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Significant correlation between serum basic fibroblast growth factor and tumor size in hepatocellular carcinoma
April 1995
• SIGNIFICANT CORRELATION BETWEEN SERUM BASIC FIBROBLAST GROWTH FACTOR AND TUMOR SIZE IN HEPATOCELLULAR CARCINOMA P.I. Hsu, K.H. Lai, N.H. Chow l, H.B. Yang l, G.H. Lo, JS. Cheng, R.L. Huang, C.F. Chang, S.M. Chen, C.K. Lin, M. Lin2, Y.F. Yang2, Divison of Gastroenterology, Department of Medicine, Department of Emergency Medicine 2, Veterans General Hospital-Kaohsiung, Department of Pathology 1, National Chang Kung University Hospital, Taiwan, R.OC. Aim: To evaluate the value of basic fibroblast growth factor (bFGF) as a diagnostic marker for hepatoceUular carcinoma (HCC) and to investigate the relationship between the serum level of bFGF and the various tumor parameters. Methods: Serum levels ofbFGF were measured in 39 patients with HCC, 16 with liver cirrhosis (LC), 22 with chronic hepatitis (CH) and 40 normal controls using an ELISA. Groups were compared by oneway ANOVA test. The relationship between serum levels ofbFGF and the clinicopathological parameters were analyzed by simple linear regression and Student's t-test. Results: Serum bFGF levels in the HCC patients (22.4+31.3 pg/ml) were significantly higher than in the patients with LC (7.9_+4. lpg/ml), CH (4.9+_2.6 pg/ml) and normal controls (5.1_+ 1.6 pg/ml). There was, however, no significant difference of serum bFGF levels among normal controls, CH and LC patients. In HCC patients, serum levels ofbFGF correlated well with tumor size (/9<0.05) and serum levels of ot-fetoprotein (p<0.05), alkaline phosphatase (p<0.01) and total bilirubin (p<0.01). There was no significant correlation between serum levels of bFGF and portal vein thrombosis, distant metastasis or serum ALT (p>0.05). Conclusions: 1. Serum levels of bFGF are significantly higher in the HCC patients as compared with the normal controls and the patients with CH and LC. 2. Serum bFGF levels significantly correlate with tumor size and serum tx-fetoprotein. These results suggest that HCC cells shed bFGF into the circulation and the determination of serum bFGF is potentially a useful supplement for diagnosis of HCC.
• E X P R E S S I O N OF M A T R I L Y S I N (MATRIX METALLOPROTEINASE-7) m R N A IS A SENSITIVE MARKER FOR LYMPH NODE M E T A S T A S E S IN COLORECTAL C A N C E R . Y lchikawa. T Ishikawa, T Chishima, N Momiyama,
H Yamaoka, A Tarnawski, IJSarfeh, MMitsuhashi, TAkitaya and H Shimada. Yokohama City University, University of California, frvine, Hitachi Chem. Res.Ctr., Irvine CA, Hitachi Chem.Co., Japan. Histopathological diagnosis of lymph node metastasis has technical limitations, because it would require numerous serial sections to examine the entire node, and carry the risk of false-negative result. Matrilysin is a matrix metalloproteinase detected in 80-90 % of colorectal cancers, but not in normal colonic mucosa. We evaluated matrilysin mRNA expression to determine whether it is useful as a marker of lymph node metastases originating from colorectal cancer. MATERIAL & METHODS; Fifteen colon cancer specimens were obtained during the operation. Five regional lymph nodes were collected from each patients. Half of each specimen was examined histologically and remaining half assessed by reverse transcription polymerase chain reaction (RT-PCR) for matrilysin mRNA expression. 1) RT-PCR mRNA assay; mP,NA captured on an oligo-d(T) immobilized plastic plate "GenePlate" (Nature 1992;357:519-520) was converted to cDNA and followed by PCR amplification with specific matrilysin and B-actin primers designed with the use of OligoProbe DesignStation described in our previous paper (Nature 1994;367.'759-761). PCR products were visualized with agarose gel electrophoresis. 2) Histology; formalin-fixed, paraffinembedded specimens were stained with Hematoxylin and Eosin and examined by 2 experienced pathologists. RESULTS; All samples including 15 primary lesions and 75 nodes showed l]-actin mRNA expression. Fourteen out of 15 primary lesions expressed matrilysin mRNA of predicted size of 420 bp. On histology, 12 positive nodes were found in 5 patients with colon cancers. The remaining 63 lymph nodes were histologically negative for metastatic cancers. However, 6 of these 63 lymph nodes were positive for matrilysin mRNA, confirming the presence of metastases. C O N C L U S I O N S ; 1) Although the prospective study is necessary, matrilysin mRNA appears to be a more sensitive marker of lymph node metastasis than the conventional histology in patients with matrilysinproducing colon cancers. 2) RT-PCR for matrilysin mRNA performed with the GenePlatc system is an easy and convenient tool for a precise assessment of lymph node metastasis which complements histological evaluation.
Gastrointestinal Oncology A483
• OPIOID GROWTH FACTOR MODULATES TUMORIGENESIS OF HUMAN COLON CARCINOMAS GROWN IN NUDE MICE. S.D. Hytrek, C.M. Lang, J.P. Smith, P.J. McLaughlin, I.S. Zagon. Depts. of Comparative Medicine, Medicine, and Neuroscience and Anatomy, The MS. Hershey Medical Center of The Pennsylvania State University, Hershey, PA. Endogenous opioids, particularly opioid growth factor (OGF) (i.e., [MetS]-enkephalin) and its receptor (i.e., zeta (~) opioid receptor) have been shown to be present and modulate the growth of a variety of tumors. To examine the effects of OGF on human colon cancer, male athymic nu/nu mice, 4 weeks of age, were injected subcutaneously over the right shoulder with lX10 ~ log phase HT-29 human colon cancer cells. Mice were randomly divided and injected daily with either 0.5, 5, or 25 mg/kg OGF or an equivalent volume of sterile water. Tumors were measured every three days using vernier calipers and tumor volumes were calculated from the two largest perpendicular dimensions. Latency until tumor appearance was significantly greater for mice treated with 0.5, 5,or 25 mg/kg OGF than for control mice. At the termination of the study (day 45) 100% of the control mice had tumors but 5 of 8 mice treated with 0.5 mg/kg OGF and 6 of 8 mice treated with 5 or 25 mg/kg OGF did not have tumors at this time. To determine if the effect of OGF on colon neoplasia was mediated by opioid receptors, some mice received 5 mg/kg OGF and 10 mg/kg naloxone (a short-acting opioid antagonist), or 10 mg/kg naloxone alone. Tumor incidence and latency in both groups of these mice were not significantly different from the controls, indicating that the inhibition occurs at the receptor level. To demonstrate the presence of OGF and ~" receptors in tumor tissue, immunocytochemical studies were performed. Specific immunoreactivity for OGF and the ~'receptor was detected associated with the cytoplasm, but not the nucleus, of adenocarcinoma cells. Specimens stained with preabsorbed antibodies or secondary antibody alone were negative. The importance of these findings to the etiology, pathogenesis, and treatment of this common cancer requires elucidation. Supported by the Laverty Foundation.
E X I S T E N C E O F PYY N E U R O N S IN T H E C A N I N E E N T E R I C NERVES SYSTEM AND ITS HISTOLOGICAL R E L A T I O N S H I P T O C H O L I N E R G I C N E U R O N S . K. Iesaki, *T. Sakai, M. Satoh, K. Tohhara, T. Yamada, Z. Itoh. GI Res. Lab., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi and *Dept. of Regulation Biology, Faculty of Science, Saitama University, Japan B a c k g r o u n d : Peptide YY (PYY) has been reported to exist in the endocrine cells of the distal gut, and to play an inhibitory role in gastrointestinal function. Previous in vivo studies have shown that exogenous PYY inhibits gastric motility and gastric emptying, and delays small intestinal transit time. Recent in vitro studies demonstrated that PYY inhibited cholinergic neurotransmission, and that PYY neurons exist in the enteric nerve system of sevral mammals. We report evidence for PYY as a neurotransmitter and its histological relationship to the cbolinergic neurons in the canine enteric nerves system. Method: An immunocytochemical study with antiserum to porcine PYY was performed in preparations from canine stomach, duodenum, jejunum, ileum and colon. To investigate the location of PYY and cholinergic neurons, double staining was carried out by with mouse anti-choline acetyltransferase antibody as a neuronal marker of cholinergic neurons. Reverse-phase HPLC elution patterns of PYY-like immunoreactivity from the mucosa and the muscle layers, including the myenteric plexus, were studied to examine the specificity of the anti PYY serum. The content of PYY-like immunoreactivity of muscle layers was determined by RIA with the same serum. Results: PYY immunoreactive nerve cell bodies and nerve fibers with varicosity were observed within the myenteric plexus, and a dense network of immunoreactive nerve fibers surrounding the non-immunoreative neurons Were also seen within the ganglia in the myenteric plexus. In the small intestine, PYY immunoreactive nerve fibers existed not only in the myenteric plexus but also in the deep muscular plexus and the submucosaI plexus. Double staining study revealed that there were no cholinergic nerve cell bodies in the dense network of PYY immunoreactive varicose nerve fibers. There was more PYY-like immunoreactivity in myenteric ganglia in the stomach and ileum than in other regions. Conclusion: It is suggested that PYY regulates gastrointestinal function indirectly by modulating enteric neurons other than cholinergic neurons as a neurotransmitter. The abundance of PYY-like immunoreactivity in the myenteric plexus may correlate with the site where PYY inhibits gut motiliy.
• SIGNIFICANT CORRELATION BETWEEN SERUM BASIC FIBROBLAST GROWTH FACTOR AND TUMOR SIZE IN HEPATOCELLULAR CARCINOMA P.I. Hsu, K.H. Lai, N.H. Chow l, H.B. Yang l, G.H. Lo, JS. Cheng, R.L. Huang, C.F. Chang, S.M. Chen, C.K. Lin, M. Lin2, Y.F. Yang2, Divison of Gastroenterology, Department of Medicine, Department of Emergency Medicine 2, Veterans General Hospital-Kaohsiung, Department of Pathology 1, National Chang Kung University Hospital, Taiwan, R.OC. Aim: To evaluate the value of basic fibroblast growth factor (bFGF) as a diagnostic marker for hepatoceUular carcinoma (HCC) and to investigate the relationship between the serum level of bFGF and the various tumor parameters. Methods: Serum levels ofbFGF were measured in 39 patients with HCC, 16 with liver cirrhosis (LC), 22 with chronic hepatitis (CH) and 40 normal controls using an ELISA. Groups were compared by oneway ANOVA test. The relationship between serum levels ofbFGF and the clinicopathological parameters were analyzed by simple linear regression and Student's t-test. Results: Serum bFGF levels in the HCC patients (22.4+31.3 pg/ml) were significantly higher than in the patients with LC (7.9_+4. lpg/ml), CH (4.9+_2.6 pg/ml) and normal controls (5.1_+ 1.6 pg/ml). There was, however, no significant difference of serum bFGF levels among normal controls, CH and LC patients. In HCC patients, serum levels ofbFGF correlated well with tumor size (/9<0.05) and serum levels of ot-fetoprotein (p<0.05), alkaline phosphatase (p<0.01) and total bilirubin (p<0.01). There was no significant correlation between serum levels of bFGF and portal vein thrombosis, distant metastasis or serum ALT (p>0.05). Conclusions: 1. Serum levels of bFGF are significantly higher in the HCC patients as compared with the normal controls and the patients with CH and LC. 2. Serum bFGF levels significantly correlate with tumor size and serum tx-fetoprotein. These results suggest that HCC cells shed bFGF into the circulation and the determination of serum bFGF is potentially a useful supplement for diagnosis of HCC.
• E X P R E S S I O N OF M A T R I L Y S I N (MATRIX METALLOPROTEINASE-7) m R N A IS A SENSITIVE MARKER FOR LYMPH NODE M E T A S T A S E S IN COLORECTAL C A N C E R . Y lchikawa. T Ishikawa, T Chishima, N Momiyama,
H Yamaoka, A Tarnawski, IJSarfeh, MMitsuhashi, TAkitaya and H Shimada. Yokohama City University, University of California, frvine, Hitachi Chem. Res.Ctr., Irvine CA, Hitachi Chem.Co., Japan. Histopathological diagnosis of lymph node metastasis has technical limitations, because it would require numerous serial sections to examine the entire node, and carry the risk of false-negative result. Matrilysin is a matrix metalloproteinase detected in 80-90 % of colorectal cancers, but not in normal colonic mucosa. We evaluated matrilysin mRNA expression to determine whether it is useful as a marker of lymph node metastases originating from colorectal cancer. MATERIAL & METHODS; Fifteen colon cancer specimens were obtained during the operation. Five regional lymph nodes were collected from each patients. Half of each specimen was examined histologically and remaining half assessed by reverse transcription polymerase chain reaction (RT-PCR) for matrilysin mRNA expression. 1) RT-PCR mRNA assay; mP,NA captured on an oligo-d(T) immobilized plastic plate "GenePlate" (Nature 1992;357:519-520) was converted to cDNA and followed by PCR amplification with specific matrilysin and B-actin primers designed with the use of OligoProbe DesignStation described in our previous paper (Nature 1994;367.'759-761). PCR products were visualized with agarose gel electrophoresis. 2) Histology; formalin-fixed, paraffinembedded specimens were stained with Hematoxylin and Eosin and examined by 2 experienced pathologists. RESULTS; All samples including 15 primary lesions and 75 nodes showed l]-actin mRNA expression. Fourteen out of 15 primary lesions expressed matrilysin mRNA of predicted size of 420 bp. On histology, 12 positive nodes were found in 5 patients with colon cancers. The remaining 63 lymph nodes were histologically negative for metastatic cancers. However, 6 of these 63 lymph nodes were positive for matrilysin mRNA, confirming the presence of metastases. C O N C L U S I O N S ; 1) Although the prospective study is necessary, matrilysin mRNA appears to be a more sensitive marker of lymph node metastasis than the conventional histology in patients with matrilysinproducing colon cancers. 2) RT-PCR for matrilysin mRNA performed with the GenePlatc system is an easy and convenient tool for a precise assessment of lymph node metastasis which complements histological evaluation.
Gastrointestinal Oncology A483
• OPIOID GROWTH FACTOR MODULATES TUMORIGENESIS OF HUMAN COLON CARCINOMAS GROWN IN NUDE MICE. S.D. Hytrek, C.M. Lang, J.P. Smith, P.J. McLaughlin, I.S. Zagon. Depts. of Comparative Medicine, Medicine, and Neuroscience and Anatomy, The MS. Hershey Medical Center of The Pennsylvania State University, Hershey, PA. Endogenous opioids, particularly opioid growth factor (OGF) (i.e., [MetS]-enkephalin) and its receptor (i.e., zeta (~) opioid receptor) have been shown to be present and modulate the growth of a variety of tumors. To examine the effects of OGF on human colon cancer, male athymic nu/nu mice, 4 weeks of age, were injected subcutaneously over the right shoulder with lX10 ~ log phase HT-29 human colon cancer cells. Mice were randomly divided and injected daily with either 0.5, 5, or 25 mg/kg OGF or an equivalent volume of sterile water. Tumors were measured every three days using vernier calipers and tumor volumes were calculated from the two largest perpendicular dimensions. Latency until tumor appearance was significantly greater for mice treated with 0.5, 5,or 25 mg/kg OGF than for control mice. At the termination of the study (day 45) 100% of the control mice had tumors but 5 of 8 mice treated with 0.5 mg/kg OGF and 6 of 8 mice treated with 5 or 25 mg/kg OGF did not have tumors at this time. To determine if the effect of OGF on colon neoplasia was mediated by opioid receptors, some mice received 5 mg/kg OGF and 10 mg/kg naloxone (a short-acting opioid antagonist), or 10 mg/kg naloxone alone. Tumor incidence and latency in both groups of these mice were not significantly different from the controls, indicating that the inhibition occurs at the receptor level. To demonstrate the presence of OGF and ~" receptors in tumor tissue, immunocytochemical studies were performed. Specific immunoreactivity for OGF and the ~'receptor was detected associated with the cytoplasm, but not the nucleus, of adenocarcinoma cells. Specimens stained with preabsorbed antibodies or secondary antibody alone were negative. The importance of these findings to the etiology, pathogenesis, and treatment of this common cancer requires elucidation. Supported by the Laverty Foundation.
E X I S T E N C E O F PYY N E U R O N S IN T H E C A N I N E E N T E R I C NERVES SYSTEM AND ITS HISTOLOGICAL R E L A T I O N S H I P T O C H O L I N E R G I C N E U R O N S . K. Iesaki, *T. Sakai, M. Satoh, K. Tohhara, T. Yamada, Z. Itoh. GI Res. Lab., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi and *Dept. of Regulation Biology, Faculty of Science, Saitama University, Japan B a c k g r o u n d : Peptide YY (PYY) has been reported to exist in the endocrine cells of the distal gut, and to play an inhibitory role in gastrointestinal function. Previous in vivo studies have shown that exogenous PYY inhibits gastric motility and gastric emptying, and delays small intestinal transit time. Recent in vitro studies demonstrated that PYY inhibited cholinergic neurotransmission, and that PYY neurons exist in the enteric nerve system of sevral mammals. We report evidence for PYY as a neurotransmitter and its histological relationship to the cbolinergic neurons in the canine enteric nerves system. Method: An immunocytochemical study with antiserum to porcine PYY was performed in preparations from canine stomach, duodenum, jejunum, ileum and colon. To investigate the location of PYY and cholinergic neurons, double staining was carried out by with mouse anti-choline acetyltransferase antibody as a neuronal marker of cholinergic neurons. Reverse-phase HPLC elution patterns of PYY-like immunoreactivity from the mucosa and the muscle layers, including the myenteric plexus, were studied to examine the specificity of the anti PYY serum. The content of PYY-like immunoreactivity of muscle layers was determined by RIA with the same serum. Results: PYY immunoreactive nerve cell bodies and nerve fibers with varicosity were observed within the myenteric plexus, and a dense network of immunoreactive nerve fibers surrounding the non-immunoreative neurons Were also seen within the ganglia in the myenteric plexus. In the small intestine, PYY immunoreactive nerve fibers existed not only in the myenteric plexus but also in the deep muscular plexus and the submucosaI plexus. Double staining study revealed that there were no cholinergic nerve cell bodies in the dense network of PYY immunoreactive varicose nerve fibers. There was more PYY-like immunoreactivity in myenteric ganglia in the stomach and ileum than in other regions. Conclusion: It is suggested that PYY regulates gastrointestinal function indirectly by modulating enteric neurons other than cholinergic neurons as a neurotransmitter. The abundance of PYY-like immunoreactivity in the myenteric plexus may correlate with the site where PYY inhibits gut motiliy.