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١ El Abd et al. Journal of the Medical Research Institute JMRI, 2006; Vol. 27 No.3: (204 -7) Prognostic Significance of Growth Hormone/Insulin Like Growth Factor-1/Insulin Axis in Colorectal Carcinoma (1) El-Abd E A, (2) EL-Kerm Y M, (3)Abdel Hammed A S, (4) El-Sewedy TS, (5)Assem N M, Kazem A, (4) El-Sewedy S M (6) (1) (5) Radiobiology unit, (2) Oncology unit, (3) Surgery department, (4) Applied Medical Chemistry department, Biochemistry department, (6) Pathology department, Medical Research Institute, Alexandria University, Egypt. Abstract Background Insulin, insulin like growth factor (IGF) system, and growth hormone (GH) play an important role in the development, proliferation, differentiation, survival, metabolism, and transformation. Aim of the work This study aimed to explore the relation between insulin, IGF-1, GH and the common prognostic factors in colorectal cancer. Subjects and methods The study included 47 subjects (30 colorectal cancer patients and 17 healthy volunteers). Serum insulin and GH were detected by immunoradiometric assay (IRMA) while, IGF-1 was detected using radioimmunoassay (RIA). Results Circulating IGF-1, insulin, and GH significantly increased in patients with colorectal (CR) neoplasms than controls. IGF-1 showed the highest diagnostic performance at cut off value of 116.5 ng/ml (area under the ROC curve = 0.993, p = 0.000). A significant decrease in GH was achieved by surgical intervention (p = 0.03). A stage dependent-significant increase was detected with IGF-1 (p = 0.02). IGF-1 significantly decreased (p = 0.02) with poorly differentiated tumors while GH significantly increased (p = 0.01). Insignificant negative correlation was observed between IGF-1 and each of GH and insulin reflecting failure of their feed back mechanism/resistance and/or immunomodulation status. Conclusion As the prognosis of patients is predominantly influenced by the anatomic extent of cancer and histologic grade, these results, provide evidence of the prognostic role of GH/IGF-1 axis in CR neoplasms. Keywords Colorectal neoplasms, IGF-1, GH, Insulin, prognosis, diagnostic performance Introduction T he colorectal cancer ranks the sixth of the male and the fifth of the female cancer sites in Egypt (1) with a mean age of 65 years.(2) Rectal carcinomas are detected in younger age than colonic carcinomas. (2) It is reported that both sexes are equally affected.(2) Colorectal cancer is responsible for about 3.9% of all cancer deaths in Egypt. (3) Dietary and genetic factors, metabolic syndrome, JMRI, 2006; Vol.27 No.3: (204 - 7) certain diseases of the colon, aging, physical inactivity, smoking, alcohol intake, night shift work are the main risk factors for colorectal cancer . (3) The insulin-like growth factor (IGF), family of ligandsbinding proteins and receptors, is an important system involved in regulation of a diverse array of biological functions both normal and pathologic including transformation. (4) Many studies have identified new signaling pathways originating from the IGF-I receptor that affect cancer cell proliferation, adhesion, migration and cell death. (4, 5) IGFs were suggested to increase colonocyte turnover and hence indicate poor prognosis in colorectal cancer.(6) IGF is also a key player in the signaling path that links insulin resistance to colon cancer. (7) Insulin resistance is characterized by compensatory hyperinsulinemia that affect the bioavailability of IGF-1 via its binding proteins. (7, 8) Currently, it has been shown that circulating insulin, at levels seen in insulin resistance, acutely increases proliferation of normal colorectal epithelial cells in vivo in a dose-dependent manner. (9) Moreover, extensive epidemiological data support a link between growth hormone (GH)/IGF-1 status and high risk for colorectal cancer.(10, 11) It is documented that IGF-1 is regulated by GH levels.(12) Experimental evidences implicate the involvement of the antiapoptotic environment created by GH/IGF-1 in maintaining a long-term survival of genetically damaged cells resulting in acceleration of colorectal carcinogenesis. (11) Altogether, this study was conducted to investigate the role of GH/IGF1/insulin axis in the prognosis of colorectal carcinoma (CRC). Materials and methods Subjects This study included 47 participants categorized as follow: 1. Thirty patients with CRC among ٢ El Abd et al. those admitted to the medical research institute and Dammanhour institute, Alexandria University, Egypt. Patients were recruited according to the ethical rules of Belmont report (13) and the following inclusion criteria: i. Age does not exceed 70 years ii. Pathologically proved adenocarcimomas of A-C Duke's stages iii. Surgically resectable non-metastatic tumors iv. No prior radiotherapy or chemotherapy 2. Seventeen healthy volunteers with no history of malignant diseases or any current clinical manifestations as control group. Pathological analysis Post-surgical specimens were sent for routine pathological assessment to determine the tumor type, grade, and stage. Treatment and follow up Treatment Surgical treatment 30 cases with colorectal neoplasms; as proved by preoperative sigmoidoscopy and biopsy were subjected to colectomy (24 cases). Abdominoperineal operation was performed in 6 cases. Adjuvant therapy Patients with CRC received adjuvant chemotherapy (Mayo Clinic regimen) according to the stage, while patients with rectal neoplasms received adjuvant radiotherapy. Follow up Patients were clinically followed up (twice per month) and subjected to: routine laboratory and radiographic investigations, and colonoscopy for suspected cases. Samples Blood samples were collected from healthy volunteers and patients (before surgery). Additional blood samples were collected after 14 days of surgical intervention and before starting any adjuvant treatment. Serum were separated and stored at -80ºC. Methods Serum GH and insulin were detected using ready-to-use IRMA (immunoradiometric assay) kits while serum IGF1 was detected using ready-to-use RIA (radio-immunoassay) kit (BioSource Europe S A, Belgium) according to the manufacturer instructions. Reference range varied for IGF-1 (ng/ml) according to age and gender. GH and insulin normal ranges are <0.2-10 µIU/ml and 4-16 µIU/ml; respectively. Statistical analysis Statistical analysis was performed using SPSS version 11.5. Paired "t" test was used to compare before with after readings, while unpaired "t" test was used to compare between the study group and control group. Alpha was set at < 0.05. Determination of the diagnostic performance of JMRI, 2006; Vol.27 No.3: (204 - 7) GH, insulin, and IGF-1 in CRC was done using the ROC curve. The cut off level for diagnosis was chosen as the point that maximizes the Youden index (Youden index = sensitivity + specificity -1). . Results The mean age of the patients and controls was 44.6 ± 12.3 and 45.3 ± 11.13 years (ranging from 23-64 and 25-62) with male to female ratio of 1:2 and 1:1; respectively. The pathological characteristics of patients with CRC are shown in table 1. A significant increase was detected in serum level of GH, IGF-1, and insulin in CRC (pre- and post-operative) compared to control group (table 2). A significant decrease in GH was achieved by surgery (table 2). Table (1): Pathological characteristics of 30 patients with CRC Parameter Histologic type Adenocarcimomas Mucinous adenocarcimomas Tumor grade II III Duke's stage B C Number (%) 24 (80) 6 (20) 13 (43.3) 17 (56.7) 19 (63.3) 11 (36.7) Table (2). Serology of the studied groups. Parameter Controls CRC Preoperative (n = 17) (n = 30) CRC Post-operative (before treatment) (n = 23) GH (µIU/ml) Range M ± SD 0-3 0.65 ± 0.02 1-40 9.4 ± 10.6^ 1-11 3.5 ± 3.0^+ IGF-1 (ng/ml) Range M ± SD 27-107 53 ± 19.4 32-157 77.7 ± 36.7^ 30.3-212 84 ± 52.2^ Insulin (µIU/ml) Range M ± SD 5.3-23.2 8.7 ± 4.6 6.5-37 13.3 ± 7.8^ 6.5-37 16.6 ± 9.4^ CRC: colorectal cancer ^ Significant when compared with control (unpaired t test) + Significant when compared with pre-operative (paired t test) The diagnostic performance of the three parameters was tested using the ROC curve. IGF-1 showed the highest diagnostic performance (table 3). IGF-1 significantly increased with higher tumor stage (table 4). GH significantly increased with higher tumor grade (poorly differentiated) while, IGF-1 significantly decreased (table 4). A negative correlation was observed between circulating IGF-1 and insulin, however it did not reach a statistical ٣ El Abd et al. level (r = - 0.481, p = 0.374). The same insignificant correlation was detected between IGF-1 and GH (- 0.481, p = 0.82). Table (3): Diagnostic performance of serology in CRC. Cut off Factor IGF-1 116.5 ng/ml GH 1.5 µIU/ml Insulin 15.55µIU/ml Area under the curve Sensitivity Specificity P value 0.993 100 85 0.000 0.88 93 70 0.002 0.86 90 79 0.003 Table (4): Relation between tumor-related factors, GH, and IGF-1 in CRC. GH IGF-1 M ± SD M ± SD 9.2 ± 12.5 9.6 ± 7.9 66.5 ± 42.6 91.5 ± 33.0* Prognostic factor Stage B C Grade II 2.7 ± 1.4 III 15.2 ± 11.8* * Significantly increased, P<0.05. + Significantly decreased, P<0.05. 98 ± 26.16 44 ± 30.48+ Discussion Although Peters et al.(14) thought that the IGF-1 expression is of limited value in CRC, the current study documents the prognostic value of GH/IGF-1 axis in CRC. Prognosis in CRC is influenced by histologic grade in most of the multivariate analyses (reviewed in 15). In our study, IGF-1 was significantly decreased with loss of differentiation (higher grade). Similar relation was detected between IGF-1 and poorly differentiated tumors in human breast cancer. (16) In contrast, a significant increase in growth hormone was observed with higher grade. Yang et al. (14) have shown a significant inverse correlation between GHR (growth hormone receptor; a member of the class I cytokine receptor family) expression and tumor differentiation (P = 0.036). Therefore, the higher GH level towards loss of differentiation parallels the increase in its receptor expression pattern indicating a role for GH in CRC physiology. In all multivariate analyses, the anatomic extent represents the most important prognostic factor after tumor resection for cure (reviewed in 15). Our results identified a significant increase in IGF-1 level in advanced stage (C). In a study of 713 CRC cases, IGF-1positive CRC reached statistical significance only in limited tumor stages (pT1/pT2; P<0.01) . (17) This may be JMRI, 2006; Vol.27 No.3: (204 - 7) explained by interobserver and technical variability [IHC (immunohistochemistry) versus RIA]. In addition, results of our study elucidated the possible diagnostic potential (as reflected by diagnostic performance tested by ROC curve analysis) of GH/IGF-1/ insulin axis in identifying patients with or more likely to develop CRC. Previous studies had contradictory results regarding the correlation between higher levels of IGF-I and increased risk of CRC (reviewed in 18). In a meta-analysis of 677 CRC patients and 1673 controls; there was a positive association between elevated levels of circulating IGF-I and CRC risk (OR: 1.58; 95% CI: 1.11–2.27). (19) However, on conducting multivariate meta-regression analysis, this positive association remained but was not statistically significant (p = 0.09). GH/IGF-1 axis presumed to play a role in neoplastic pathology. (20., 21) More persuasive evidence came from the association of polymorphic variation in the growth hormone gene (GH1) and risk of colorectal cancer, suggesting that lifetime exposure may be an important determinant of risk. (22) In addition, several studies have shown increased colon cancer in acromegaly (reviewed in 7). It is also evident that in response to growth factors such as IGF-I, insulin, and heregulin, HIF-1 (hypoxia inducible factor-1) protein synthesis is increased. (23, 24) HIF-1 activates more than 100 genes bridging the IGF-1 signaling pathways in cell cycle, angiogenesis, motility, invasion, drug resistance, growth, proliferation, apoptosis and survival. (25) The insulin resistance-colon cancer hypothesis was also supported by several studies (reviewed in 7). However, the results did not establish causality, but they have advanced this hypothesis to a level that justifies rigorous studies to provide proof of it. Modulation of GH by surgery might indicate that the tumor represents the major source of circulating GH and the optimum time to detect this modulation effect is 14 days after surgery. The absence of negative feedback mechanism (as indicated by insignificant negative relation) may indicate a hormone resistance status. During the past 20 years, it was clear that there is a rout of communication between protein hormones and immunity (reviewed in 26). This communication is activated when inflammatory processes induced by pro-inflammatory cytokines which antagonize the function of a variety of hormones in cancer cells, leading to endocrine resistance. In conclusion, the GH/IGF-1 axis has diagnostic and prognostic value in CRC. Moreover, it represents a new therapeutic target that should be considered. More detailed well-designed studies to validate relation between inflammations, immunity, hormone resistance and molecular changes during transformation are mandatory. El Abd et al. Acknowledgement We wish to thank Al-Hanash A and El-Tieb M for collecting samples, data and financing part of this work. References 1. 2. Statistical report for NCI 2004. El-Bolkainy T N, Sakr M A, Nouh A A, Ali El-Din N H. A Comparative Study of Rectal and Colonic Carcinoma: Demographic, Pathologic and TNM Staging Analysis. 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