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Cancer Biomarkers 14 (2014) 457–467 DOI 10.3233/CBM-140424 IOS Press

Clinical significance of putative cancer stem cell marker CD44 in different histological subtypes of lung cancer Raheleh Roudia,b , Zahra Madjda,b,c,∗ , Alireza Korouriana, Mitra Mehrazmab,c, Saadat Molanaed, Mehrdad Nasrollahzadeh Sabeta,b and Ahmad Shariftabrizie a

Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran b Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran c Department of Pathology, Iran University of Medical Sciences, Tehran, Iran d Department of Pathology, Milad Hospital, Tehran, Iran e Department of Pathology, Tufts University School of Medicine, Boston, MA, USA

Abstract. BACKGROUND: According to the cancer stem cell theory, tumors originate from a subset of cells known as cancer stem cells (CSCs) that are responsible for tumor initiation, resistance and relapse. CD44 is a cell adhesion molecule that can aid in the identification of CSCs in various malignancies. OBJECTIVE: The purpose of the current study is to evaluate the expression level and clinical significance of CD44 in lung cancer samples. METHODS: One hundred and ninety-five lung tumor samples including 74 (38%) squamous cell carcinomas (SCC), 61 (31%) adenocarcinomas (ADC), 23 (12%) large cell carcinoma (LCC) in non-small cell lung cancer (NSCLC) group and 37 (19%) small cell lung cancer (SCLC) samples were examined for the expression of CD44 using immunohistochemistry method. The correlation of CD44 expression with clinicopathological parameters as well as Ki-67 status was also assessed. RESULTS: Univariate analysis demonstrated that CD44 expression was significantly higher in NSCLC compared to SCLC (P < 0.001). Among NSCLC, higher level of CD44 expression was found in SCC compared to ADC (P < 0.001) and LCC (P = 0.046). Increased expression of CD44 was significantly correlated with higher grade tumors which correspond to poor prognosis in SCC (P = 0.012) and the lower level of CD44 expression was more often found in well differentiated ADC tumors (P = 0.03). In addition, high expression of CD44 was significantly associated with decreased level of proliferative marker Ki-67 (P = 0.04). CONCLUSIONS: CD44 could be a valuable tool for the study of lung CSCs and provide a novel therapeutic target for treatment of the patients with lung cancer in combination with conventional therapy. Keywords: Cancer stem cells, non-small cell lung cancer, small cell lung cancer, CD44, Ki-67

1. Introduction

∗ Corresponding

author: Zahra Madjd, Oncopathology Research Center, Iran University of Medical Sciences (IUMS), Hemmat Street (Highway), Next to Milad Tower, Tehran 14496-14530, Iran. Tel.: +98 2186703212; Fax: +98 2188622608; E-mail: Zahra.madjd@ yahoo.com.

Lung cancer is the leading cause of cancer related death all over the world. Based on the histopathology, lung cancer can be broadly categorized as: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is a heterogeneous group of cancers

c 2014 – IOS Press and the authors. All rights reserved ISSN 1574-0153/14/$27.50 

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that include squamous cell carcinoma (SCC) and adenocarcinoma (ADC) with similar frequencies (30–40% each), and the large cell carcinoma (LCC) with a lower frequency (< 10%) [46]. SCLC, another group of lung cancer, comprises nearly 20% of all lung cancer cases and shows dramatically more invasive and metastatic potential compared to NSCLC [17]. Five year relative survival rate is 15% for NSCLC, whereas it is only 6.9% for SCLC [51]. According to the cancer stem cell hypothesis, there is a distinct population of cells in the bulk of the tumor known as cancer stem cells (CSCs), harboring various genetic abnormalities that are responsible for tumor initiation, maintenance, metastasis and relapse [39]. Successful treatment and prevention of lung cancer recurrence requires more detailed knowledge of the properties of these cells which could result in effective therapeutic targeting of this subpopulation. Many putative stem cell markers including CD44 have been identified for isolating CSCs from various malignancies including breast [1], brain [40], prostate [5] and melanoma [9]. CD44 is a multifunctional class 1 transmembrane glycoprotein that is involved in cell adhesion, proliferation, motility and differentiation; and alone or combination with other markers is used for isolating CSCs [18,34]. Previous immunohistochemical studies of lung carcinomas showed that SCC tumors express a significantly higher level of CD44 compared to ADC cases [10,13,21,41]. Some of studies indicated that higher expression of CD44 was a negative prognostic marker in patients with SCC and ADC [13,21, 50], whereas other recent studies showed that higher level of expression of CD44 was associated with a better prognosis in SCC [41] and ADC [24]. Recently, in vitro and in vivo tumorigenecity assessment of isolated cells from NSCLC cell lines indicated that CD44-expressing subpopulations were enriched of CSCs properties [24]. Also in a comprehensive study on a panel of primary lung cancer cell lines (PLCCLs, representative of all lung cancer subtypes), flow cytometric analysis was used to determine the stem celllike properties. The findings indicated that stem celllike features were observed in a small subset of cells that expressed CD44high /CD90+ in SCLC and LCC and CD44high in SCC subtype [49]. In addition, evaluation of some putative CSCs markers including CD44 in stage-1 ADC by immunohistochemistry suggested that high expression of CD44 increased the risk of the recurrence compared with cases with low expression of CD44 [50].

The other putative stem cell marker used for identification of lung CSCs is CD133, a five transmembrane glycoprotein with unknown function [37]. Early studies showed that CD133 may be a reliable lung CSC marker [4,8,44], but subsequent studies did not confirm this notion [6,42]. Considering the lack of consensus in results of previous studies concerning the expression patterns and pathological significance of CD44 as CSC marker in various types of lung carcinomas, herein we aimed to investigate the immunohistochemical expression and clinicopathologic significance of CD44 in a well characterized series of lung tumor specimens including NSCLC (SCC, ADC and LCC) and SCLC using tissue microarray (TMA) technique. The prognostic value of nuclear protein Ki-67 in NSCLC has been shown in a very recent study, although the role of Ki-67 as a predictive factor needs further investigation [19]. Therefore, for the first time, we sought to determine whether the expression level of CD44 as a putative CSC marker could be correlated with Ki-67 as a prognostic value.

2. Materials and methods 2.1. Experimental subjects A total of 195 paraffin-embedded tumor tissues from patients with primary lung cancers who underwent either surgery or biopsy between 2004 and 2012 at Milad Hospital, one of the major public referral centers in Tehran, Iran, were used in the current study. All of specimens were obtained before any systematic treatment and paraffin-embedding was performed within the framework of diagnostic procedures. These series consisted of different subtypes of lung cancer including NSCLC (SCC, ADC and LCC) and SCLC samples. Recently, the pathological classification of lung ADC was revised by integrating the novel clinical and biological knowledge concerning this tumor type [47]. In our study, diagnosis of ADC was performed based on the new classification [47]. Patients’ information including age, gender and available pathological parameters including information on tumor types, histological grade (in SCC and ADC), nuclear grade (only in ADC) and inflammation (only in SCC) were collected and recorded in a database (Table 1). This research project was approved by Iran University of Medical Sciences (IUMS) Research Ethics Committee. Patients’ data were kept fully anonymous.

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Table 1 Patients’ demographic and pathological characteristic of various subtypes of lung cancer Patients and Tumor characteristics Number of tumor samples Median of age (years), (range)  67 > 67 Gender (Male/Female) Histological Grade Well Moderately Poorly Expression of CD44 Intensity of staining No staining Weak Moderate Strong Percentage of positive cells 0 1–50% 50–75% > 75% Median of H-score Low ( 10) High (> 10) Expression of Ki-67 (n = 104, Cut-off=15%) Low (< 15%) High (> 15%) ∗ Not

Total samples 195 67 (37–89) 115 (59) 80 (41) 150/45

SCC 74 (38) 68 (45–89) 36(49) 38 (51) 68/6

NSCLC ADC 61 (31) 67 (38–89) 40(66) 21 (34) 35/26

42 (31) 50 (37) 43 (32)

33 (44) 22 (30) 19 (26)

61 (31) 83 (43) 27 (14) 24 (12)

SCLC

p value

LCC 23 (12) 67 (43–75) 16(70) 7 (30) 15/8

37 (19) 67 (37–84) 23(62) 14 (38) 32/5

< 0.001

9 (15) 28 (46) 24 (39)

NA∗

NA∗

< 0.001

15 (20) 24 (33) 18 (24) 17 (23)

34 (56) 25 (41) 2 (3) 0 (0)

7 (30) 4 (18) 5 (22) 7 (30)

5 (14) 30 (81) 2 (5) 0 (0)

< 0.001

118 (61) 24 (12) 18 (9) 35 (18)

23 (31) 9 (12) 17 (23) 25 (34)

49 (80) 10 (17) 0 (0) 2 (3)

10 (44) 5 (22) 1 (4) 7 (30)

36 (97) 0 (0) 0 (0) 1 (3)

< 0.001

111 (57) 84 (43)

19 (26) 55 (74)

46 (75) 15 (25)

11 (48) 12 (52)

35 (95) 2 (5)

< 0.001

83 (80) 21 (20)

36 (77) 11 (23)

24 (92) 2 (8)

11 (85) 2 (15)

12 (67) 6 (33)

0.31

0.17

available; Numbers in parentheses are percentage values. The values which are shown in bold, are statistically significant.

2.2. Tissue microarray construction Tissue microarrays (TMA) from paraffin-embedded blocks of lung carcinomas were constructed as described previously [22,30]. All samples were reviewed by a pathologist (A.K.) and the best tumor region for preparing TMAs was marked on their H&E stained slides.The TMA blocks were then constructed in three copies, each containing one sample from a different area of the tumor using tissue-arraying instrument (Minicore; ALPHELYS, Plaisir, France). The mean scoring of three cores was then calculated as the final score. The normal lung tissues adjacent to tumors were also included in TMAs to compare the staining patterns of CD44 in a range of different tissue samples. 2.3. Immunohistochemistry staining Immunohistochemical staining of CD44 and ki67 were conducted on TMA slides (Superfrost plus, Thermo Scientific, Germany) using a standard chain polymer-conjugated (Envision) technique as described previously [31] applying mouse monoclonal antihuman CD44 (Product code = NCL-CD44-2, Novocastra -UK) and Ki-67 (Dako, Denmark) as primary anti-

bodies. After deparaffinization in xylene and rehydration through graded alcohol, slides were immersed in methanol/hydrogen peroxide for 20 min to block endogenous peroxidase activity. Antigen retrieval was performed by microwaving tissue sections for 20 minutes in sodium citrate buffer (pH = 6.0). The sections were then incubated with primary antibodies with optimal dilution of 1:40 and 1:30 for 1 hour at room temperature. After washing, the sections were incubated with EnVisionTM + /HRP, Dual Link Rabbit/Mouse (Dako, Denmark) secondary antibody for 1 hour. Color was developed with addition of 3, 3’diaminobenzidine (DAB, Dako) to achieve visualization of the antigen. In the final step, sections were lightly counter stained with haematoxylin (Dako), dehydrated in alcohol, cleared in xylene and mounted for examination. Human tonsil tissues were used as positive control for both CD44 and Ki-67 antibodies. The omission of primary antibody and its replacement with TBS (Tris Buffered Saline) was used as negative reagent control. 2.4. Evaluation of immunohistochemical staining Semi-quantitative scoring system which relies on the subjective assessment of multiple independent ob-

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servers was used in this study as described previously [28,31]. All immunostained TMAs were evaluated by two investigators (ZM and AK) on two separate occasions after the series were examined on a double-headed microscope (on a blind basis). The controversial cases were reviewed by the third independent pathologist (MM) to achieve a final consensus. Initially, slides were scanned at 10x magnification to obtain a general impression of the overall distribution of the tumor cells and then positive cores were assessed semi-quantitatively for expression level of markers at higher magnifications and the final scores were given. 2.5. Scoring system The intensity of the CD44 staining was scored on a scale as 0 (absent), 1 (weak), 2 (moderate) or 3 (strong). The pattern of CD44 expression was mainly cell membranous. The percentage of CD44 positive cells was graded as: 0 (no staining), 1 (< 50% positive cells), 2 (50–75% positive cells), 3 (> 75% positive cells). The overall score was obtained by H-score (Histochemical score) for each case by multiplying the intensity of staining by the percentage of positive cells and a final score of 0 to 300 was given [29]. The median of H-scores was chosen as cut-off value to classify the samples as high or low expression of CD44; which was found to be 10. The level of Ki-67 expression was scored as percentage of positive cells and based on cut-off value of 15%, was categorized as low (< 15% of cells are positive) and high (> 15% of cells are positive) expression. This cut-off value was chosen because the lowest p-values were obtained in the evaluation of the correlation between the level of CD44 expression and Ki-67 status [23,33]. 2.6. Statistical analysis Statistical analysis of data was performed using SPSS software version 20 (SPSS, Chicago, IL, USA). Pearson’s χ2 and Pearson’s R tests were used to analyze the significance of correlation between CD44 expression and clinicopathological parameters, also ki67 status. Moreover, the comparisons of CD44 expression in various subtypes of NSCLC (including SCC, ADC and LCC) were performed using Mann-Whitney U test. A p-value of < 0.05 was considered to be statistically significant.

3. Results 3.1. Study population One hundred and ninety-five paraffin blocks of lung cancer samples were used in the current study. Overall, the median age of patients was 67 years (ranged 37– 89); 115 (59%) of cases were younger than 67, and 80 (41%) of patients were over 67 years of age. The median age of SCC patients was 68 years (ranged 45–89), the median age of ADC patients was 67 years (ranged 38–89), in LCC cases was 67 years (ranged 43–75) and SCLC cases had a median age of 67 years (ranged 37–84). The study population consisted of 150 (77%) male and 45 (23%) female with a male/female (M/F) ratio of 3.33. While M/F ratio in SCC was 11.33 (68/6) and in SCLC was 6.4 (32/5), in LCC decreased to 2 (15/8) and in ADC, the M/F ratio was 1.34 (35/26). NSCLC with a total of 158 (81%) of cases included 74 (38%) SCC, 61 (31%) ADC and 23 (12%) LCC, whereas 37 (19%) of cases were SCLC. Of SCC samples 33 (44%) had well differentiated, 22 (30%) had moderately differentiated and 19 (26%) had poorly differentiated carcinoma. Nine (15%), 28 (46%) and 24 (39%) of ADC samples reported to have well, moderately and poorly differentiation. Of 104 samples for whom nuclear Ki-67 status were evaluated, 83 (80%) of cases showed low expression (< 15% positive cells); whereas 21 (20%) of samples showed high expression (> 15% positive cells) of Ki-67. Patients’ and pathological parameters are summarized in Table 1. 3.2. CD44 expression in normal lung epithelium Human lung is composed of main bronchi, bronchioles and the most distal area; a complex system of alveoli that consist of two types of epithelial cells: alveolar type 1 cells that are involved in gas exchange and alveolar type 2 cells that secrete surfactant protein C. In our experiments studying the CD44 staining of normal lung tissue, bronchioles displayed no staining, however some basal cells present in normal human bronchi and bronchioles expressed high level of CD44 protein. Immunohistochemical staining of CD44 marker in normal alveolar type 2 cells and epithelial stroma exhibited strong staining pattern, but alveolar type 1 cells showed only a faint staining. 3.3. Expression of CD44 in lung cancer The expression of putative CSC marker CD44 was assessed using immunohistochemistry on tissue mi-

R. Roudi et al. / Clinical significance of putative cancer stem cell marker

SCC

LCC

ADC

461

SCLC

a

b

c

d

Fig. 1. Representative photographs of the immunohistochemical expression of CD44 in different subtypes of lung tumor. Squamous cell carcinoma (SCC) and large cell carcinoma (LCC) expressed CD44 at various levels; (a) strong, (b) moderate, (c) weak and (d) negative, but adenocarcinoma (ADC) and small cell lung carcinoma (SCLC) expressed CD44 at various levels; (b) moderate, (c) weak and (d) negative. (All figures are shown with a magnification of × 200). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-140424)

croarrays containing various types of lung cancer. The level of expression was examined by three scoring methods, namely, the intensity of the staining, the percentage of positive cells, and H-score. The human tonsil tissues and infiltrating lymphocytes within the tumor (internal positive control) showed strong and uniform staining of CD44 on cell membrane, with no background staining of stroma or nuclei. CD44 was expressed with a variety of intensities in cell membrane of epithelial of lung tumor cells (Fig. 1). Of 195 lung tumor samples stained for CD44, 61 (31%) cases did not show any staining, whereas weak, moderate and strong intensity were seen in 83 (43%), 27 (14%) and 24 (12%) of cases, respectively. Of 158 NSCLC samples, no staining was found in 56 (35%), whereas weak, moderate and strong patterns were de-

tected in 53 (34%), 25 (16%) and 24 (15%) of cases, respectively. Of 74 SCCs, no staining, weak, moderate and strong staining were observed in 15 (20%), 24 (33%), 18 (24%) and 17 (23%) of samples, respectively (Fig. 1, SCC, a-d). Of 23 evaluated LCC samples, 7 (30%) showed no staining, while 4 (18%), 5 (22%) and 7 (30%) of cases were weak, moderate and strong staining, respectively (Fig. 1, LCC, a-d). Of 61 ADC specimens evaluated for expression of CD44, no staining, weak and moderate staining were found in 34 (56%), 25 (41%) and 2 (3%) of cases, respectively, whereas any of cores did not display strong staining pattern (Fig. 1, ADC, b-d). Of 37 SCLC samples, no staining, weak and moderate staining were found in 5 (14%), 30 (81%) and 2 (5%), whereas there was no case with strong staining pattern (Fig. 1, SCLC, b-

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Table 2 Association between the expression patterns of CD44 (intensity, percentage of positive cells and H-score) based on histological subtypes; SCC, ADC and LCC (Mann-Whitney U Test) NSCLC subtypes Intensity of staining SCC with ADC LCC ADC with LCC

CD44 expression (P value, 2-tailed) Percentage of positive cells

H-score

< 0.001 0.944

< 0.001 0.447

< 0.001 0.046

< 0.001

< 0.001

0.016

The values which are shown in bold, are statistically significant.

d). Although the majority of cases in SCC and SCLC showed weak expression of CD44, in SCC moderate and strong expression were observed in 18 (24%) and 17 (23%) respectively; in SCLC they were 2 (5%) and 0 (0%), respectively. Moreover, although most cases of ADC (56%) showed no staining pattern, in LCC, 7 (30%) of cases showed no staining and 7 (30%) expressed CD44 strongly. Expression of CD44 (in terms of intensity) in different subtypes of lung tumor is shown in Table 1. For overall staining in terms of H-score, the cutoff value for each tumor type was calculated based on median of H-score to define groups showing low and high expression of CD44 which was 10. The evaluation of CD44 expression in patients with lung cancer demonstrated that 111 (57%) of cases displayed low expression (H-score  10), while 84 (43%) of tumors expressed higher level of CD44 marker (H-score > 10). Of 158 NSCLC, low expression was observed in 76 (48%) (H-score  10), whereas high expression was detected in 82 (52%) (H-score > 10). Among SCC tumors, 19 (26%) of cases showed low expression of CD44, whereas 55 (74%) of evaluated cases exhibited high expression of CD44. Of 61 ADC tumors stained for CD44, low expression was observed in 46 (75%), while high expression was detected in 15 (25%) of cases. In LCC tumors, 11 (48%) of cases exhibited low expression of CD44 and 12 (52%) of tumors showed high expression of CD44. In contrast, of 37 SCLC samples, as a major group of lung cancer, low expression was observed in 35 (95%), whereas high expression was detected in 2 (5%) of cases. Expression of CD44 (in terms of H-score) in different subtypes of lung tumor is shown in Table 1. A significant difference was evident in the level of expression of CD44 (either intensity or Hscore) between NSCLC and SCLC cases, indicating higher level of expression in NSCLC compared to SCLC (P < 0.001). Moreover, among NSCLC the level of expression of CD44 (either intensity or H-score) was significantly higher in SCC and LCC compared to ADC tumors (P
10) 11 (33) 8 (36) 0 (0)

22 (67) 14 (64) 19 (100) 0.012

8 (30) 9 (19)

7 (26) 12 (25)

0.47

20 (74) 35 (75) 0.97

Numbers in the parentheses are percentage values. The values which are shown in bold, are statistically significant. Table 4 Association between level of CD44 expression (intensity and H-score) and pathological characteristic of ADC patients (P value; Pearson χ2 ) Tumor characteristic

Histological Grade Well Moderately Poorly p value Tumor Differentiation∗ Well (Lepidic, microinvasive, atypical adenomatous hyperplasia (AAH) and mucinous tumors) Moderately (Acinar and papillary tumors) Poorly (solid, invasive mucinous, micropapillary and solid-acinar tumors) p value Nuclear Grade Low Moderately High p value

Expression of CD44 Intensity of staining Median of H-score = 10 No expression Weak Moderate Low ( 10) High (> 10) 3 (33) 15 (54) 16 (67)

6 (67) 11 (39) 8 (33) 0.23

0 (0) 2 (7) 0 (0)

9 (100) 17 (61) 20 (83)

4 (40)

6 (60)

0 (0)

9 (90)

1 (10)

14 (52) 16 (67)

11 (41) 8 (33)

2 (7) 0 (0)

16 (59) 21 (88)

11 (41) 3 (12)

0.03

0.31 5 (50) 19 (58) 10 (56)

5 (50) 13 (39) 7 (39) 0.91

0 (0) 11 (39) 4 (17)

0.033 0 (0) 1 (3) 1 (5)

10 (100) 22 (67) 14 (78)

0 (0) 11 (33) 4 (22) 0.09

∗ Tumor

differentiation based on the new diagnostic classification of ADC; Numbers in parentheses are percentage values. The values which are shown in bold, are statistically significant.

Although, the intensity of CD44 expression was correlated with gender (P = 0.046), age was not significantly correlated with the level of expression of CD44 (either intensity or H-score, P = 0.6 and P = 0.18, respectively). 3.5. Distribution of ki-67 in various subtypes of lung carcinomas and its association with CD44 expression Of 47 SCC cases for whom the Ki-67 data were available, 36 (77%) cases had low expression of Ki67 (< 15%); whereas 11 (23%) of cases showed high expression of Ki-67 (> 15%). Of 26 ADC cases for which Ki-67 data were available, low expression of Ki-

67 was detected in 24 (92%); whereas 2 (8%) of cases had strong expression of Ki-67. Of 13 LCC tumors low and high expression of Ki-67 were found in 11 (85%) and 2 (15%) of cases, respectively. Of 18 SCLC samples, 12 (67%) cases showed low expression of Ki-67; whereas the frequency of high expression of Ki-67 was 6 (33%) (Table 1). Univariate analysis showed that the higher level of CD44 expression (in terms of H-score) in lung tumors was significantly associated with low expression of Ki-67 (P = 0.04). Similarly in SCC tumors, increased expression of CD44 (in terms of Hscore) was significantly correlated with decreased level of Ki-67 (P = 0.001), whereas no significant association was found between expression of CD44 and Ki67 status in ADC (P = 0.36), LCC (P = 0.25) and

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Table 5 Association between level of Ki-67 expression and expression of CD44 (intensity and H-score) in various subtypes of lung cancer (P value; Pearson χ2 ) Tumor characteristic

Expression of CD44 Intensity of staining Negative Weak Moderate Strong p value Median of H-score Low ( 10) High (> 10) p value Total No.

SCC Ki-67 expression Low∗ High∗∗

ADC Ki-67 expression Low High

3 (9) 10 (30) 9 (27) 11 (34)

9 (48) 9 (48) 1 (4) 0 (0)

4 (37) 4 (37) 2 (18) 1 (8) 0.11

3 (9) 30 (91)

1 (50) 1 (50) 0 (0) 0 (0)

LCC Ki-67 expression Low High

3 (30) 1 (10) 1 (10) 5 (50)

0.94 6 (55) 5 (45)

15 (79) 4 (21)

0.001 44

0 (0) 9 (90) 1 (10) 0 (0)

0.17 1 (50) 1 (50)

0.36 21

0 (0) 1 (100) 0 (0) 0 (0)

SCLC Ki-67 expression Low High

4 (40) 6 (60)

0.46 1 (100) 0 (0)

0.25 11

0 (0) 5 (100) 0 (0) 0 (0)

9 (90) 1 (10)

5 (100) 0 (0) 0.46 15

∗ /∗∗ Cut-off of Ki-67 expression = 15%, Low (< 15%) and High (> 15%); Numbers in parentheses are percentage values. The values which are shown in bold, are statistically significant.

SCLC (P = 0.46) tumors. All data are summarized in Table 5.

4. Discussion CSCs are considered as cancer initiating cells that possess the ability to initiate and sustain growth of the bulk of the tumor, resistance to conventional chemo/radio therapy, metastasis and relapse [20,39]. Enhancing the methods of cancer treatment requires a deep understanding of the cellular and molecular aspects of CSCs and targeting them either at the cell surface marker level or their signaling pathways [7,15, 27]. CD44 is a transmembrane glycoprotein that plays role in cell adhesion, proliferation, migration and angiogenesis [34,38]. These functions were suggestive of the fact that CD44 may be a potential CSC marker and thus has been studied in various tumors [1,16,25]. In the present study, the expression of CD44 as a prospective CSC marker was investigated in a well characterized series of 195 lung carcinomas consisting of SCC, ADC and LCC in addition to SCLC which were assembled in tissue microarray. Our study was included larger number of lung carcinoma patients compared to some of previous studies on expression of putative CSC markers, CD44 or CD133 in lung carcinomas [4,21,44,50]. TMA technique was used in order to examine the level of expression of CD44 in different types of lung tumor with various degrees of differentiation and to correlate CD44 expression level with clinicopathological parameters, and also the Ki-67 status. Indeed, tu-

mor profiling by tissue microarray technology has been established to overcome the limitations of conventional immunohistochemical studies and allows the simultaneous analysis of target protein expression for hundreds of tumor samples [22]. Consistent with other studies, the most common tumor type of our series was NSCLC and among them, SCC (38%) and LCC (12%) were the most and least common types of lung cancer, respectively [46]. We observed various expression patterns of CD44 in different histological types of lung cancer. In total, NSCLC tumors expressed significantly higher level of CD44 expression compared to SCLC samples. Among NSCLC group, SCC and LCC showed a statistically significant higher level of CD44 expression than ADC cases. Analysis of the intensity of staining in each subtype demonstrated that the pattern of expression of CD44 in SCC and LCC were heterogeneous, with a range of intensities from weak to strong. In ADC samples, the majority of cases showed no staining or weak expression of CD44 with nearly similar frequencies, but strong staining pattern was not seen. In SCLC, weak staining was the dominant expression pattern of CD44; whereas there was no strong expression pattern. In agreement with previous literature, our findings indicated that SCC samples expressed higher level of CD44 expression than other histological types of NSCLC [2,10,13,21,24,36,41,45]. We showed that the majority of SCLC samples expressed low level of CD44; whereas previous reports demonstrated no expression of CD44 in SCLC cases [2,10,36]. Furthermore, exploration of the relationship between expression of CD44 and tumor differentiation showed a sig-

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nificant correlation between expression of CD44 and histological grade in SCC, indicating that increased level of CD44 was more often found in poorly differentiated SCC. Therefore, in line with previous studies, we showed that higher expression of CD44 was associated with more aggressive behavior in SCC patients [21], but an independent study demonstrated that high expression of CD44 is a favorable prognostic factor in SCC [41]. Also Leung et al. showed that expression of CD44 marker was not correlated with survival in SCC cases [24]. The discrepancy could result from different experimental conditions including application of different clones of antibodies, scoring methods and selection of different cut-off points. In this study all TMA slides were evaluated independently by at least two observers using three scoring systems including intensity of scoring, percentage of positive cells and overall scoring (H-score). Moreover, lung tumor samples assessed in various studies have different pathological stages, even tumors in a group may have very different stages and grades. In ADC cases, the lower level of CD44 expression demonstrated a significant association with well histological grade, and a trend with lower nuclear grade. Although some recent studies showed higher expression of CD44 was associated with more aggressive behavior in ADC tumors [21,35], another studies reported a longer survival in CD44-expressing ADC [24,41]. The general concept suggested that more aggressive tumors express higher level of CSCs markers and this discrepancy between results can be attributed to expression of variant isoforms of CD44 and its different biological functions and also to the controversial nature of the cell of origin and histogenesis of ADC [3]. We also performed a further analysis to investigate correlation of CD44 expression with Ki-67 status as another prognostic factor. Ki-67 is a nuclear protein that can be detected during all active phases of the cell cycle (G1 , S, G2 and mitosis), but it is absent in quiescent stage of the cell cycle- namely G0 ; therefore Ki67 can be used as a valuable proliferative biomarker to define the proliferative fraction of a cell population [11,12]. The prognostic importance of Ki-67 in lung cancer has been previously shown, but the predictive value of Ki-67 is still controversial [19]. Our analysis reflected an inverse significant association between expression of CD44 and Ki-67 status, indicating that high expression of CD44 was found in tumors with low expression of Ki-67. This observation suggested that majority of CD44+ lung tumor cells are in slow-cycling or non-dividing Go state known as quies-

465

cence [26]; thus the level of proliferative marker Ki67 decreased. This finding may indicate that CD44+ cells are not intensively proliferating, while it does not provide a definitive indication that they are CSC. Although there is no data in the literature regarding the correlation of CD44 expression and Ki-67 in lung cancer samples, a recent study in breast cancer showed an inverse correlation between expression of CSC marker ALDH1 and Ki-67 [33]. Emerging evidence indicated that quiescent stem cells are often resistant to conventional chemo/radio therapy and play an important role in treatment failure and tumor recurrence; thus identification and targeting of quiescent stem cells will have clinical applications [26,32]. In addition, in parallel with previous studies, we showed that SCC tumors expressed higher level of Ki-67 compared to ADC cases [14,43,48]. In agreement with previous reports, the expression of CD44 was not correlated with age, gender and inflammation [21,41].

5. Conclusions In summary, our findings reveal that expression of CD44 as a putative CSC marker is significantly higher in in SCC and LCC groups compared to the ADC, also compared to SCLC. The expression of CD44 was significantly higher in poorly differentiated SCC and decreased expression of CD44 was found more often in well differentiated ADC. Moreover, CD44 was inversely correlated with proliferative marker ki-67, supporting the notion that CD44+ lung tumors cells might be in the quiescence state. These results are therefore particularly applicable to the development of new treatment strategies for lung cancer.

Acknowledgement This study was conducted as a research project and supported by a grant from Iran University of Medical Sciences (Grant #15998).

Conflict of interest The authors declare that they have no conflict of interest.

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R. Roudi et al. / Clinical significance of putative cancer stem cell marker

References [1]

Al-Hajj, M., M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, M. F. Clarke. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003 Apr 1; 100(7): 3983-8. [2] Ariza, A., J. L. Mate, M. Isamat, D. Lpez, V. UexkllGldeband, R. Rosell, A. Fernndez-Vasalo, J. J. NavasPalacios. Standard and variant CD44 isoforms are commonly expressed in lung cancer of the non-small cell type but not of the small cell type. J Pathol. 1995 Dec; 177(4): 363-8. [3] Asselin-Labat, M.-L., C. E. Filby. Adult lung stem cells and their contribution to lung tumourigenesis. Open Biol. 2012 Aug; 2(8): 120094. [4] Bertolini, G., L. Roz, P. Perego, M. Tortoreto, E. Fontanella, L. Gatti, G. Pratesi, A. Fabbri, F. Andriani , S. Tinelli. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci U S A. 2009 Sep 22; 106(38): 16281-6. [5] Collins, A. T., P. A. Berry, C. Hyde, M. J. Stower, N. J. Maitland. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 2005 Dec 1; 65(23): 10946-51. [6] Cui, F., J. Wang, D. Chen, Y.-J. Chen. CD133 is a temporary marker of cancer stem cells in small cell lung cancer, but not in non-small cell lung cancer. Oncol Rep. 2011 Mar; 25(3): 701-8. [7] Eramo, A., T. Haas, R. De Maria. Lung cancer stem cells: tools and targets to fight lung cancer. Oncogene. 2010 Aug 19; 29(33): 4625-35. [8] Eramo, A., F. Lotti, G. Sette, E. Pilozzi, M. Biffoni, A. Di Virgilio, C. Conticello, L. Ruco, C. Peschle, R. De Maria. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008 Mar; 15(3): 504-14. [9] Fang, D., T. K. Nguyen, K. Leishear, R. Finko, A. N. Kulp, S. Hotz, P. A. Van Belle, X. Xu, D. E. Elder, M. Herlyn. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res. 2005 Oct 15; 65(20): 9328-37. [10] Fasano, M., M. T. Sabatini, R. Wieczorek, G. Sidhu, S. Goswami, J. Jagirdar. CD44 and its v6 spliced variant in lung tumors. Cancer. 1997 Jul 1; 80(1): 34-41. [11] Gerdes, J., H. Lemke, H. Baisch, H.-H. Wacker, U. Schwab, H. Stein. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984 Oct; 133(4): 1710-5. [12] Gerdes, J., U. Schwab, H. Lemke, H. Stein. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983 Jan 15; 31(1): 13-20. [13] Hirata, T., T. Fukuse, H. Naiki, S. Hitomi, H. Wada. Expression of CD44 variant exon 6 in stage I non-small cell lung carcinoma as a prognostic factor. Cancer Res. 1998 Mar 15; 58(6): 1108-10. [14] Hommura, F., H. Dosaka-Akita, T. Mishina, M. Nishi, T. Kojima, H. Hiroumi, S. Ogura, M. Shimizu, H. Katoh and Y. Kawakami (2000). Prognostic significance of p27KIP1 protein and ki-67 growth fraction in non-small cell lung cancers. Clinical Cancer Research 6(10): 4073-4081. [15] Hu, Y., L. Fu. Targeting cancer stem cells: A new therapy to cure cancer patients. Am J Cancer Res. 2012; 2(3): 340-56. [16] Hurt, E. M., B. T. Kawasaki, G. J. Klarmann, S. B. Thomas, W. L. Farrar. CD44+ CD24 – prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br J Cancer. 2008 Feb 26; 98(4): 756-65.

[17]

Jackman, D. M., B. E. Johnson. Small-cell lung cancer. Lancet. 2005; 366(9494): 1385-1396. [18] Jaggupilli, A., E. Elkord. Significance of CD44 and CD24 as Cancer Stem Cell Markers: An Enduring Ambiguity. Clin Dev Immunol. 2012; 2012: 708036. [19] Jakobsen, J. N.„ Jens Benn Sørensen. Clinical impact of ki67 labeling index in non-small cell lung cancer. Lung Cancer. 2013 Jan; 79(1): 1-7. [20] Jordan, C. T., M. L. Guzman, M. Noble. Cancer stem cells. N Engl J Med. 2006 Sep 21; 355(12): 1253-61. [21] Ko, Y. H., H. S. Won, E. K. Jeon, S. H. Hong, S. Y. Roh, Y. S. Hong, J. H. Byun, C. K. Jung, J. H. Kang. Prognostic significance of CD44s expression in resected non-small cell lung cancer. BMC Cancer. 2011 Aug 7; 11: 340. [22] Kononen, J., L. Bubendorf, A. Kallionimeni, M. Brlund, P. Schraml, S. Leighton, J. Torhorst, M. J. Mihatsch, G. Sauter, O.-P. Kallionimeni. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998 Jul; 4(7): 844-7. [23] Krüger, S., C. Thorns, W. Stöcker, E. Müller-Kunert, A. Böhle, A. C. Feller. Prognostic value of MCM2 immunoreactivity in stage T1 transitional cell carcinoma of the bladder. Eur Urol. 2003 Feb; 43(2): 138-45. [24] Leung, E. L., R. R. Fiscus, J. W. Tung, V. P. Tin, L. C. Cheng, A. D. Sihoe, L. M. Fink, Y. Ma and M. P. Wong. Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One. 2010 Nov 19; 5(11): e14062. [25] Li, C., D. G. Heidt, P. Dalerba, C. F. Burant, L. Zhang, V. Adsay, M. Wicha, M. F. Clarke, D. M. Simeone. Identification of pancreatic cancer stem cells. Cancer Res. 2007 Feb 1; 67(3): 1030-7. [26] Li, L., R. Bhatia. Stem cell quiescence. Clin Cancer Res. 2011 Aug 1; 17(15): 4936-41. [27] Madjd, Z., E. Gheytanchi, E. Erfani, M. Asadi-Lari. Application of Stem Cells in Targeted Therapy of Breast Cancer: A Systematic Review. Asian Pac J Cancer Prev. 2013; 14(5): 2789-800. [28] Madjd, Z., I. Spendlove, S. E. Pinder, I. O. Ellis, L. G. Durrant. Total loss of MHC class I is an independent indicator of good prognosis in breast cancer. Int J Cancer. 2005 Nov 1; 117(2): 248-55. [29] McCarty Jr, K., L. Miller, E. Cox, J. Konrath, K. McCarty Sr. Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med. 1985 Aug; 109(8): 71621. [30] Mehrazma, M., Z. Madjd, E. Kalantari, M. Panahi, A. Hendi, A. Shariftabrizi. Expression of Stem Cell Markers, CD133 and CD44, in Pediatric Solid Tumors: A Study Using Tissue Microarray. Fetal Pediatr Pathol. 2013 Jun; 32(3): 192-204. [31] Mohsenzadegan, M., Z. Madjd, M. Asgari, M. Abolhasani, M. Shekarabi, J. Taeb, A. Shariftabrizi. Reduced expression of NGEP is associated with high-grade prostate cancers: A tissue microarray analysis. Cancer Immunol Immunother. 2013 Oct; 62(10): 1609-18. [32] Moore, N., S. Lyle. Quiescent, slow-cycling stem cell populations in cancer: A review of the evidence and discussion of significance. J Oncol. 2011; 2011. [33] Morimoto, K., S. J. Kim, T. Tanei, K. Shimazu, Y. Tanji, T. Taguchi, Y. Tamaki, N. Terada, S. Noguchi. Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009 Jun; 100(6): 1062-8.

R. Roudi et al. / Clinical significance of putative cancer stem cell marker [34]

[35]

[36]

[37] [38]

[39]

[40]

[41]

[42]

[43]

Naor, D., S. B. Wallach-Dayan, M. A. Zahalka, R. V. Sionov .Involvement of CD44, a molecule with a thousand faces, in cancer dissemination. Semin Cancer Biol. 2008 Aug; 18(4): 260-7. Okudela, K., T. Woo, H. Mitsui, M. Tajiri, M. Masuda, K. Ohashi. Expression of the potential cancer stem cell markers, CD133, CD44, ALDH1, and β-catenin, in primary lung adenocarcinoma – their prognostic significance. Pathol Int. 2012 Dec; 62(12): 792-801. Penno, M. B., J. T. August, S. B. Baylin, M. Mabry, R. I. Linnoila, V. S. Lee, D. Croteau, X. L. Yang, C. Rosada. Expression of CD44 in human lung tumors. Cancer Res. 1994 Mar 1; 54(5): 1381-7. Piechaczek, C.. CD133. J Biol Regul Homeost Agents. 2001 Jan-Mar; 15(1): 101-2. Ponta, H., L. Sherman, P. A. Herrlich. CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol. 2003 Jan; 4(1): 33-45. Reya, T., S. J. Morrison, M. F. Clarke, I. L. Weissman. Stem cells, cancer, and cancer stem cells. Nature. 2001 Nov 1; 414(6859): 105-11. Singh, S. K., I. D. Clarke, M. Terasaki, V. E. Bonn, C. Hawkins, J. Squire, P. B. Dirks. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003 Sep 15; 63(18): 5821-8. Situ, D., H. Long, P. Lin, Z. Zhu, J. Wang, X. Zhang, Z. Xie, T. Rong. Expression and prognostic relevance of CD44v6 in stage I non-small cell lung carcinoma. J Cancer Res Clin Oncol. 2010 Aug; 136(8): 1213-9. Sullivan, J. P., J. D. Minna, J. W. Shay. Evidence for selfrenewing lung cancer stem cells and their implications in tumor initiation, progression, and targeted therapy. Cancer Metastasis Rev. 2010 Mar; 29(1): 61-72. Takahashi, S., Y. Kamata, W. Tamo, M. Koyanagi, R. Hatanaka, Y. Yamada, T. Tsushima, S. Takaya, I. Fukuda.

[44]

[45]

[46] [47]

[48]

[49]

[50]

[51]

467

Relationship between postoperative recurrence and expression of cyclin E, p27, and Ki-67 in non-small cell lung cancer without lymph node metastases. Int J Clin Oncol. 2002 Dec; 7(6): 349-55. Tirino, V., R. Camerlingo, R. Franco, D. Malanga, A. La Rocca, G. Viglietto, G. Rocco, G. Pirozzi. The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer. Eur J Cardiothorac Surg. 2009 Sep; 36(3): 446-53. Tran, T. A., B. V. Kallakury, C. E. Sheehan, J. S. Ross. Expression of CD44 standard form and variant isoforms in non-small cell lung carcinomas. Hum Pathol. 1997 Jul; 28(7): 809-14. Travis, W. D. Pathology of lung cancer. Clin Chest Med. 2002; 23(1): 65. Travis, W. D., E. Brambilla, M. Noguchi, A. G. Nicholson, K. R. Geisinger, Y. Yatabe, D. G. Beer, C. A. Powell, G. J. Riely, P. E. Van Schil. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011 Feb; 6(2): 244-85. Tsubochi, H., N. Sato, M. Hiyama, M. Kaimori, S. Endo, Y. Sohara, T. Imai. Combined analysis of cyclooxygenase-2 expression with p53 and Ki-67 in nonsmall cell lung cancer. Ann Thorac Surg. 2006 Oct; 82(4): 1198-204. Wang, P., Q. Gao, Z. Suo, E. Munthe, S. Solberg, L. Ma, M. Wang, N. A. C. Westerdaal, G. Kvalheim, G. Gaudernack. Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines. PLoS One. 2013; 8(3): e57020. Woo, T., K. Okudela, H. Mitsui, T. Yazawa. Prognostic value of CD133 expression in stage I lung adenocarcinomas. Int J Clin Exp Pathol. 2010 Dec 4; 4(1): 32-42. Youlden, D. R., S. M. Cramb, P. D. Baade. The International Epidemiology of Lung Cancer: geographical distribution and secular trends. J Thorac Oncol. 2008 Aug; 3(8): 819-31.

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