Research Article, J Clin Exp Oncol Vol: 6 Issue: 2
Cripto-1 and RUNX2 Expressions in Non-small Cell Lung Cancer, their Roles in its Progression and Patients Outcome
|Ola A Harb1, Shereen El shorbagy2*, Nehal S Abouhashem1, Ola M Elfarargy2, Safa A Balata3, Loay M gertallah4, Mohammed M N Abozaid5, Walid Galal6 and Sameh Saber7|
|1Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|2Department of Medical Oncology, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|3Department of Clinical oncology and Nuclear medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|4Department of general surgery, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|5Department of Chest, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|6Department of cardiothoracic surgery, Faculty of Medicine, Zagazig University, Zagazig, Egypt|
|7Department of Radiology, Faculty of Medicine, Zagazig University, Zagazig|
|Corresponding author : Shereen El shorbagy
Department of Medical Oncology, Faculty of Medicine, Zagazig University, 44519, Zagazig, Egypt
E-mail: [email protected]
|Received: February 15, 2017 Accepted: March 15, 2017 Published: March 22, 2017|
|Citation: Harb OA, El shorbagy S, Abouhashem NS, Elfarargy OM, Balata SA, et al. (2017) Cripto-1 and RUNX2 Expressions in Non-small Cell Lung Cancer, their Roles in its Progression and Patients’ Outcome. J Clin Exp Oncol 6:2. doi: 10.4172/2324-9110.1000182|
Background: Non-small cell carcinoma of lung (NSCLC) is the commonest and most lethal lung cancer type; it includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma subtypes. The five-year survival rate in NSCLC patients is still very low although improvements in treatment modalities are still emerging. Hence, new prognostic markers and therapies need to be brought to light aiming to improve patients' outcome. Cripto-1 (CR-1) is one of the family members of epidermal-growth-factor; cripto FRL1 cryptic-(EGF-CFC) is needed for embryogenesis. Runt-related-transcription-factor [RUNX] family members make core-binding factor complex (CBFC) that attach to DNA to stimulate or inhibit many genes transcription, regulate the survival, differentiation and maturation of many tissues. The aim of this work is to detect the clinical significance and prognostic role of CR-1 and RUNX2 expressions in NSCLC using immunohistochemistry.
Method: CR-1 and RUNX2 expressions were evaluated in 59 paraffin blocks sections of NSCLC. The relationship between their level of expressions and patient's prognosis was analyzed.
Results: CR-1 and RUNX2 were highly expressed in NSCLC patients, 59.3% and 67.8%, respectively. There was a significant positive association between their expressions in NSCLC patients (p=0.015). Both markers were significantly correlated with size, grade, stage, site of the tumor within the lung, malignant (pleural and/or pericardial) effusion, presence of distant metastases, ECOG performance status of the patients (p<0.001) and existence of hepatic metastases (p=0.004). Both markers expressions were significantly correlated with poor response to treatment (p<0.001). After a median follow up of 30 months, mean PFS of NSCLC patients having elevated CR-1 and RUNX2 expressions was shorter (p<0.001). Patients with high RUNX2 expressing have significantly shorter mean OS (p=0.025). High CR-1 expression negatively affected OS but that was not statistically significant (p=0.2).
Conclusion: NSCLC patients with elevated CR-1 and RUNX2 expression values had unfavorable prognosis.
Keywords: Non-small cell lung cancer; CR-1; RUNX2; Progression; Prognosis
|Non-small cell lung cancer; CR-1; RUNX2; Prognosis|
|Globally, lung cancer is the commonest lethal malignancy. The most common type of lung cancer is non-small cell lungcarcinoma (NSCLC) (80-85%) , the major subtypes of NSCLC are:adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Although improvements in the treatment modalities, the fiveyear overall- survival rate in patients having NSCLC still very low(15%) . Low survival rate of those patients can be attributed todelay in diagnosis and lack of suitable therapy at the proper timeleading to increase the metastasis risk. Many factors that can assessthe patient prognosis and survival have been explored including:ECOG performance status, grade and stage of cancer  but thesewere found to be un-satisfactory, so the treatment and prognosisof every patient should be individualized. New prognostic markersand therapies are needed to be discovered to improve outcome ofpatient by comprehensive studying of the molecular pathogenesisof cancer lung. Cripto-1 (CR-1) is one of the family members ofepidermal-growth-factor; cripto FRL1 cryptic (EGF-CFC) is neededfor embryogenesis [6,7]. Recent studies stated that CR-1 also controlsthe initiation and progression of many human cancers . Previousstudies had correlated the levels of CR-1 expression with cancerinitiation, invasive ability, metastatic potential, and tumor prognosisof many organs, [9-11], but researches which studied its expressionpattern and prognostic role in NSCLC were insufficient.|
|The progression from early to advanced stage NSCLC is controlledby many genes; which were under research to understand the initiationand progression of NSCLC. The RUNX family contains 3 members(RUNX1-3), forming the core-binding factor (CBF) complex whichattaches to DNA; stimulating or inhibiting transcription of manygenes  which in turn regulates the survival, differentiation andmaturation of many tissues . RUNX2 plays an essential rolein osteogenesis during embryogenesis . RUNX2 controls thedeposition of bone matrix, mainly collagen I, by osteoblasts afterbirth . RUNX2 may either stimulates or suppresses the process ofcarcinogenesis depending on the type and site of cancer , but onlyfew reports focused on the role of RUNX2 in NSCLC.|
|The aim of this study was to assess the clinical relevance andprognostic role of CR-1 and RUNX2 expressions in NSCLC byimmunohistochemistry as the prognostic significance of theirexpressions in NSCLC is still unclear.|
Patients and Methods
|This study was carried out at Zagazig University Hospitals. Thestudy protocol was approved by the Ethical Committee of Facultyof Medicine, Zagazig University. It comprised of 59 previouslydiagnosed NSCLC patients. Patient’s data including: age, gender,smoking history, tumor size, tumor differentiation, lymph nodalstatus, pathological stage and follow up data were obtained frompatients’ hospital records in Medical Oncology and Clinical oncology& Nuclear Medicine Departments. The 7th TNM staging system fornon-small cell lung cancer (NSCLC) was used for pathologic staging. Patients were treated according to their stage either by surgery,chemotherapy (platinum-based chemotherapy), and radiotherapy orcombined modalities.|
|Expressions of CR-1and RUNX2 were evaluated in 59 paraffinblocks sections of NSCLC that were obtained from Pathologydepartment, Faculty of Medicine, Zagazig University archives in theperiod from September 2013 to September 2016.|
|Four-unthick paraffin-embedded sections had been deparaffinizedthen rehydrated. For antigen retrieval, tissues were heated for 10 minin sodium citrate, addition of 3 % hydrogen peroxide blocked theactivity of endogenous peroxidase then incubation with polyclonalrabbit anti-RUNX2 antibody ab23981 and monoclonal rabbit anti-CR-1 antibody ab108391 (1:100 dilutions) (Abcam. Cambridge, MA,USA) was done. Colorectal carcinoma was used as positive controls forboth CR-1& RUNX2. Secondary antibody was added for half an hourfollowed by using chromogenic for five minutes. We counterstainedthe slides with hematoxylin.|
|Determination of CR-1Expression by ImmunohistochemicalAssay|
|The expression of CR-1 was assessed based on the extent of stain(E) (positive cells were graded from 0 to 3: 0<1 %, 1=1-33 %, 2=33-67 %, and 3>67 %) and the intensity of stain (I) (graded from 0-3:0=none, one=weak stain, two=moderate stain, and three=strongstain). The final score of stain was calculated by the product of extent× intensity, resulting in points zero to nine . We use the cut pointof 3 above which is considered high expression.|
|Determination of RUNX2 Expression by ImmunohistochemicalAssay|
|The expression results were done by multiplying intensity of stainby stain area. We calculated stain intensity by the following score: nostain (scored zero), weak stain (scored 1), moderate stain (scored 2),or strong stain (scored 3). We calculated stain areas by the followingscore: less than 25 percent (scored 1), 25 to 50 percent (scored 2), 50to 75 percent (scored 3), or more than 75 percent (scored 4) of cancercells. Final scores of; 0,1, 2,3,4,6,8,9, and 12 were reached [19,20]. Weuse the cut point of 4 above which is considered high expression.|
|We expressed the categorical variables as a number& percentage,but the continuous variables as the mean ± standard deviation&median (range). We calculated Progression-Free-Survival rate (PFS);the time from treatment initiation to time of disease progression.Overall survival (OS) and PFS rates were calculated in comparisonwith all clinicopathological features and Immunohistochemicalmarkers. Time-to-mortality distributions were calculated by usingof the Kaplan-Meier method, with considering the p-value of lessthan 0.05 as significant value. Statistics were made by using SPSS 22.0windows (SPSS Inc., IL, and USA) and windows (MedCalc Softwarebvba 13, Belgium).|
|The clinical data of our patients are summarized in Table 1.|
|Table 1: Clinicopathological features, Immunohistochemical markers and outcome of our patients.|
|Fifty nine patients were included in the study, in which there were39(66.1%) men and 20 (33.9%) women, aged from (45-77) years (themean: 62.42 ± 8.45 years), 38 (64.4%) cases were adenocarcinoma,and 21 (35.6) were squamous cell carcinoma.|
|CR-1 expression and its relation to clinicopathological dataof our patients (Tables 2 and 3) (Figure 1)|
|High CR-1 expression in the cytoplasm was present in thirtyfiveof fifty-nine (59.3%) patients, and it was significantly positivelycorrelated with: the presence of co-morbid conditions, weight loss,age of the patients (p=0.018), poor performance status (p=0.005),tumor stage, grade of the tumor, distant metastases, lymph nodemetastases, malignant (pleural and/or pericardial) effusion (p<0.001),liver metastases (p=0.004), brain metastases (p=0.016), and site ofthe tumor within the lung (p=0.030), but we found non- significantcorrelation with sex, smoking history of the patients, tumor size,histopathological type of the tumor or number of distant metastases.|
|Figure 1: Immunohistochemical expression of Cripto-1(CR1) in non-small cell lung cancer (NSCLC).
*Note: (A) High expression in the cytoplasm of poorly differentiated squamous cell carcinoma x400 (B) High expression in the cytoplasm of poorly differentiated adenocarcinoma x400 (C) Low expression in cytoplasm of moderately differentiated squamous cell carcinoma stage IIx400 (D) Low expression in the cytoplasm of moderately differentiated adenocarcinoma x400 (E) Negative expression in the cytoplasm of well differentiated squamous cell carcinoma stage IIx400(F) Negative expression in the cytoplasm of well differentiated adeno-carcinoma x400.
|Table 2: Correlation between immune-histochemical expressions of Cripto-1& RUNX-2 with clinicopathological features of our patients.|
|Table 3: Correlation between immune-histochemical expressions of both markers together with clinicopathological features of our patients.|
|RUNX2 expression and its relation to clinicopathologicaldata of our patients (Tables 2 and 3) (Figure 2)|
|High nuclear expression of RUNX2 was demonstrated in fortyof fifty-nine (67.8%) patients, and it was significantlypositivelycorrelated with the presence of co-morbid conditions, age of thepatients, weight loss, stage, size, location of the tumor within thelung, presence of distant metastases, poor performance status of thepatients(p<0.001), grade of the tumor(p=0.003), malignant (pleuraland/or pericardial) effusion (p=0.006), liver metastases (p=0.022) andbrain metastases (p=0.045), no significant correlations were foundbetween RUNX2 expression and sex, smoking history of the patients,number of distant metastases or histopathological type of the tumor.|
|Figure 2: Immunohistochemical expression of RUNX2 in non-small cell lung cancer (NSCLC).
*Note: (A) High expression in nucleus of poorly differentiated squamous cell carcinoma stage IVx400 (B) High expression in nucleus of poorly differentiated adenocarcinoma stage IVx400 (C) High expression in nucleus of moderately differentiated squamous cell carcinoma stage IIIx400. (D) High expression in nucleus of moderately differentiated adenocarcinoma x400 (E) Low expression in nucleus of well differentiated squamous cell carcinoma stage IIx400 (F) Low expressions in nucleus of well differentiated adenocarcinoma x400.
|There was a significant positive association between both markersexpressions (p=0.015) and high expressions of both markers togetherwere significantly correlated with tumor size, grade, stage, site ofthe tumor within the lung, malignant (pleural and/or pericardial)effusion, presence of distant metastases, ECOG performance statusof the patients(p<0.001) and presence of liver metastases (p=0.004).|
|Relations between CR-1 &RUNX2 expressions and patientsurvival (Tables 4 and 5) (Figure 3)|
|The high expressions of both markers expressions weresignificantly correlated with poor treatment outcome (p<0.001).After median follow up of 30 months (range: 5-35 months),the 2yearprogression free survival (PFS) of all patients was 69.1%, patientswith CR-1 high and/or high RUNX2 expressions had shorter PFSthan those with low expressions (p<0.001). OS rate was significantlyshorter in patients with high expressions of RUNX2 in comparisonto those with low expression (p=0.025), patients with high CR-1expressing tumors had shorter OS but that was statistically nonsignificant(p=0.2).|
|Figure 3: Kaplan Meier Survival plots; Left Panel: Progression Free Survival (PFS); Right Panel: Overall Survival.
*Note: (A) & (E): All studied NSCLC patients; (B) & (F) Stratified by RUNX-2 IHC staining; (C) & (G) Stratified by Cripto-1 IHC staining; (D) & (H) Stratified by RUNX-2/Cripto-1 IHC staining.
|Table 4: Correlation between immune-histochemical expressions of Cripto-1& RUNX-2 with outcome of our patients.|
|Table 5: Correlation between immune-histochemical expressions of both markers together with outcome of our patients.|
|There are no applicable guidelines for early detection of NSCLCwhich result in poor prognosis and low survival of patients. Hence, it isextremely important to discover new biomarkers for determining theprogression, metastasis and treatment outcomes. In this study, CR-1expression in NSCLC patients was positively correlated with grade,stage of the tumor, lymph node and distant metastases which supportour hypothesis that CR-1 could have a highly important role in NSCLCOncogenesis and progression. These results were in agreement with Xuet al.  who verified that CR-1 has an essential role in increasing lungcancer cell proliferation and allow early progression. We also foundthat patients who have elevated CR-1 immuno-expressions have poorsurvival. Our results showed and explained the promising use of CR-1as a recent prognostic marker and added a spot light on the relation between its biological function and NSCLC carcinogenesis. Thesefindings were supported by Bianco et al., 2013 and Nagaoka et al.[6,7] who showed that CR-1 which is EGF-CFC family member; isessential for angiogenesis, cell migration and maintenance of stemcells. Sun et al.  reported that CR-1 regulates EMT (EpithelialMesenchymal Transition) and invasiveness of HCC. Also Wei etal.  demonstrated the role of CR-1 in bladder cancer growth,proliferation, recurrence and metastasis. Moreover, similar resultsfrom Wang et al., 2015 , Zhong et al. , Yoon et al.  andWu et al., 2009  who proved the association between CR-1over expression, tumor recurrence, lower 5-year survival rates andthe histological differentiation in oral squamous cell carcinoma;and the carcinogenesis and progression of nasopharyngeal cancer.|
|Regarding RUNX2 expression in NSCLC, we found that highRUNX2 immuno-expressions were correlated significantly withtumor size, tumor stage, tumor grade, lymph node and distantmetastases. Furthermore, PFS and OS rates of NSCLC patientswith high RUNX2 immuno-expression were shorter than thosewith low RUNX2 expression. So we clarify that, RUNX2 elevatedexpression may be considered a substantial factor in expecting the poor prognosis of NSCLC patients; hence, it may be considereda novel prognostic marker for NSCLC patients. Hong et al., 2013, Yang et al., 2015 , Chua et al.  and Tonomoto etal., 2007  reported similar results in NSCLC, colon cancer,prostatic cancer and esophageal squamous cell carcinoma,respectively.|
|High expression of RUNX2 is markedly related to metastasis ofosteosarcoma  that may suggests that RUNX2 up-regulation islinked to the invasiveness of malignant cells . RUNX2 enhancesendothelial cell proliferation, invasion, and tube formation andactivate vascular endothelial growth factor gene expression; allof which may lead to stimulation of angiogenesis in cancer cellswhich facilitate their growth and spread . RUNX2 stimulatestranscription of osteopontin, which increases the metastatic ability incancer cells . Also RUNX2 increases the metastasis of carcinomamainly by increasing the expression of matrix metalloproteinase and bone sialoprotein . RUNX2 can be considered a potentprognostic factor for NSCLC patients as it plays important roles inthe growth and metastasis of cancer cells through wide spectrums ofits biological functions.|
|In this study, we found a significant positive association betweenthe levels of expression of both CR-1 and RUNX2 in NSCLC cases(p=0.015), and high expressions of both markers together weresignificantly correlated with poor performance status, poor PFS,shorter OS and subsequently bad prognosis.|
|These results indicated that CR-1 could serve as a feasibleprognostic biomarker of NSCLC. RUNX2 plays an important role inthe tumorigenesis and progression of NSCLC; hence both markerstogether may provide a chance for discovering recent-therapeutictargets, as well as prognostic markers in NSCLC. Further studies arerecommended on large number of cases of NSCLC and other types ofcancers to clarify the precise molecular functions of these markers andthe value of using them together in assessment of NSCLC prognosisand as therapeutic targets.|
|Previous studies have investigated a panel of prognostic markersfor NSCLC but most of them were investigated as serum markerswith low sensitivity and specificity conveying conflicting results; hence, our study assessed the tissue expressions of both Cripto-1 andRUNX2 using immunohistochemistry which demonstrated moresensitive and specific results for assessment of NSCLC prognosis [36-39].|
|Our research confirmed that CR-1 and RUNX2 high expressionshad an essential role in tumor aggression and poor NSCLC patients’prognosis. Also, their levels appear to be an important predictor forNSCLC patient’s survival. Nonetheless, further studies are needed toelucidate the mechanisms by which both markers facilitate NSCLCdevelopment and progression and to address whether one of them orboth together could be used as targets for new therapeutic approaches.|
Limitations of our Work
|• Small number of patients.|
|• Assessment of Cripto-1& RUNX2 expression was only by immunohistochemistry without any assessment of serumlevels or genetic analysis by FISH or real time-PCR|
|• Cripto-1 and RUNX2 together may provide a chance fordiscovering recent-therapeutic targets as well as prognosticmarkers in NSCLC.|
|• Further studies are recommended on large number of casesof NSCLC and other types of cancers to clarify the precisemolecular functions of these markers and the values of usingthem together in assessment of NSCLC prognosis, and astherapeutic targets.|
|• To use different methods of assessment of both markers;serum levels or gene analysis.|
|• to compare between both markers expression with levels ofCEA, TPA, SCC-Ag, CYFRA 21-1, ferritin, CA19-9, CA50,CA242, H-K-N-ras mutations and p53 mutation seemwhich were previously used as the most specific biomarkersin N-SCLC|