed a positive correlation between pleural fluid YKL-40 and LDH point more to the possible value of increased YKL-40 level in malignant effusion. The increased levels of both markers (LDH and YKL-40) in pleural fluid may be secondary to increased local production either through increased cellular destruction, local invasion or local tissue irritation. The current study had shown a significant increase in pleural fluid lymphocytic count in patients with malignant pleural effusion, and a good AUC for pleural lymphocytic count. However increased pleural fluid lymphocytic count may be found in other disease states (e.g. Tuberculosis, pulmonary embolism) [30]. Pleural YKL-40/serum YKL-40 ratio >1.6 yielded 71% sensitivity, 73% specificity and likelihood ratio 2.8 for diagnosing TBPE, with an area under the curve of 0.785 (95% CI: 0.711–0.890). The use of pleural fluid/serum ratio to enhance the diagnostic sensitivity of tuberculous effusion was evaluated by Kayhan et al. [31] who found, Pleural YKL-40/serum YKL-40 ratio >1.5 yielded 75% sensitivity, 72% specificity and likelihood ratio 2.6 for diagnosing TBPE, with an area under the curve of 0.825 (95% CI: 0.710–0.940). The current study did not find a significant difference in pleural fluid/serum YKL-40 ratio between the transudative and exudative effusions. The use of pleural fluid/ serum ratio to enhance the diagnostic sensitivity of tumor markers was evaluated by Trape´ et al. [32] who classified tumor markers into two categories: released/secreted by normal pleura (e.g. CA-125 and cytokeratin) and non released/ non-secreted tumor markers (e.g. CEA and CA-19.9) by
494 normal pleura. Trape´ et al. showed clearly that the fluid/serum ratio was only useful for non-secreted tumor markers while for secreted tumor markers (i.e. those that may be secreted by normal respiratory epithelium) the ratio did not improve their sensitivity and specificity. As YKL-40 has been previously shown to be expressed by normal respiratory epithelium, it may be considered as a secreted/released tumor marker and it would not be surprising that pleural/fluid serum YKL-40 ratio did not enhance its diagnostic sensitivity and specificity due to possible concomitant increase in serum and pleural fluid YKL-40 with the net result of a normal or reduced ratio of pleural fluid/serum YKL-40 ratio. An important finding of the current study is the superior performance of pleural effusion YKL-40 levels in detecting malignant pleural effusions with a value of 256 ng/mL as the best cut-off value to suggest a malignant pleural effusion with a diagnostic sensitivity of 85.3% and specificity of 90.7%. Even when the cut-off point was increased to 286 ng/mL to achieve 100% specificity, the marker still predicted malignancy with a sensitivity of 45%. This result favors comparably with other similar studies examining the utility of single tumor markers where the achieved sensitivity was very low when a 100% specificity cut-off was chosen. This is the first study to examine the role of YKL-40 in the diagnosis of malignancy in pleural effusions, and it was limited by the relatively small number of subjects, and therefore the utility of this marker will have little clinical benefit currently. Conclusion However this novel finding of increased pleural fluid YKL-40 levels in malignant pleural effusions compared to either transudative or non-malignant exudative effusions will add to our scientific knowledge, and set the stage for larger studies of this marker in pleural effusions, or the inclusion of this marker in combination with other panels to strengthen the diagnostic performance of tumor markers in detecting malignant pleural effusions. Conflict of interest The authors declare no conflict of interests. References [1] M.J. Murphy, F. Jenkinson, Categorisation of pleural fluids in routine clinical practice: analysis of pleural fluid protein and lactate dehydrogenase alone compared with modified Light’s criteria, J. Clin. Pathol. 61 (2008) 684–685. [2] R.W. Light, M.I. Mac Gregor, P.C. Luchsinger, W.C. Ball, Pleural effusions: the diagnostic separation of transudates and exudates, Ann. Intern. Med. 77 (1972) 507–513. [3] J.E. Heffner, L.K. Brown, C.A. Barbieri, Diagnostic value of tests that discriminate between exudative and transudative pleural effusions. Primary study investigators, Chest 111 (1997) 970–980. [4] K.V. Nance, R.W. Shermer, F.B. Askin, Diagnostic efficacy of pleural biopsy as compared with that of pleural fluid examination, Mod. Pathol. 4 (1991) 320–324. [5] L. Garcia, The value of multiple fluid specimens in the cytological diagnosis of malignancy, Mod. Pathol. 7 (1994) 665–668.
A. Attia et al. [6] W.W. Huang, S.M. Tsao, C.L. Lai, C.C. Su, C.E. Tseng, Diagnostic value of Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen levels in malignant pleural effusions of lung adenocarcinoma, Pathology 42 (2010) 224–228. [7] J.S. Hackbarth, K. Murata, W.M. Reilly, A. AlgecirasSchimnich, Performance of CEA and CA19-9 in identifying pleural effusions caused by specific malignancies, Clin. Biochem. 43 (2010) 1051–1055. [8] J.M. Porcel, M. Vives, A. Esquerda, A. Salud, B. Perez, F. Rodriguez-Panadero, Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3, and cytokeratin 19 fragments) in pleural fluid for the differential diagnosis of benign and malignant effusions, Chest 126 (2004) 1757–1763. [9] G.L. Chupp, C.G. Lee, N. Jarjour, Y.M. Shim, C.T. Holm, J.D. Dziura, J. Reed, A.J. Coyle, et al, A chitinase-like protein in the lung and circulation of patients with severe asthma, N. Engl. J. Med. 357 (2007) 2016–2027. [10] S. Letuve, A. Kozhich, N. Arouche, M. Grandsaigne, et al, YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages, J. Immunol. 181 (2008) 5167–5173. [11] J.S. Johansen, B.V. Jensen, A. Roslind, D. Nielsen, P.A. Price, Serum YKL-40, a new prognostic biomarker in cancer patients?, Cancer Epidemiol Biomarkers Prev. 15 (2) (2006 Feb) 194–202. [12] I.K. Choi, Y.H. Kim, J.S. Kim, J.H. Seo, High serum YKL-40 is a poor prognostic marker in patients with advanced non-small cell lung cancer, Acta Oncol. 49 (6) (2010 Aug) 861–864. [13] I. Tho¨m, B. Andritzky, G. Schuch, I. Burkholder, L. Edler, J.S. Johansen, C. Bokemeyer, U. Schumacher, E. Laack, Elevated pretreatment serum concentration of YKL-40 – an independent prognostic biomarker for poor survival in patients with metastatic nonsmall cell lung cancer, Cancer 116 (17) (2010 Sep 1) 4114–4121. [14] A. Roslind, J.S. Johansen, I.J. Christensen, K. Kiss, E. Balslev, D.L. Nielsen, J. Bentzen, P.A. Price, E. Andersen, High serum levels of YKL-40 in patients with squamous cell carcinoma of the head and neck are associated with short survival, Int. J. Cancer 122 (4) (2008 Feb 15) 857–863. [15] B. Volck, J.S. Johansen, M. Stoltenberg, C. Garbarsch, P.A. Price, M. Østergaard, et al, Studies on YKL-40 in knee joints of patients with rheumatoid arthritis and osteoarthritis. Involvement of YKL-40 in the joint pathology, Osteoarthr. Cartilage 9 (2001) 203–214. [16] J.S. Johansen, P. Christoffersen, S. Møller, P.A. Price, J.H. Henriksen, C. Garbarsch, et al, Serum YKL-40 is increased in patients with hepatic fibrosis, J. Hepatol. 32 (2000) 911–920. [17] G. Kronborg, C. Østergaard, N. Weis, H. Nielsen, N. Obel, S.S. Pedersen, et al, Serum level of YKL-40 is elevated in patients with Streptococcus pneumoniae bacteremia and is associated to the outcome of the disease, Scand. J. Infect. Dis. 34 (2002) 323– 326. [18] J. Kzhyshkowska, A. Gratchev, S. Goerdt, Human chitinases and chitinase-like proteins as indicators for inflammation and cancer, Biomark Insights 2 (2007) 128–146. [19] M.S. Niederman, L.A. Mandell, A. Anzueto, et al, Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention, Am. J. Respir. Crit. Care Med. 163 (2001) 1730–1754. [20] C.A. Burtis, E.R. Ashwood, Tietz Fundamental of Clinical Chemistry, fourth ed., WB Saunders, Philadelphia, 1996. [21] S. Harvey, M. Weisman, J. O’Dell, et al, Chondrex: new marker of joint disease, Clin. Chem. 44 (1998) 509–516. [22] W.J. Youden, Index for rating diagnostic tests, Cancer 3 (1950) 32–35. [23] P. Korczynski, R. Krenke, A. Safianowska, K. Gorska, M.B. Abou Chaz, M. Maskey-Warzechowska, A. Kondracka, J.
Evaluation of pleural fluid YKL-40 Nasilowski, R. Chazan, Diagnostic utility of pleural fluid and serum markers in differentiation between malignant and nonmalignant pleural effusions, Eur. J. Med. Res. 14 (Suppl. 4) (2009) 128–133. [24] M.J. Gaspar, J. De Miguel, J.D. Garcı´ a Dı´ az, M. Dı´ ez, Clinical utility of a combination of tumour markers in the diagnosis of malignant pleural effusions, Anticancer Res. 28 (2008) 2947– 2952. [25] B. Grigoriu, B. Chahine, F. Zerimech, M. Gre´goire, M. Balduyck, M.C. Copin, P. Devos, P. Lassalle, A. Scherpereel, Serum mesothelin has a higher diagnostic utility than hyaluronic acid in malignant mesothelioma, Clin. Biochem. 42 (2009) 1046– 1050. [26] C.G. Lee, D. Hartl, G.R. Lee, et al, Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis, J. Exp. Med. 206 (2009) 1149– 1166.
495 [27] J.D. Funkhouser, N.N. Aronson Jr., Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family, BMC Evol. Biol. 7 (2007) 96. [28] H.R. Kim, C.D. Jun, K.S. Lee, et al, Levels of YKL-40 in pleural effusions and blood from patients with pulmonary or pleural disease, Cytokine 58 (2012) 336–343. [29] L. Bouzas, E. San Jose´, J.C. Tutor, Chitotriosidase activity in pleural effusions, Clin. Lab. 53 (2007) 449–452. [30] J.M. Porcel, R.W. Light, Diagnostic approach to pleural effusion in adults, Am. Fam. Physician 73 (2006) 1211–1220. [31] S. Kayhan, A. Gumus, H. Cinarka, N. Murat, A. Yilmaz, R. Bedir, U. Sahin, The clinical utility of pleural YKL-40 in diagnosing pleural effusion, J. Thorac. Dis. 5 (5) (2013) 634–640. [32] J. Trape´, R. Molina, F. Sant, Clinical evaluation of the simultaneous determination of tumor markers in fluid and serum and their ratio in the differential diagnosis of serous effusions, Tumour Biol. 25 (2004) 276–281.
H O S T E D BY
The Egyptian Society of Chest Diseases and Tuberculosis
Egyptian Journal of Chest Diseases and Tuberculosis www.elsevier.com/locate/ejcdt www.sciencedirect.com
ORIGINAL ARTICLE
Evaluation of pleural fluid YKL-40 as a marker of malignant pleural effusionq Adel Attia
a,*
, Ayman Rasmy b, Amal Amin c, Manal Alanazi
d
a
Chest Department, Faculty of Medicine, Zagazig University, Egypt Oncology Department, Faculty of Medicine, Zagazig University, Egypt c Microbiology Department, Faculty of Medicine, Fayoum University, Egypt d Internal Medicicne Department, King Fahad Hospital Dammam, Saudi Arabia b
Received 19 December 2014; accepted 8 January 2015 Available online 16 March 2015
KEYWORDS YKL-40; Pleural effusion; Malignant pleural effusion
Abstract The glycoprotein YKL-40 is synthesized both by cancer cells and by tumor-associated macrophages and plays a functional role in tumor progression. Consequently, high serum YKL40 levels have been associated with a poor prognosis in patients with several cancer types. The aim of this study is to assess pleural effusion and serum concentrations of YKL-40 in patients with different types of pleural effusions and to evaluate the diagnostic performance of YKL-40 in detecting malignant pleural effusion. Patients and methods: A prospective cohort study was carried out of 88 consecutive patients presenting with pleural effusions. The patients were divided into four groups: 22 patients with transudative effusions, 24 patients with parapneumonic pleural effusion and 8 patients with tuberculous pleural effusion and 34 patients with malignant pleural effusions. Blood and pleural fluid YKL-40 levels were measured using enzyme-linked immunosorbent assay. Results: Both serum HE4 levels and pleural effusion YKL-40 levels were significantly higher in patients with malignant effusions than in patients with transudative or non-malignant exudative effusions. A pleural fluid YKL-40 cut-off value of 256 ng/mL was found to predict malignant pleural effusions with a diagnostic sensitivity of 85.3% and specificity of 90.7%. Conclusion: The current study reports a finding of increased serum and pleural fluid YKL-40 levels in patients with malignant pleural effusions compared to non-malignant effusions. ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction q
All the authors contributed to research design. Participated in the writing of the manuscript and data analysis, performed research, and contributed with analytical tools. * Corresponding author. Tel.: +20 1224733108, +966502591539. E-mail address: [email protected] (A. Attia). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.
Pleural effusion is a commonly encountered presentation in clinical practice, with a large number of possible causes, including disease localized to the lung, primary or metastatic pleural disease or a manifestation of systemic disease. The differential diagnosis of a pleural effusion is wide, and pleural
http://dx.doi.org/10.1016/j.ejcdt.2015.01.003 0422-7638 ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
490 fluid analysis is usually the first step toward identifying the cause of a pleural effusion. It gives an indication about the underlying pathophysiologic process and the need for further investigations [1]. Light’s criteria is the most commonly used parameter for the differentiation between exudative and transudative effusions [2] as it is a very robust test with a diagnostic accuracy of 93–96% [3]. Malignant pleural effusions are a frequent cause of exudative effusions, and their diagnosis often poses a clinical challenge as pleural fluid cytology is only positive in around 60% of cases on average [4,5]. More invasive procedures such as thoracoscopy or thoracotomy may be required to reach a diagnosis, but at the expense of higher morbidity [6]. Numerous studies have been published analyzing the potential utility of different tumor markers in pleural effusions [7,8]. The chitinase-like protein YLK-40, also known as human cartilage glycoprotein 39, is a glycoprotein produced in association with various inflammatory and malignant processes [9,10]. It is mainly secreted by macrophages, synovial cells, chondrocytes, and neutrophils [11,10]. Although the exact physiological function of YKL-40 protein is unknown, the pattern of its expression suggests a role in inflammation and tissue remodeling. Elevated serum YKL-40 levels have been found in a wide range of inflammatory diseases including asthma, cirrhosis, sarcoidosis, osteoarthritis, cardiovascular disease, diabetes mellitus, rheumatoid arthritis, chronic obstructive pulmonary disease, and inflammatory bowel disease. High serum concentration of YKL-40 has also been found in various malignancies and seems to be a marker for decreased survival [12–14]. YKL-40 is not specific for SCLC. It is secreted by many different types of solid carcinomas and also by non-malignant cells in tissues characterized by inflammation, tissue degradation/remodeling or ongoing fibrogenesis. YKL-40 is produced by macrophages, neutrophils, malignant tumor cells, mesothelial cells and arthritic chondrocytes [15], and has been found in the fibrotic liver [16]. Elevated serum YKL-40 levels are found in patients with non-malignant diseases such as severe active rheumatoid arthritis, severe bacterial infections and liver fibrosis [17]. YKL-40 has been shown to be associated with lung injury pathogenesis contributing to inflammation, remodeling and cellular proliferation [18]. The aim of the current study was to assess pleural effusion and serum concentrations of YKL-40 in patients with pleural effusions of varying characteristics, and to evaluate the diagnostic performance of YKL-40 in detecting malignant pleural effusions. Patients and methods A prospective cohort study was carried out of 88 consecutive patients with pleural effusions seen in the pulmonary and Oncology Departments of King Fahad Hospital Dammam. The patients were subsequently divided into four groups according to the type of effusion: transudative effusion (TPE), parapneumonic pleural effusion (PPPE), tuberculous pleural effusion (TBPE), and malignant pleural effusion (MPE). Light’s criteria were calculated to differentiate exudative from transudative effusions. An effusion was classified as malignant if tumor cells were found in the pleural fluid or pleural biopsy, or in a patient with disseminated malignancy and no alternative explanation for an exudative effusion.
A. Attia et al. Parapneumonic effusion was defined as; the presence of pleural effusion in patients with community-acquired pneumonia. We used The American Thoracic Society criteria for defining the pneumonia [newly occurred respiratory symptoms, fever and abnormal breath sounds, together with a new pulmonary infiltration on chest X-ray, and consistent laboratory findings (leukocytosis count and high serum CRP levels) and neutrophilic pleural effusion] [19]. Pleural fluid samples were diagnosed as tuberculous effusion by pleural biopsy specimen showing granulomas with caseification necrosis, or pleural fluid positive on Ziehl-Nielsen stain or Lowenstein-Jensen culture, or pleural fluid consistent with clinical signs and symptoms of tuberculosis together with lymphocyte predominance in pleural effusion and good response to anti-tuberculosis treatment. The study thereafter comprised twenty-two patients with transudative effusions, twenty-four patients with parapneumonic effusions, eight patients with tuberculous effusion and thirty-four patients with malignant effusions. Patients with transudative effusions comprised 12 women and 10 men and were aged (Mean ± SD) 60.2 ± 6.5 years. Patients with parapneumonic effusions comprised 13 women and 11 men and were aged (Mean ± SD) 59.8 ± 5.4 years. Patients with tuberculous effusions comprised 3 women and 5 men and were aged (Mean ± SD) 58.9 ± 8.9 years. Patients with malignant effusions comprised of 18 males and 16 females and were aged; (Mean ± SD) 63.5 ± 8.3 years (Table 1). Twenty-three patients had lung cancer, 2 metastatic colon adenocarcinoma, 2 patients with non Hodgkin lymphoma, 2 patients with metastatic gastric cancer and 5 patients with metastatic breast cancer. Of the 23 patients with bronchial carcinoma, histopathologically the cases comprised of 11 adenocarcinoma (3 of them were well differentiated and 8 were moderately differentiated), 12 cases of squamous cell carcinoma (3 of them were moderately differentiated and the other 9 poorly differentiated). Five out of the 11 distant metastatic cases originated from breast cancer, and they were microscopically invasive ductal carcinoma not otherwise specified (NOS), ranging from grade II/III to grade III/IV. All patients included in this study had unilateral pleural effusion except 2 patients of the transudative group with congestive heart failure. As per institution guidelines, the study was approved by King Fahad Hospital Ethics Committee. Informed consent was obtained from each patient included in the study. Thoracocentesis was performed under the aseptic technique, and pleural effusion and serum samples were collected at the same time. The pleural fluid was collected in tubes containing EDTA for determination of the cellular content, in plain tubes for determination of LDH, protein, albumin and YKL-40 levels. Paired serum samples were analyzed for urea, creatinine, albumin, total protein, and LDH levels. These parameters were measured using Siemens Diagnostics (Marburg GmbH, Germany) (RxL Dimension Clinical Chemistry analyzer) [20]. Pleural fluid pH was measured using blood gas analyzer, GEM premier 3000 (instrumentation laboratory, USA). Sample for pleural fluid pH was collected anaerobically, kept on ice and analyzed immediately within five minutes of collection. Pleural adenosine deaminase (ADA) levels were also measured as UI/mL. For serum YKL-40 analysis, the blood samples were centrifuged at 3000g for 10 min at 4 C. After the centrifugation, the serum was removed and transferred into a clean test tube. All pleural and serum samples were stored in a refrigerator at 80 C until YKL-40 analysis and they were dissolved and stored for 24 h at
Evaluation of pleural fluid YKL-40 Table 1
491
Demographic data, clinical and biochemical criteria of the studied groups.
Parameters
TPE N = 22
PPPE N = 24
TBPE N=8
MPE N = 34
Age Sex BMI BUN (mmol) S. creatinine (lmol/L) S. protein (g/L) S. albumin (g/L) Blood WBCs · 103/mm3 Pl. WBCs Pl. lymphocytic count Pl. neutrophils count Pl. PH
60.2 ± 6.5 M,10/F,12 20.8 ± 1.6 6.9 ± 4.7 79 (42–45) 62 ± 0.6 30 ± 0.34 5.5 ± 1.3 45.50(1–314) 23.18 ± 10.95 23.2 ± 11.1 8 ± 0.26
59.8 ± 5.4 M,11/F,13 25.3 ± 3.4 4.9 ± 2.7 79.05(42–43) 71 ± 0.9 36 ± 0.45 12.7 ± 3.2 980(345–4610) 17.72 ± 9.36 72.5 ± 13.05 7.21 ± 0.03
58.9 ± 8.9 M,5/F,3 25.3 ± 3.4 6.9 ± 4.7 79(42–39) 71 ± 0.7 36 ± 0.58 11.2 ± 2.4 950(339–3245) 57.54 ± 8.6 25.76 ± 11.5 7.19 ± 0.04
63.5 ± 8.3 M,18/F,16 23.9 ± 3.4 6.19 ± 4.7 79(42–41) 71 ± 0.5 37 ± 0.98 5.7 ± 1.9 1025(475–5346) 64.3 ± 9.7 24.7 ± 9.4 7.22 ± 0.108
TPE: transudative pleural effusion, PPPE: parapneumonic pleural effusion, TBPE: tuberculous pleural effusion, MPE: malignant pleural effusion, BMI: body mass index, BUN: blood urea nitrogen.
2–8 C before the study. YKL-40 protein concentrations were determined in serum and pleural fluids using a specific kit (MICROVUE YKL-40 EIA Kit, Quidel, San Diego, USA) by an enzyme-binding immunosorbent test (ELISA). Standards, controls and the concentration of YKL-40 presented in the test specimens were measured by spectrophotometer (PerkinElmer LAMBDA UV/Vis Spectrophotometer, Massachusetts, USA) and the results are calculated from the generated standard curve using linear regression analysis according to the producer’s instructions [21]. Statistical analysis Comparison among the groups was conducted using analysis of variance (ANOVA) test for normally distributed variables. The non-parametric Mann–Whitney U test was used for variables with non-Gaussian distribution. For the choice of the optimal cut-off, Receiver Operating Characteristic (ROC) curves were constructed and the Youden index was calculated [22]. The Youden index is defined as follows: (sensitivity + specificity) 1. The best cut-off has the highest Youden index. The commercial statistical software package used was SPSS11.0 (SPSS, Inc., Chicago, IL, USA). Results Demographic data, clinical and biochemical criteria of the studied groups are illustrated in Table 1. Both serum and pleural fluid levels YKL-40 were significantly higher in the exudative pleural effusions group (i.e. parapneumonic effusion, tuberculous effusion and malignant effusion) than the transudative group (p < 0.01). Median (range) values of serum YKL-40 were 154(56–237.3) & 123(45–187) ng/mL and pleural fluid YKL-40 values were 194(85–265) & 141(78–187) ng/mL in exudative and transudative groups respectively: (Fig. 1a). In the exudative pleural effusion group pleural fluid YKL-40 positively correlated with serum YKL-40 (r: 0.49 & p < 0.001). Similarly serum YKL-40 positively correlated with serum LDH (r: 0.383 & p = 0.002). Fig. 1b and d illustrates serum and pleural effusion levels of YKL-40. Both serum YKL-40 levels and effusion YKL-40 levels were significantly
higher in malignant effusions than in transudative or parapneumonic effusion, and tuberculous effusion (p < 0.05) while YKL-40 pleural fluid/serum ratio did not show significant difference between the four groups. When malignant effusions were classified according to the histopathological diagnosis, there was no significant difference between pleural and serum YKL-40 levels in the different tumor types (p > 0.05). The pleural effusion/serum ratio of LDH and albumin, pleural effusion LDH level, YKL-40 serum level and YKL-40 pleural effusion levels were evaluated as indicators for malignant effusion using Receiver Operating Characteristic curve (ROC). Effusion YKL-40 outperformed the other markers followed by serum YKL-40, pleural fluid LDH, Pleural fluid/serum LDH ratio then finally pleural fluid/serum albumin ratio (Fig. 1c). Pleural YKL-40/serum YKL-40 ratio >1.6 yielded 71% sensitivity, 73% specificity and likelihood ratio 2.8 for diagnosing TBPE, with an area under the curve of 0.785 (95% CI: 0.711–0.890). The results of pleural ADA levels were found to be high in pleural tuberculosis compared to other pleural fluids (p < 0.001) although we did not find such a correlation between serum and pleural YKL-40 and ADA levels. The area under the curve (AUC) for fluid YKL-40 was 0.886 (95% confidence interval [CI] was 0.8–0.973). The AUC for the serum YKL-40 was 0.798 (95% CI was 0.689– 0.907), while the AUC for the pleural fluid LDH was 0.722 (95% CI was 0.617–0.827). The AUC for pleural fluid LDH/ serum ratio was 0.615 (95% CI was 0.499–0.731). The AUC for pleural fluid lymphocyte count was 0.69 (95% CI was 0.544–0.836). The AUC for pleural fluid neutrophil count was 0.22 (and 95% CI was 0.123–0.317). Using the Youden index, the best cut-off value for pleural fluid YKL-40 to predict malignant effusions was 256 ng/mL. This cut-off value had a diagnostic sensitivity of 85.3% and specificity of 90.7%. The best cut-off value for pleural fluid YKL-40 to differentiate between exudative and transudative effusions was 212 ng/mL. This cut-off value had a diagnostic sensitivity of 74.3% and specificity of 75.7% (see Tables 2 and 3). Discussion The diagnosis of malignancy in pleural effusion is a challenging clinical problem. Several studies have been published
492
A. Attia et al.
Figure 1 HE4 levels in the different groups and YKL-40 AUC under a ROC curve. Boxplots (a, b and d) illustrate YKL-40 values in serum, and pleural effusion in patients with transudative effusion (TPE), parapneumonic pleural effusion (PPPE), tuberculous pleural effusion (TBPE) and malignant pleural effusions (MPE). The boxplots represent the interquartile range from the 25th to the 75th percentiles. The whiskers below and above the boxes represent the minimum and maximum values. The line across each box represents the median value. (c) ROC curves were constructed by plotting the sensitivity versus 1-specificity at different cut-off values for each analyte. EPE: Exudative pleural effusion, P: Pleural, S: Serum.
Table 2 Pleural, serum YKL-40 and pleural/serum ratio values in the transudative and exudative effusion patients. Parameters
TPE (n = 22)
EPE (n = 66)
p value
Pleural YKL-40 ng/mL Serum YKL-40 ng/mL PL/S YKL-40 ratio
141(78–187) 123(45–187) 1.05(0.8–5.5)
194(85–265) 154(56.7–237.3) 2.16(1.01–6.02)
<0.001 <0.05 <0.001
TPE: transudative pleural effusion, EPE: exudative pleural effusion, PL/S: pleural/serum.
reporting the presence of elevated levels of markers in malignant pleural effusions [23], however, none of these markers gained consensus as a highly sensitive marker for neoplastic effusions due to varied sensitivity and specificity [24] or specificity for certain neoplasms (e.g. mesothelioma) [25]. The search therefore continues for a marker or combination of markers that will increase the ability to diagnose malignancy
in a pleural effusion, and to select those patients who would benefit from more extensive invasive investigation when the pleural fluid cytology is negative. Proposed cut-off values for pleural tumor markers have varied in several studies; however some authors have suggested that cut-off points for a tumor marker that allows 100% specificity are necessary to substantiate a diagnosis of a malignant pleural effusion. Adopting such high cut-off points has resulted in the sensitivity of single tumor markers to predict malignancy to be as low as 17–30% [8]. The current study found a significantly higher level of YKL-40 in serum of patients with exudative effusion than those with transudative ones. The best cut-off value for pleural fluid YKL-40 to differentiate between exudative and transudative effusions was 212 ng/mL. This cut-off value had a diagnostic sensitivity of 74.3% and specificity of 75.7%. Most importantly, a marked significant increase was found in serum of patients with neoplastic pleural effusions than those with non-malignant exudative effusions and transudative effusions. This finding was also mirrored by an increased
Evaluation of pleural fluid YKL-40 Table 3
493
Pleural, serum, and pleural/serum ratio values of some biochemical parameters evaluated in the study.
Parameters
TPE (n = 22)
PPPE (n = 24)
TBPE (n = 8)
MPE (n = 34)
Protein(g/L) Pleural Serum PL/S ratio
22.7 ± 2.7 62 ± 5.9 1.9 ± 0.21
39 ± 2.4 70.2 ± 5.7 1.8 ± 0.17
43 ± 1.9 70.3 ± 5.9 1.8 ± 0.21
41.6 ± 6.4 70.1 ± 5.2 1.7 ± 0.23
Albumin(g/L) Pleural Serum PL/S ratio
16 ± 4.1 30 ± 4.8 0.52
32.29 ± 1.5 36 ± 3.5 0.84 ± 0.09
28.11 ± 1.8 36.2 ± 1.4 0.81 ± 0.08
29 ± 4.9 37.4 ± 3.6 0.80 ± 0.09
LDH(U/L) Pleural Serum PL/S ratio
59.68 ± 19.9 151 ± 47.9 0.42 ± 0.14
755 ± 274.3 209.2 ± 86.1 4.16 ± 2.4
721 ± 187.46 301.8 ± 84 5.12 ± 3.5
843.1 ± 501.3 332.8 ± 214 3.03 ± 1.19
Pleural ADA(U/mL)
12(1–25)
16(1–39)
48(23–100)
14(1–37)
YKL-40 (ng/mL) Pleural Serum PL/S ratio
141(78–187) 123(45–187) 1.05(0.8–5.5)
154(55–224) 149(59–200) 1.09(0.81–5.44)
182(65–235) 109(52–200) 4.11(1.3–6.7)
255(135–335) 205(59–312) 1.28(0.91–5.92)
TPE: transudative pleural effusion, PPPE: parapneumonic pleural effusion, TBPE: tuberculous pleural effusion, MPE: malignant pleural effusion, PL/S: pleural/serum, ADA: adenosine deaminase, LDH: lactic dehydrogenase.
pleural fluid YKL-40 level in patients with malignant effusions compared to those with transudative or non-malignant exudative effusions. This finding may be explained by increased local secretion of YKL-40 that is produced by the neoplastic tissues with subsequent overload to the systemic circulation resulting in increased serum YKL-40. The current study did not find a significant difference in pleural fluid YKL-40 between primary pulmonary neoplasm and secondary metastatic pleural effusions. This increase in pleural fluid YKL-40 may be due to increased secretion by the malignant cells in cases of primary pulmonary neoplasm or local irritation of respiratory epithelial cells with subsequent increase in YKL-40 local production in secondary metastatic pleural effusions. Furthermore the observed lack of correlation between serum or pleural fluid YKL-40 and the histopathological type of the tumor may be explained by the fact that all patients with malignant pleural effusion were advance metastatic stage IV patients according to TNM staging system. The metastasis may have caused increased production of YKL-40 either through local irritation or cellular destruction irrespective of the histopathological tumor origin. The biologic functions of YKL-40 in cancer cells are unknown, and very few studies have evaluated the functional role of YKL-40 expression in cancer cells. It has been suggested that YKL-40 plays a role in the proliferation and differentiation of malignant cells, protects the cells from undergoing apoptosis, YKL-40 is a relatively new biomarker of inflammation and it has been further investigated in several pathologic conditions [26]. Funkhouser et al. [27] reported that the levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders correlating with disease severity. Kim et al. [28] studied YKL-40 levels in different lung diseases and pleural effusions, they found that the results of serum YKL-40 were the highest in tuberculosis compared with other pleural effusions including parapneumonic,
and cardiogenic effusions. Bouzas et al. [29] demonstrated a significant relation between chitotriosidase and ADA activities, although we did not find such a correlation between serum and pleural YKL-40 and ADA levels. Contrary to our results, Kim et al. [28] reported that serum YKL-40 level of the patients with pleural tuberculous was higher than that of the patients with effusions due to malignant or cardiac causes. The current study showed a positive correlation between pleural fluid YKL-40 and LDH point more to the possible value of increased YKL-40 level in malignant effusion. The increased levels of both markers (LDH and YKL-40) in pleural fluid may be secondary to increased local production either through increased cellular destruction, local invasion or local tissue irritation. The current study had shown a significant increase in pleural fluid lymphocytic count in patients with malignant pleural effusion, and a good AUC for pleural lymphocytic count. However increased pleural fluid lymphocytic count may be found in other disease states (e.g. Tuberculosis, pulmonary embolism) [30]. Pleural YKL-40/serum YKL-40 ratio >1.6 yielded 71% sensitivity, 73% specificity and likelihood ratio 2.8 for diagnosing TBPE, with an area under the curve of 0.785 (95% CI: 0.711–0.890). The use of pleural fluid/serum ratio to enhance the diagnostic sensitivity of tuberculous effusion was evaluated by Kayhan et al. [31] who found, Pleural YKL-40/serum YKL-40 ratio >1.5 yielded 75% sensitivity, 72% specificity and likelihood ratio 2.6 for diagnosing TBPE, with an area under the curve of 0.825 (95% CI: 0.710–0.940). The current study did not find a significant difference in pleural fluid/serum YKL-40 ratio between the transudative and exudative effusions. The use of pleural fluid/ serum ratio to enhance the diagnostic sensitivity of tumor markers was evaluated by Trape´ et al. [32] who classified tumor markers into two categories: released/secreted by normal pleura (e.g. CA-125 and cytokeratin) and non released/ non-secreted tumor markers (e.g. CEA and CA-19.9) by
494 normal pleura. Trape´ et al. showed clearly that the fluid/serum ratio was only useful for non-secreted tumor markers while for secreted tumor markers (i.e. those that may be secreted by normal respiratory epithelium) the ratio did not improve their sensitivity and specificity. As YKL-40 has been previously shown to be expressed by normal respiratory epithelium, it may be considered as a secreted/released tumor marker and it would not be surprising that pleural/fluid serum YKL-40 ratio did not enhance its diagnostic sensitivity and specificity due to possible concomitant increase in serum and pleural fluid YKL-40 with the net result of a normal or reduced ratio of pleural fluid/serum YKL-40 ratio. An important finding of the current study is the superior performance of pleural effusion YKL-40 levels in detecting malignant pleural effusions with a value of 256 ng/mL as the best cut-off value to suggest a malignant pleural effusion with a diagnostic sensitivity of 85.3% and specificity of 90.7%. Even when the cut-off point was increased to 286 ng/mL to achieve 100% specificity, the marker still predicted malignancy with a sensitivity of 45%. This result favors comparably with other similar studies examining the utility of single tumor markers where the achieved sensitivity was very low when a 100% specificity cut-off was chosen. This is the first study to examine the role of YKL-40 in the diagnosis of malignancy in pleural effusions, and it was limited by the relatively small number of subjects, and therefore the utility of this marker will have little clinical benefit currently. Conclusion However this novel finding of increased pleural fluid YKL-40 levels in malignant pleural effusions compared to either transudative or non-malignant exudative effusions will add to our scientific knowledge, and set the stage for larger studies of this marker in pleural effusions, or the inclusion of this marker in combination with other panels to strengthen the diagnostic performance of tumor markers in detecting malignant pleural effusions. Conflict of interest The authors declare no conflict of interests. References [1] M.J. Murphy, F. Jenkinson, Categorisation of pleural fluids in routine clinical practice: analysis of pleural fluid protein and lactate dehydrogenase alone compared with modified Light’s criteria, J. Clin. Pathol. 61 (2008) 684–685. [2] R.W. Light, M.I. Mac Gregor, P.C. Luchsinger, W.C. Ball, Pleural effusions: the diagnostic separation of transudates and exudates, Ann. Intern. Med. 77 (1972) 507–513. [3] J.E. Heffner, L.K. Brown, C.A. Barbieri, Diagnostic value of tests that discriminate between exudative and transudative pleural effusions. Primary study investigators, Chest 111 (1997) 970–980. [4] K.V. Nance, R.W. Shermer, F.B. Askin, Diagnostic efficacy of pleural biopsy as compared with that of pleural fluid examination, Mod. Pathol. 4 (1991) 320–324. [5] L. Garcia, The value of multiple fluid specimens in the cytological diagnosis of malignancy, Mod. Pathol. 7 (1994) 665–668.
A. Attia et al. [6] W.W. Huang, S.M. Tsao, C.L. Lai, C.C. Su, C.E. Tseng, Diagnostic value of Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen levels in malignant pleural effusions of lung adenocarcinoma, Pathology 42 (2010) 224–228. [7] J.S. Hackbarth, K. Murata, W.M. Reilly, A. AlgecirasSchimnich, Performance of CEA and CA19-9 in identifying pleural effusions caused by specific malignancies, Clin. Biochem. 43 (2010) 1051–1055. [8] J.M. Porcel, M. Vives, A. Esquerda, A. Salud, B. Perez, F. Rodriguez-Panadero, Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3, and cytokeratin 19 fragments) in pleural fluid for the differential diagnosis of benign and malignant effusions, Chest 126 (2004) 1757–1763. [9] G.L. Chupp, C.G. Lee, N. Jarjour, Y.M. Shim, C.T. Holm, J.D. Dziura, J. Reed, A.J. Coyle, et al, A chitinase-like protein in the lung and circulation of patients with severe asthma, N. Engl. J. Med. 357 (2007) 2016–2027. [10] S. Letuve, A. Kozhich, N. Arouche, M. Grandsaigne, et al, YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages, J. Immunol. 181 (2008) 5167–5173. [11] J.S. Johansen, B.V. Jensen, A. Roslind, D. Nielsen, P.A. Price, Serum YKL-40, a new prognostic biomarker in cancer patients?, Cancer Epidemiol Biomarkers Prev. 15 (2) (2006 Feb) 194–202. [12] I.K. Choi, Y.H. Kim, J.S. Kim, J.H. Seo, High serum YKL-40 is a poor prognostic marker in patients with advanced non-small cell lung cancer, Acta Oncol. 49 (6) (2010 Aug) 861–864. [13] I. Tho¨m, B. Andritzky, G. Schuch, I. Burkholder, L. Edler, J.S. Johansen, C. Bokemeyer, U. Schumacher, E. Laack, Elevated pretreatment serum concentration of YKL-40 – an independent prognostic biomarker for poor survival in patients with metastatic nonsmall cell lung cancer, Cancer 116 (17) (2010 Sep 1) 4114–4121. [14] A. Roslind, J.S. Johansen, I.J. Christensen, K. Kiss, E. Balslev, D.L. Nielsen, J. Bentzen, P.A. Price, E. Andersen, High serum levels of YKL-40 in patients with squamous cell carcinoma of the head and neck are associated with short survival, Int. J. Cancer 122 (4) (2008 Feb 15) 857–863. [15] B. Volck, J.S. Johansen, M. Stoltenberg, C. Garbarsch, P.A. Price, M. Østergaard, et al, Studies on YKL-40 in knee joints of patients with rheumatoid arthritis and osteoarthritis. Involvement of YKL-40 in the joint pathology, Osteoarthr. Cartilage 9 (2001) 203–214. [16] J.S. Johansen, P. Christoffersen, S. Møller, P.A. Price, J.H. Henriksen, C. Garbarsch, et al, Serum YKL-40 is increased in patients with hepatic fibrosis, J. Hepatol. 32 (2000) 911–920. [17] G. Kronborg, C. Østergaard, N. Weis, H. Nielsen, N. Obel, S.S. Pedersen, et al, Serum level of YKL-40 is elevated in patients with Streptococcus pneumoniae bacteremia and is associated to the outcome of the disease, Scand. J. Infect. Dis. 34 (2002) 323– 326. [18] J. Kzhyshkowska, A. Gratchev, S. Goerdt, Human chitinases and chitinase-like proteins as indicators for inflammation and cancer, Biomark Insights 2 (2007) 128–146. [19] M.S. Niederman, L.A. Mandell, A. Anzueto, et al, Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention, Am. J. Respir. Crit. Care Med. 163 (2001) 1730–1754. [20] C.A. Burtis, E.R. Ashwood, Tietz Fundamental of Clinical Chemistry, fourth ed., WB Saunders, Philadelphia, 1996. [21] S. Harvey, M. Weisman, J. O’Dell, et al, Chondrex: new marker of joint disease, Clin. Chem. 44 (1998) 509–516. [22] W.J. Youden, Index for rating diagnostic tests, Cancer 3 (1950) 32–35. [23] P. Korczynski, R. Krenke, A. Safianowska, K. Gorska, M.B. Abou Chaz, M. Maskey-Warzechowska, A. Kondracka, J.
Evaluation of pleural fluid YKL-40 Nasilowski, R. Chazan, Diagnostic utility of pleural fluid and serum markers in differentiation between malignant and nonmalignant pleural effusions, Eur. J. Med. Res. 14 (Suppl. 4) (2009) 128–133. [24] M.J. Gaspar, J. De Miguel, J.D. Garcı´ a Dı´ az, M. Dı´ ez, Clinical utility of a combination of tumour markers in the diagnosis of malignant pleural effusions, Anticancer Res. 28 (2008) 2947– 2952. [25] B. Grigoriu, B. Chahine, F. Zerimech, M. Gre´goire, M. Balduyck, M.C. Copin, P. Devos, P. Lassalle, A. Scherpereel, Serum mesothelin has a higher diagnostic utility than hyaluronic acid in malignant mesothelioma, Clin. Biochem. 42 (2009) 1046– 1050. [26] C.G. Lee, D. Hartl, G.R. Lee, et al, Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis, J. Exp. Med. 206 (2009) 1149– 1166.
495 [27] J.D. Funkhouser, N.N. Aronson Jr., Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family, BMC Evol. Biol. 7 (2007) 96. [28] H.R. Kim, C.D. Jun, K.S. Lee, et al, Levels of YKL-40 in pleural effusions and blood from patients with pulmonary or pleural disease, Cytokine 58 (2012) 336–343. [29] L. Bouzas, E. San Jose´, J.C. Tutor, Chitotriosidase activity in pleural effusions, Clin. Lab. 53 (2007) 449–452. [30] J.M. Porcel, R.W. Light, Diagnostic approach to pleural effusion in adults, Am. Fam. Physician 73 (2006) 1211–1220. [31] S. Kayhan, A. Gumus, H. Cinarka, N. Murat, A. Yilmaz, R. Bedir, U. Sahin, The clinical utility of pleural YKL-40 in diagnosing pleural effusion, J. Thorac. Dis. 5 (5) (2013) 634–640. [32] J. Trape´, R. Molina, F. Sant, Clinical evaluation of the simultaneous determination of tumor markers in fluid and serum and their ratio in the differential diagnosis of serous effusions, Tumour Biol. 25 (2004) 276–281.