Identification of potential pharmacogenomic markers of clinical efficacy of 5-fluorouracil in colorectal cancer.

Although adjuvant chemotherapy has significantly increased overall survival in resected Stage III colorectal cancer, disease recurrence is still high (30-40%). 20-25% of Stage II patients also develop recurrent disease. Thus, high-risk patients may benefit from chemotherapy. As patient response to standard chemotherapy varies, the study of molecular differences in the expression of pharmacologically relevant genes may help clinicians to understand variability and tailor therapy. The expression of 5-fluorouracil (5-FU) pathway genes in tumors from 53 Stages II-III colorectal cancer patients who underwent 5-FU adjuvant chemotherapy was investigated by reverse transcription quantitative real-time polymerase chain reaction. Patients were dichotomized into high- and low-mRNA expression level groups using median values of gene mRNA levels. Then, a threshold analysis to identify a cut-off distinguishing recurrent- or nonrecurrent-disease was used. A high degree of interpatient variation in relative tumor expression of study genes was observed. Multiple gene correlations were found, which suggest possible coregulation mechanisms. No statistically significant relationship between experimental data and baseline clinical/pathological characteristics or clinical outcome was observed using gene expression median values. Threshold analysis indicated significant inverse relationships between deoxyuridine triphosphatase (DUT), ferrodoxin reductase (FDXR) or tumor protein p53 (TP53) and disease-free survival (DFS) in the entire case series and between DUT or NM23-H1 and DFS in Stage III patients: higher gene expression was associated with shorter DFS. This study provides data on relationships between expression of 5-FU pathway genes and clinical outcome of colorectal cancer patients undergoing 5-FU adjuvant chemotherapy and underscores the predictive role of specific genes. Validation in an independent case series is warranted.

[Benefits and limits of minimally invasive techniques in thyroid surgery]. / Vantaggi e limiti delle tecniche mini-invasive nella chirurgia della tiroide.

The present study was designed to investigate the potential benefits and limits of two minimally invasive thyroidectomy procedures, namely minimally invasive video-assisted thyroidectomy (MIVAT) and open minimal-incision thyroidectomy (MIT). From May 2000 to June 2006, a prospective, non-randomised study was performed on 957 consecutive patients undergoing thyroid surgery. Fifty-six (5.8%) underwent MIVAT, 214 (22.4%) MIT and 687 (71.8%) conventional thyroidectomy (CT). Patients were selected for MIVAT when total thyroid volume was < or = 30 mL and for MIT when total thyroid volume was > 30 but < or = 80 mL, as determined by ultrasonography. The length of the central neck skin incision was 1.5-2 cm for MIVAT, 2.5-3.5 cm for MIT and 6-10 cm for CT. The incidence of definitive hypoparathyroidism or recurrent laryngeal palsy after MIVAT or MIT was comparable to that occurring after CT. Patients undergoing MIVAT or MIT experienced significantly less postoperative pain than those undergoing CT. Less pain was also registered in the MIVAT patient cohort as compared to the MIT group. Patients undergoing MIVAT or MIT were more satisfied with the cosmetic result as compared to those undergoing CT, whereas no significant differences were found between the MIVAT and MIT groups. As compared to CT, MIVAT and MIT provided a significant improvement in terms of cosmetic results and postoperative pain. Nevertheless, the main limiting factor for minimally invasive thyroid surgery still remains the size of the thyroid.

DNA ploidy and S-phase fraction as prognostic factors in surgically resected gastric carcinoma: a 7-year prospective study.

BACKGROUND: DNA ploidy and S-phase fraction (SPF) measured by DNA flow cytometry (FC) have been previously shown to correlate with several clinicopathological variables in several types of tumours. PATIENTS AND METHODS: DNA FC was performed on multiple frozen tumour samples obtained from 115 patients undergoing curative surgery for gastric cancer (GC). The findings were prospectively tested for correlation with traditional clinicopathological indicators of prognosis. RESULTS: Overall, 20 tumours (17.4%) were diploid, 46 (40.0%) monoclonal and 49 (42.6%) multiclonal. Excluding 4 patients who died within 1 month of surgery, high SPF (>9.6%) was detected in 55 patients (49.6%) and was found to be significantly associated with vascular invasion and multiclonality (p=0.02). An association of borderline statistical significance emerged with macroscopic type (p=0.06) and pN and pM status (p=0.07). Multivariate regression analysis did not show a significant effect of SPF (p=0.11) or DNA ploidy (p=0.28) on 7-year survival. CONCLUSION: Aneuploidy appears to be a prognostic factor of low penetrance, whereas SPF is a more promising parameter of tumour aggressiveness in patients with GC.

Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells.

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E(2) and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 micromol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer.

Incidence and prognostic significance of occult tumor cells in lymph nodes from patients with stage IIA colorectal carcinoma.

Approximately 30% of patients with lymph node (LN)-negative colorectal carcinoma (CRC) die of tumor recurrence, which can be related to the presence of tumor cells in LNs not detected by conventional histopathologic analysis. However, the prognostic significance of occult cancer cells still remains uncertain. We evaluated the incidence and the prognostic significance of occult cancer cells in LNs from 395 consecutive patients with curatively resected stage IIA CRC using immunohistochemistry for cytokeratin 20. Immunostained tumor cells were categorized as micrometastases (MCMs) or isolated tumor cells (ITCs) according to the American Joint Committee on Cancer criteria. The detection rates were compared with the clinicopathologic characteristics of the patients and with cancer-specific survival. The median follow-up time was 128 months. Micrometastases were detected in 39 patients (9.9%), whereas ITCs were found in 112 (28.4%), for an overall frequency of 38.2%. None of the clinicopathologic parameters examined was correlated with the presence of occult cancer cells. Patients with ITCs and those with negative LNs showed a similar survival rate (77.7% and 78.3%, respectively), whereas patients with MCMs had a lower survival rate (64.1%). At the univariate analysis, MCMs, tumor growth pattern, extent of tumor spread, and Crohn's-like lymphoid reaction influenced the survival rate significantly. Nevertheless, at the multivariate analysis, only the pattern of tumor growth and the extent of tumor spread were independent prognostic factors. The detection of immunostained tumor cells in the LNs of patients with stage IIA CRC occurs relatively frequently but has no significant effect on prognosis.

The role of cyclooxygenase-2 in mediating the effects of histamine on cell proliferation and vascular endothelial growth factor production in colorectal cancer.

PURPOSE: Activity of histidine decarboxylase, the key enzyme in the synthesis of histamine, has been shown to be increased in several types of human tumors. We attempted to establish whether the possible involvement of histidine decarboxylase and histamine in colorectal carcinogenesis might be mediated by the activation of the cyclooxygenase-2 (COX-2) pathway. EXPERIMENTAL DESIGN: Expression/activity of histidine decarboxylase, histamine content, and prostaglandin E2 (PGE2) production were analyzed in 33 colorectal cancer samples and in the HT29, Caco-2, and HCT116 colon cancer cell lines. The effects of histamine, celecoxib, and H1, H2, and H4 receptor antagonists on COX-2 expression/activity, cell proliferation, and vascular endothelial growth factor (VEGF) production were assessed in the three colon cancer lines that showed different constitutive COX-2 expression. RESULTS: We showed the up-regulation of histidine decarboxylase protein expression and activity in the tumor specimens when compared with normal colonic mucosa. Histidine decarboxylase activity and histamine content were also significantly higher in metastatic tumors than in nonmetastatic ones. These variables significantly correlated with tumor PGE(2) production. The administration of histamine increased COX-2 expression/activity, cell proliferation, and VEGF production in the COX-2-positive HT29 and Caco-2 cells. Treatment with either H2/H4 receptor antagonists or celecoxib prevented these effects. Histamine had no effect on both the COX-2 pathway and VEGF production in the COX-2-negative HCT116 cells. CONCLUSIONS: Our data showed that histamine exerts both a proproliferative and a proangiogenic effect via H2/H4 receptor activation. These effects are likely to be mediated by increasing COX-2-related PGE2 production in COX-2-expressing colon cancer cells.

Relationships between promoter polymorphisms in the thymidylate synthase gene and mRNA levels in colorectal cancers.

Thymidylate synthase (TS) intratumoural expression may be a prognostic marker and predict outcome of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer patients. The TS gene promoter enhancer region contains two different polymorphisms which can influence TS mRNA transcriptional and translational efficiency: a polymorphic tandem repeat sequence (2 or 3 repeats; 2R and 3R) and a single nucleotide polymorphism (SNP), G > C, within the second repeat of the 3R alleles. We studied the relationship between tumoural TS mRNA expression levels and TS gene polymorphisms in the colonic mucosa of 48 colorectal cancer patients. The 3R/3R genotype was characterised by higher TS mRNA levels in the tumour than the 2R/2R-2R/3R genotypes (P = 0.071). Regarding the relationship with the SNP polymorphism, a statistically significant difference in TS gene expression between the 3RG/3RG genotype and 2R/2R-2R/3RC-2R/3RG genotype subset was observed (P = 0.017). No statistically significant correlation was observed between experimental data and baseline clinical-pathological characteristics as well as clinical outcome in the relatively small patient series investigated. This is the first study reporting an association between the TS intra-repeat SNP and gene expression levels in colorectal cancer patients. These results suggest that in 3R/3R patients, the G > C polymorphism may be an important factor in determining TS mRNA expression levels, and warrant further investigation of the role of TS promoter polymorphisms as predictors of sensitivity to 5-FU-based chemotherapy in larger case series.

Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.

PURPOSE: Up-regulation of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes has been reported in colorectal cancer. We aimed at evaluating the possible interaction between the nitric oxide and COX-2 pathways, and its effect on promoting tumor angiogenesis. EXPERIMENTAL DESIGN: Expression of iNOS, COX-2, vascular endothelial growth factor (VEGF), and CD31 was analyzed in tumor samples and corresponding normal mucosa obtained from 46 surgical specimens. We also evaluated iNOS activity, prostaglandin E(2) (PGE(2)), cyclic GMP and cyclic AMP production in the same specimens. Nitrite/nitrate levels, and PGE(2) and VEGF production were assessed in HCT116 and HT29 colon cancer cell lines after induction and selective inhibition of the two enzyme pathways. RESULTS: A significant correlation was found between iNOS and COX-2 immunohistochemical expression. PGE(2) production significantly correlated with iNOS activity and cGMP levels. A significant correlation was also found among PGE(2) production, microvessel density, and VEGF expression. Coinduction of both iNOS and COX-2 activities occurred after lipopolysaccharide (LPS) and epidermal growth factor (EGF) treatment in HCT116 and HT29 cells. Inhibition of iNOS by 1400W significantly reduced both LPS- and EGF-induced PGE(2) production. Treatment with LPS, EGF, and arachidonic acid significantly increased VEGF production in the iNOS-negative/COX-2-positive HT29 cells. This effect was completely reversed by treatment with the selective COX-2 inhibitor celecoxib. CONCLUSIONS: Our data showed a prominent role of nitric oxide in stimulating COX-2 activity in colorectal cancer. This interaction is likely to produce a cooperative effect in promoting angiogenesis through PGE(2)-mediated increase in VEGF production.

Quantitative evaluation of somatostatin receptor subtype 2 expression in sporadic colorectal tumor and in the corresponding normal mucosa.

PURPOSE: The somatostatin (SS) receptor subtype 2 (sst2) is the principal mediator of the antiproliferative effects of SS and has the highest affinity for the commercially available SS analogues. The purpose of this study was to evaluate sst2 mRNA expression by quantitative reverse transcription-PCR (RT-PCR) in colon cancers and in corresponding normal tissues. EXPERIMENTAL DESIGN: The expression of sst2 mRNA was measured with a quantitative method based on real time RT-PCR with TaqMan assay in 100 colon cancers and in the corresponding normal tissues. In a limited number of patients, these results were compared with those obtained by in situ hybridization (n = 26) and by in vivo imaging with (111)In-pentetreotide (n = 17). RESULTS: Results obtained by quantitative RT-PCR on sst2 expression in colorectal cancer were significantly related to those obtained by in situ hybridization and (111)In-pentetreotide scintigraphy. Sst2 was expressed in all of the tumors investigated without any relationship with localization, grading, and stage of disease. Although the paired, unaffected mucosa tends to express a higher abundance of sst2 than the corresponding cancer samples, this difference did not reach a statistical significance. However, in patients with elevated carcinoembryonic antigen levels (>5 ng/ml) there was a significant loss of sst2 mRNA in the tumor when compared with its paired normal tissue. CONCLUSIONS: In this study we confirmed, by a quantitative method, that colorectal cancer does not express higher concentrations of sst2 mRNA than the corresponding unaffected tissue. Conversely, a loss of sst2 was found in patients with elevated preoperative concentrations of carcinoembryonic antigen, an unfavorable prognostic marker for colorectal cancer.

Long-term results of Heller myotomy without an antireflux procedure in achalasic patients.

Both open and laparoscopic myotomies have been used in the treatment of achalasia. Postoperative gastro-oesophageal reflux is among the commonly reported side effects of myotomy. The addition of an antireflux procedure to the standard surgical approach has given rise to controversy. The objective of our study was to determine whether or not an antireflux procedure should be used in addition to Heller myotomy. Over the period from 1980 to 1990, 94 patients (mean age: 47.9 years) with achalasia underwent Heller myotomy calibrated by intraoperative oesophageal manometry without fundoplication. In 1999-2000, all patients filled in a clinical questionnaire: all underwent radiographic oesophageal imaging, oesophageal manometry, ambulatory 24-h oesophageal pH monitoring, and oesophagogastroduodenoscopy, when necessary. Ten healthy age-matched subjects were compared in the manometric and radiological studies. Myotomy improved the clinical profiles and instrumental data results in all patients. Gastro-oesophageal reflux was present in 10 patients (10.6%); none of these 10 subjects presented oesophagitis. Heller open myotomy yields good long-term results. Intraoperative manometric calibration reduces the side effects of myotomy, such as gastro-oesophageal reflux. The addition of fundoplication is not justified in all patients.

The ultrastructure of the muscle coat of human gastro-oesophageal junction, with special reference to "interstitial cells of Cajal".

The muscle coat of the human lower oesophageal sphincter and stomach was studied 5 cm above and 4 cm below the gastro-oesophageal junction. Four subjects were operated on for motility disorders of the esophagus, two for a hypertensive lower oesophageal sphincter and two for an epiphrenic diverticulum; six subjects were operated on for oesophageal or gastric carcinomas. Specimens were fixed in phosphate-buffered OsO4, embedded in Epon, contrasted with uranyl acetate and lead citrate and observed under a Siemens Elmiskop Ia electron microscope. Both the oesophageal and gastric muscle cells, which showed features typical of this cell type, were innervated by multiple varicosities that were rich in synaptic vesicles; these varicosities were generally rarely encountered at distances less than 1000 Å from muscle cells. Only a very few, close neuromuscular junctions were detected. Special cells, which correspond to the "interstitial cells of Cajal" as reported by other authors, were discerned at the periphery of muscle cell bundles. These cells were characterized by an elongated cell body with many thin branches and an oval, sometimes indented nucleus. Some pinocytotic vesicles were located at the cell periphery. These cells were surrounded by a discontinuous basal lamina and were seen in close contact with each other and with muscle cells; the close contact areas were often very wide. The cytoplasm contained variable amounts of mitochondria, a well-developed smooth endoplasmic reticulum and a Golgi complex. As a characteristic feature, bundles of thin filaments were located at the cell periphery and were attached to electron-dense areas of the cell membrane. Morphologically, these filaments resembled myofilaments; they were present in variable amounts and were sometimes very numerous. The observation that the cytoplasmic organelles and filaments varied in number, is probably related to the different functional properties of these cells. Interstitial cells were richly innervated by varicose nerve fibers that were densely packed with synaptic vesicles; many close junctions to nerve endings were also detected. These morphological data lead us to assume that the interstitial cells demonstrated by the electron microscope do not correspond to the cells initially identified by Cajal and cannot even be considered connective tissue cells. We propose that they are specialized smooth muscle cells that are involved in generating spontaneous, myogenic electrical activity in the gastrointestinal tract.

Adequacy of lymphadenectomy in laparoscopic colorectal cancer surgery: a single-centre, retrospective study.

PURPOSE: This study aimed at evaluating the lymph node (LN) harvest after both open and laparoscopic colorectal cancer surgery. METHODS: In the period between 1996 and 2009, 404 patients with colorectal cancer underwent open resection, whereas 147 patients underwent laparoscopic surgery. RESULTS: The overall number of harvested LNs was significantly higher in the laparoscopic group than in the open one (16.5 vs. 14.3, P<0.001). A higher number of LNs was found in moderately differentiated tumors of the laparoscopic group when compared with the open surgery group (16.7 vs. 14.2, P<0.01). The numbers of harvested LNs in the proximal tumors and in stage II and III tumors were higher in the laparoscopic group than in the open group (18.9 vs. 15.4, P<0.001; 17.9 vs. 14.2, P=0.002; 17.3 vs. 15.3, P=0.02, respectively). CONCLUSIONS: Laparoscopic surgery for colorectal cancer can achieve LN retrieval similar to that achieved by the open approach.

Clinical benefits of minimally invasive techniques in thyroid surgery.

BACKGROUND: Recently there has been a strong impetus to develop minimally invasive techniques in endocrine neck surgery. This study was designed to investigate the potential benefits of two minimally invasive thyroidectomy procedures, namely video-assisted and open minimal-incision thyroidectomy (VAT and MIT, respectively) when compared with conventional thyroidectomy. METHODS: Between May 2000 and June 2006, a prospective, nonrandomized study was performed on 957 consecutive patients undergoing thyroid surgery. Fifty-six (5.8%) patients underwent VAT, 214 (22.4%) underwent MIT, and 687 (71.8%) underwent a conventional procedure. RESULTS: Patients were selected for VAT when total thyroid volume was < or =30 ml and for MIT when total thyroid volume was >30 but < or =80 ml as determined by ultrasonography. The length of the central neck skin incision was 1.5-2 cm for VAT, 2.5-3.5 cm for MIT, and 6-10 cm for the conventional operation. The incidence of definitive hypoparathyroidism or recurrent laryngeal palsy after VAT or MIT was comparable with that occurring after conventional treatment. Patients having VAT or MIT experienced significantly less postoperative pain than patients undergoing conventional treatment. Less pain was also registered in the VAT patient cohort when compared with the MIT cohort. Patients having VAT or MIT were more satisfied with the cosmetic result than patients who underwent conventional treatment, but no significant differences in patient satisfaction were found between the VAT and MIT groups. CONCLUSIONS: When compared with conventional treatment, VAT and MIT provided significant benefit in terms of cosmetic results and postoperative pain. Nevertheless, the main limiting factor for minimally invasive thyroid surgery still remains the size of the thyroid.

Pathologic determinants of survival after resection of T3N0 (Stage IIA) colorectal cancer: proposal for a new prognostic model.

PURPOSE: There is an increasing need for accurate prognostic stratification of patients with Stage II colorectal cancer to identify a subgroup of high-risk patients who may benefit from adjuvant therapies. This study was designed to evaluate the prognostic impact of a wide spectrum of pathologic parameters in a consecutive series of homogenously treated and well-characterized patients with Stage IIA (T3N0M0) colorectal cancer. METHODS: The study included 238 patients operated on by a single surgeon for Stage IIA colorectal tumors. The median postoperative follow-up was 110 (range, 96-120) months. At least 12 lymph nodes were harvested and examined in all the resection specimens. The prognostic value of 13 pathologic parameters, including lymph node occult disease (micrometastases) detected by immunohistochemistry, was investigated. RESULTS: Multivariate analysis identified tumor growth pattern (expanding or infiltrating; P = 0.01) and extent of tumor spread beyond muscularis propria (< or =5 mm or >5 mm; P = 0.04) as the only factors having independent prognostic value. The combination of these two easily determined parameters allowed us to identify two groups of patients at low risk or high risk of tumor recurrence. The eight-year survival rates were 83.3 and 53.4 percent for the two groups, respectively. The high-risk group comprised those patients with infiltrating tumors and extramural tumor spread > 5 mm. CONCLUSIONS: We propose a new and simple prognostic model to identify patients with high-risk Stage IIA colorectal cancer for whom adjuvant therapies may be justified and effective.

Lymph node recovery from colorectal tumor specimens: recommendation for a minimum number of lymph nodes to be examined.

Lymph node involvement is the most important prognostic factor for patients who have undergone radical surgery for colorectal carcinoma. An accurate examination of the surgical specimens is mandatory for the correct assessment of the lymph node status of the tumor. The risk of understaging is particularly high for patients with tumors classified as Dukes B (TNM stage II). The aim of this study was to determine if a specified minimum number of lymph nodes examined per surgical specimen could have any effect on the prognosis of patients who had undergone radical surgery for Dukes B colorectal cancer. Between 1988 and 1995 a total of 140 patients underwent radical resection of Dukes B colorectal cancer by the same surgeon (C.C.). The relation between clinicopathologic variables and survival was estimated using the Kaplan-Meier method. The Cox proportional hazard regression model was used to identify the variables that can independently influence survival. A median of 12 lymph nodes (range 3-38) was examined per tumor specimen. The 5-year survival rate of Dukes B patients who had had eight or fewer lymph nodes examined after surgery was 54.9%, whereas the survival rate for those who had had nine or more lymph nodes examined was 79.9% (p < 0.001). Cox regression analysis identified the number of lymph nodes as the only independent prognostic factor (p = 0.01). Seventy patients with one to four metastatic lymph nodes (Dukes C patients) who had been operated on during the same period were included in the survival analysis for comparison. The 5-year survival rate of the Dukes B patients with eight or fewer lymph nodes examined was similar to that of the 70 Dukes C patients (54.9% and 51.8%, respectively). Examination of eight or fewer lymph nodes in Dukes B colorectal patients may be considered a high risk factor for missing positive lymph nodes in the surgical specimens. Our results suggest that harvesting and examining a minimum of nine lymph nodes per surgical specimen may be sufficient for reliable staging of lymph node-negative tumors.

Tumor angiogenesis in lymph node-negative rectal cancer: correlation with clinicopathological parameters and prognosis.

BACKGROUND: Intratumoral microvessel density (MVD) could be used as a prognostic factor in colorectal cancer. We retrospectively analyzed the value of microvessel count in predicting the clinical outcome of stage I and II (Dukes A and B) rectal cancer patients. METHODS: Eighty-four patients who had undergone curative resection of lymph node-negative rectal cancer were included. Tumor type and differentiation, the depth of local invasion, venous invasion, the character of the invasive margin, and the degree of lymphocytic infiltration were evaluated for each tumor specimen. Immunohistochemical staining for the CD31 endothelial antigen was performed to highlight the microvessels. RESULTS: The median value of MVD was 45 microvessels. Low MVD (microvessels < or = 45) was observed in 41 patients (48.8%), and high MVD (>45) was found in 43 (51.2%). The presence of conspicuous lymphocytic infiltration was significantly associated with increased vessel density. With uni- and multivariate survival analysis MVD did not show any prognostic significance. The character of the invasive margin was the only parameter with independent prognostic value. CONCLUSIONS: MVD does not seem to provide any additional prognostic information when compared with standard histopathological parameters in lymph node-negative rectal cancer. It is likely that the strong association between MVD and the presence of conspicuous lymphocytic infiltration may interfere with its predictive value.

Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis.

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.