Hans U. Graber

Nerve Growth Factor Expression Correlates With Perineural Invasion and Pain in Human Pancreatic Cancer

PURPOSE The reasons for the high frequency of perineural invasion and the presence of pain in pancreatic cancer are still not clear. Nerve growth factor (NGF) and its high-affinity receptor TrkA are involved in stimulating epithelial cancer cell growth and perineural invasion, as well as in pain generation in chronic benign disorders. PATIENTS AND METHODS NGF and TrkA were examined by Northern blot analysis, in situ hybridization, and immunohistochemistry in 27 normal and 37 pancreatic cancer tissue samples. The molecular findings were correlated with the degree of perineural invasion, pain, and histopathologic tumor characteristics. RESULTS Northern blot analysis indicated that NGF and TrkA mRNA levels were increased 2.7-fold and 5.6-fold, respectively (P <.05 and P <.05), in pancreatic cancer tissues compared with the normal pancreas tissue. As shown by in situ hybridization and immunohistochemistry, NGF was strongly present in the cytoplasm of pancreatic cancer cells. TrkA was intensely present in the perineurium of pancreatic nerves but not in the cancer cells. There was no difference in NGF and TrkA expression between early (stages I and II) and advanced (stage III) tumor stages and between well-/moderately differentiated (grades 1 and 2) and poorly differentiated (grade 3) tumors. However, tumors with high NGF/TrkA expression levels exhibited more frequent perineural invasion (P <.01). Furthermore, increased NGF/TrkA expression levels were associated with a higher degree of pain (P <. 01). CONCLUSION Enhanced expression of the NGF/TrkA system may influence perineural invasion and may contribute to the pain syndrome in human pancreatic cancer.

Nerve Growth Factor and Its High-Affinity Receptor in Chronic Pancreatitis

OBJECTIVE To study the mechanisms that are involved in nerve growth and contribute to pain generation in chronic pancreatitis (CP). SUMMARY BACKGROUND DATA Chronic pancreatitis is a painful disease associated with characteristic nerve changes, including an increase in nerve number and diameter. The mechanisms that influence nerve growth are not known. Nerve growth factor (NGF) and its high-affinity tyrosine kinase receptor A (TrkA) are involved in neural development and survival and growth of central and peripheral nerves. METHODS Nerve growth factor and TrkA were investigated by Northern blot analysis, in situ hybridization, and immunohistochemical staining in the pancreases of 24 patients with CP, and the findings were correlated with clinical parameters. RESULTS By Northern blot analysis, NGF and TrkA mRNA expression were increased in 42% (13.1-fold) and 54% (5.5-fold) of the CP samples (p < 0.01), respectively. In situ hybridization revealed that in CP, enhanced NGF mRNA expression was present in metaplastic ductal cells, in degenerating acinar cells, and in acinar cells dedifferentiating into tubular structures. TrkA mRNA was intensely present in the perineurium. Further, enhanced NGF and TrkA mRNA signals were also present in intrapancreatic ganglia cells in CP samples. Immunohistochemistry confirmed the in situ hybridization findings. Analysis of the molecular findings with clinical parameters revealed a significant relation (p < 0.05) between NGF mRNA levels and pancreatic fibrosis (r = 0.64) and acinar cell damage (r = 0.74) and between TrkA mRNA and pain intensity (r = 0.84). CONCLUSION Activation of the NGF/TrkA pathway occurs in CP. It might influence neural morphologic changes and the pain syndrome in this disorder.

bax , but not bcl-2 , influences the prognosis of human pancreatic cancer

Background—bcl-2and bax belong to thebcl-2-related gene family, which marks a new class of genes that influence apoptosis. Thebcl-2 oncogene acts as a broad antiapoptotic factor and extends both normal and tumour cell survival. In contrast, the bax gene is a promoter of apoptosis. Aims—To analyse the expression of bcl-2 andbax in pancreatic cancer and correlate the results with clinical parameters. Patients—Pancreatic cancer tissue samples were obtained from 28 female and 32 male patients (median age 63, range 43–79 years) having surgery for pancreatic cancer. Normal pancreatic tissues obtained from 18 previously healthy organ donors served as controls. Methods—The levels ofbcl-2 and baxmRNA expression were analysed by northern blot and the exact site of mRNA transcription was determined by in situ hybridisation. The presence of the corresponding proteins was determined by immunohistochemistry. Results—Northern blot analysis indicated that, in comparison with the normal pancreas,bcl-2 mRNA was overexpressed in 30% andbax mRNA in 61% of the pancreatic cancer samples. Concomitant overexpression ofbcl-2 and bax was present in 26% of the cancer samples. Pancreatic adenocarcinomas exhibited 3.7-fold and 5.4-fold increases (p<0.001) inbcl-2 and baxmRNA levels respectively. In situ hybridisation showed that bothbcl-2 and baxmRNA were expressed in the cancer cells. Immunohistochemical analysis showed positive Bcl-2 and Bax immunostaining in 28 and 83% of the cancer samples respectively. In multivariate analysis (Cox regression model), bax expression was found to be a strong indicator of survival (p<0.001). Patients whose tumours exhibited Bax immunostaining lived significantly longer (12 months) than those whose tumours were Bax negative (five months) (p<0.039). In contrast, no relation was found between Bcl-2 and survival time. Conclusions—The data indicate that genes that are involved in the regulation of apoptosis are upregulated in human pancreatic cancer cells. Prolonged survival times in patients in whom apoptosis promoting factors are upregulated indicate that apoptotic pathways are of biological significance in pancreatic cancer.

Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma.

BACKGROUND Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC). METHODS HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouins solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins. RESULTS All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues. CONCLUSION The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm.

Connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis

OBJECTIVE To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP). BACKGROUND Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis. PATIENTS AND METHODS CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples. RESULTS Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues. CONCLUSION The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP.

Over-expression of ICAM-1, VCAM-1 and ELAM-1 might influence tumor progression in colorectal cancer

Adhesion molecules might play a role in tumor progression. We investigated expression of the adhesion molecules ICAM‐1, VCAM‐1 and ELAM‐1 in 24 primary colorectal carcinomas using immuno‐histochemistry and Northern blot analysis. Normal colonic tissue from the same patients served as controls. ICAM‐1 immunostaining was restricted to the intercellular matrix and vascular endothelial cells. The vast majority of normal tissue samples revealed only faint ICAM‐1 immunoreactivity. However, moderate to strong immunostaining was found in 86% of cancerous sections. The ICAM‐1 immunoreaction was more intense in well‐differentiated carcinomas as well as in the adenomatous parts and transition zones of cancers. Similarly, the cancers exhibited markedly enhanced VCAM‐1 and ELAM‐1 immunostaining in the endothelial cells of small blood vessels. The intense vascular immunostaining by ICAM‐1 and VCAM‐1 was associated with a strong presence of CD3‐positive T lymphocytes, whereas ELAM‐1 immunoreactivity did not correlate with round cell infiltration. On Northern blot analysis, ICAM‐1, VCAM‐1 and ELAM‐1 mRNA levels were increased in 67%, 57% and 63% of carcinomas, respectively, in comparison with normal tissue samples. Densitometric analysis of Northern blots revealed an increase in ICAM‐1 by 2.1‐fold, an increase in VCAM‐1 by 3.4‐fold and an increase in ELAM‐1 by 2.2‐fold in cancerous tissues compared to normal controls. Over‐expression of ICAM‐1 might prevent cell–cell disruption and, hence, tumor dissemination. Furthermore, over‐expression of ICAM‐1 and VCAM‐1, but not ELAM‐1, might favor host anti‐tumor defense by trafficking of lymphocytes. Int. J. Cancer (Pred. Oncol.) 79:76–81, 1998.

Presence of two signaling TGF-β receptors in human pancreatic cancer correlates with advanced tumor stage

Transforming growth factor-β (TGF-β)signal transduction is mediated via specific cellsurface signaling TGF-β receptors, most notably thetype I ALK5 (TβR-IALK5)and the type II(TβR-II). We evaluated TβR-IALK5 andTβR-II expression in 41 human pancreatic cancertissue samples and correlated these findings withclinical data of the patients. Northern blot analysisindicated that, in comparison with the normal pancreas,pancreatic adenocarcinomas exhibited 8.0-fold and4.5-fold increases (P < 0.01), respectively, in mRNAlevels encoding TβR-IALK5 andTβR-II. In situ hybridization showed that both TβR-IALK5 mRNAwere highly expressed in the majority of pancreaticcancer cells. Immunohistochemical analysis ofTβR-IALK5 and TβR-II revealedpositive immunostaining in 73% and 56% of the tumors, respectively. Both receptorswere concomitantly present in 54% of the pancreaticcancer samples. The presence ofTβR-IALK5 or TβR-II and theconcomitant presence of TβR-IALK5 and TβR-II in the cancer cells was associatedwith advanced tumor stage (P < 0.01). These findingsshow that in many human pancreatic cancers, increasedlevels of the two signaling TβRs are present. The presence of the signaling TβRs inadvanced tumor stages indicates a role in diseaseprogression.

Reduced KAI1 expression in pancreatic cancer is associated with lymph node and distant metastases.

KAI1 belongs to a structurally distinct family of membrane glycoproteins, which function via cell‐cell and cell‐extracellular matrix interactions, thereby potentially influencing the ability of cancer cells to invade tissues and to metastasize into lymph nodes and distant organs. In the present study, we examined KAI1 expression in lymph node and liver metastases in comparison with primary pancreatic cancer to evaluate its influence on metastasis. KAI1 mRNA analysis was performed by Northern blot analysis and in situ hybridization. In addition, the respective protein was studied by immunostaining. Fourteen primary pancreatic cancer samples in which no lymph node metastases were present and 25 primary pancreatic cancer samples in which lymph node metastases were present at the time of tumor resection were included. In 20 of these cases, primary pancreatic cancer tissues and corresponding lymph node metastases from the same patient were studied. Furthermore, 11 liver metastases were available for KAI1 analysis. Increased steady‐state levels of KAI1 mRNA were found in 33/39 (85%) primary pancreatic cancers in comparison with normal controls. Statistical analysis of KAI1 mRNA levels and clinical parameters of the patients revealed that KAI1 mRNA levels were significantly higher in non‐metastasized tumors compared with tumors in which lymph node or distant metastases were present. In lymph node metastases KAI1 mRNA expression was lower than in the corresponding primary tumors: In 14 of 20 lymph node metastases no KAI1 mRNA expression and in 6 of 20 lymph node metastases only weak KAI1 mRNA levels were present in some cancer cells. Cancer cells of distant metastases were devoid of or exhibited low KAI1 mRNA levels compared with those of primary pancreatic cancers. A similar pattern was observed by immunostaining. These data support the hypothesis that KAI1 gene expression might influence the metastatic ability of pancreatic cancer cells in vivo. Reduction of KAI1 appears to promote cancer cell spread in lymph nodes and distant organs. Int. J. Cancer (Pred. Oncol.) 79:349–355, 1998.

Apoptosis Inhibiting Factor Bcl-xL Might Be the Crucial Member of the Bcl-2 Gene Family in Colorectal Cancer

Since the role of the Bcl-2 gene family has beenonly poorly investigated in colorectal cancer, we haveexamined the expression of the apoptosis blockersBcl-xL and Bcl-2, as well as the proapoptoticfactors Bax and Bak. Northern blot analysis andimmunohistochemistry were performed on normal andcancerous colonic tissue from 12 patients. In colorectalcancer, Bcl-xL immunoreaction was strongerthan in normal controls, and 83% of the cancers had increasedBcl-xL mRNA expression. The mediandensitometric Bcl-xL values were 3.4-foldhigher in carcinomas (P < 0.005). In contrast to thenormal colon, colorectal carcinomas often lack any Bcl-2 immunostaining,and Bcl-2 mRNA was not detectable by Northern blotseither. Bax was not obviously altered in colorectalcancer, either at the protein level or at the mRNA level compared to the normal control colon. BakmRNA expression exhibited a wide variation incarcinomas, but was somewhat decreased in comparison tothe controls. Of these members of the Bcl-2 gene family, Bcl-xL seems to play a major role incolorectal tumori genesis and disease progression. Anagonistic effect might have caused the tendency forreduced Bak expression. The Bcl-2/Bax regulation systemof cell homeostasis seems to be of lesserimportance.

ErbB-4 mRNA expression is decreased in non-metastatic pancreatic cancer

The erbB‐4 gene encodes a detected receptor protein that possesses intrinsic tyrosine kinase activity and belongs to the family of the epidermal growth factor receptor (EGFR); erbB‐4 is stimulated by the heregulins and betacellulin, which enables this receptor to form heterodimers with erbB‐2, a prerequisite for erbB‐2 activation. Because the expression of erbB‐4 mRNA is generally low in the pancreas, quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) was used to determine the erbB‐4 levels in human normal and cancerous pancreatic tissue. Our results show that the mRNA expression of this receptor is 6‐fold decreased in the non‐metastatic stages of pancreatic cancer when compared to tumors with lymph node or distant metastases or to the normal pancreas. In addition, immunohistochemistry demonstrated that in the normal pancreas, the erbB‐4 antigen was predominantly present in the cell membrane and cytoplasm of the ductal and acinar cells and at a much lower level, in islet cells. In pancreatic cancer, 61 of 75 samples exhibited weak to moderate immunoreactivity for erbB‐4 in the tumor cells. Moreover, in the peri‐tumorous region with chronic pancreatitis‐like morphological changes, there was weak‐to‐moderate erbB‐4 immunostaining in small ductules and degenerating acinar cells. Uni‐ and multivariate survival analyses using as variables age, sex, stage of cancer, histo‐pathological grading, and erbB‐4 immunoreactivity, revealed a significant effect for stage of cancer (p < 0.01) whereby the risk of dying was 2.3 times higher in patients with metastases than in patients without. However, the level of erbB‐4 immunoreactivity in pancreatic cancer cells had no influence on patient survival. Int. J. Cancer (Pred. Oncol.) 84:24–27, 1999.