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Purpose: Some 10% of familial adenomatous polyposis (FAP) and 80% of attenuated polyposis (AFAP) cases remain molecularly unexplained. We scrutinized such cases by exome-wide and targeted methods to search for novel susceptibility genes. Methods: Exome sequencing was conducted on 40 unexplained (mainly sporadic) cases with FAP or AFAP from Finland. The DNA mismatch repair (MMR) gene MLH3 (MutL Homolog 3) was pinpointed and prompted a subsequent screen of similar to 1000 Swedish patients referred to clinical panel sequencing for colon tumor susceptibility. Results: Three homozygous carriers of a truncating variant in MLH3, c.3563C>G, p.Ser1188Ter, were identified among the index cases from the Finnish series. An additional biallelic carrier of the same variant was present in the Swedish series. All four patients shared a 0.8-Mb core haplotype around MLH3, suggesting a founder variant. Colorectal polyps from variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats, in agreement with previous findings of a minor role of MLH3 in MMR. Multiple loci were affected by loss of heterozygosity, suggesting chromosomal instability. Conclusion: Our results show that a biallelic nonsense variant of MLH3 underlies a novel syndrome with susceptibility to classical or attenuated adenomatous polyposis and possibly extracolonic tumors, including breast cancer.

Hydroxamic acid derivatives constitute an interesting novel class of antitumor agents. Three of them, including vorinostat, are approved drugs for the treatment of malignancies, while several others are currently under clinical trials. In this work, we present new vorinostat analogs containing the benzoxazole ring as the cap group and various linkers. The benzoxazole-based analogs were synthesized starting either from 2-aminobenzoxazole, through conventional coupling, or from benzoxazole, through a metal-free oxidative amination. All the synthesized compounds were evaluated for their antiproliferative activity on three diverse human cancer cell lines (A549, Caco-2 and SF268), in comparison to vorinostat. Compound 12 (GK601), carrying a benzoxazole ring replacement for the phenyl ring of vorinostat, was the most potent inhibitor of the growth of three cell lines (IC50 1.2-2.1 μΜ), similar in potency to vorinostat. Compound 12 also inhibited human HDAC1, HDAC2 and HDAC6 like vorinostat. This new analog also showed antiproliferative activity against two colon cancer cell lines genetically resembling pseudomyxoma peritonei (PMP), namely HCT116 GNAS R201C/+ and LS174T (IC50 0.6 and 1.4 μΜ, respectively) with potency comparable to vorinostat (IC50 1.1 and 2.1 μΜ, respectively).

A 19-year-old man noticed blurred vision in his right eye. He had an intraocular pressure of 60 versus 12 mmHg in the fellow eye. He was initially diagnosed with an atypical, advanced pigmentary glaucoma. The intraocular pressure did not respond to maximal medication, deep sclerectomy, goniopuncture, and 2 cyclophotocoagulations. Sixteen months after presentation, malignancy was first suspected, and the eye was enucleated. A ring melanoma of the anterior chamber angle was confirmed by the histopathologic examination. Normal nuclear staining for breast cancer 1 gene (BRCA1)-associated protein 1 suggested that the tumor was likely of disomy 3 type with a favorable prognosis. No local or systemic recurrence has developed within 4 years. A literature review of this rare type of minimal volume diffuse uveal melanoma identified 18 additional patients. The initial diagnosis in 18 of the 19 patients with a ring melanoma of the anterior chamber angle was unilateral glaucoma with a median intraocular pressure of 40 mmHg and an age range of 16–76 years. Liver metastasis developed in 5 of 12 patients older than 45 years. This rare subtype is estimated to account for 0.05%–0.16% of all uveal melanomas.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.

Introduction Pseudomyxoma peritonei (PMP) is a fatal clinical syndrome, where most often appendix-originating mucinous adenocarcinoma cells grow in the peritoneal cavity filling it with mucinous ascites. KRAS mutations are found in nearly all PMPs and GNAS mutations in over half of the cases, but other common driver mutations are infrequent. As glycosylation alterations can also contribute to carcinogenesis, our aim was to compare the N-linked glycan profiles of PMP tissue specimens to those of normal appendices. Material and methods N-glycans were detached from formalin-fixed, paraffin-embedded tissue specimens of 8 normal appendices and 8 low-grade and 8 high-grade PMPs, and analysed by mass spectrometry. The expression levels of glycosylation-related genes were further analysed from PMP microarray data, and the expression of fucosylation-related enzymes and fucosylated glycans was localised by immunohistochemistry and lectin histochemistry. Finally, cell culture experiments were used to study the relationship between fucosylation and mucin expression. Results and discussions The N-glycan profiles, especially those of neutral glycans, clearly differed between PMPs and normal control samples. The most prominent alteration in PMP was highly increased fucosylation and multifucosylation further showed correlation with the proportion of high-grade morphology in the sample. From microarray data we could demonstrate upregulated mRNA expression of four fucosylation-related enzymes (FUT8, GMDS, GMPPA, and TSTA3) in PMP specimens, and by immunohistochemistry we localised these enzymes into PMP cells. Finally, by using colon carcinoma cell line HCT116 with inherently defective fucosylation, we demonstrated that restoration of fucosylation enhanced GNAS mutation-induced upregulation of MUC2 expression. Conclusion PMPs show highly increased N-glycan fucosylation as compared to normal appendices, and this glycosylation alteration may be linked to the characteristic mucin overexpression of the disease. Most importantly, multifucosylation correlates with the proportion of high-grade morphology in the specimen and this glycan class may thus provide structures bearing prognostic potential.

Cyclooxygenase-2 (COX-2) expression is a marker of poor prognosis in gastric cancer patients, and its inhibition suppresses gastric tumorigenesis in experimental animal models. The mechanism that leads to COX-2 overexpression in this tumor type is unknown. We have now shown that inhibition of phosphatidylinositol 3-kinase by LY294002 suppresses both basal and phorbol myristate acetate-induced COX-2 expression in TMK-1 and MKN-28 gastric cancer cells. Furthermore, inhibition of glycogen synthase kinase-3beta (GSK-3beta) by SB415286 induced expression of COX-2 mRNA and protein as well as the enzyme activity in the gastric cancer cells. The effect of SB415286 was confirmed by the use of two additional GSK-3beta inhibitors, lithium chloride and SB216763. SB415286 had a modest 1.6-fold stimulatory effect on a 2-kb COX-2 promoter reporter construct, but more importantly, it was shown to block the decay of COX-2 mRNA. In contrast to modulation of phosphatidylinositol 3-kinase/Akt/GSK-3beta pathway, inhibitors of mitogen-activated protein kinases (MEK 1/2, p38, JNK) or the mammalian target of rapamycin did not alter COX-2 expression in gastric cancer cells. Our data show that inhibition of GSK-3beta stimulates COX-2 expression in gastric cancer cells, which seems to be primarily facilitated via an increase in mRNA stability and to a lesser extent through enhanced transcription.

Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (

Cyclooxygenase-2 (COX-2) expression has been found to associate with poor prognosis in several types of carcinomas. HuR is an mRNA stability protein and it regulates the expression of COX-2. Objectives and methods. We analyzed the expression of COX-2 and HuR in 64 mucinous ovarian carcinoma specimens by immunohistochemistry. Results. In mucinous tumors, high COX-2 protein expression was found in epithelial cancer cells in 39% (22/56) and in stromal cells in 24% (13/55) of the specimens. The expression of COX-2 in cancer cells correlated with high grade ( P = 0.0285), but stromal COX-2 expression had no correlation with any clinical parameter tested. Cytoplasmic HuR protein expression was observed in cancer cells in 47% (27/57) and in stromal cells in 7% (4/56) of the mucinous tumors, and it correlated with COX-2 expression in the cancer cells ( P = 0.0162) but not in the stroma. Conclusion. Our results support the hypothesis that cytoplasmic HuR is connected to COX-2 expression in ovarian carcinoma, but that its role is restricted to the transformed epithelial cancer cells.

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.

Background & Aims: Use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of cancer in the digestive tract. Cyclooxygenase (COX) is the best-known target of NSAIDs, and expression of the COX-2 isoform is elevated in esophageal carcinomas but its clinical significance remains unclear. We examined COX-2 expression in esophageal adenocarcinomas and its relation to clinicopathologic parameters. Methods: Tumor sections from 145 consecutive patients undergoing intentionally curative surgery for an adenocarcinoma arising from a Barrett's esophagus were immunohistochemically stained using a COX-2–specific anti-human monoclonal antibody. The specimens were scored based on the intensity and extent of COX-2 immunopositivity. Results: COX-2 immunoreactivity was negative to weak in 21% (COX-2 low) and moderate to strong in 79% (COX-2 high) of the carcinomas. Patients with high COX-2 expression were more likely to develop distant metastases ( P = 0.02) and local recurrences ( P = 0.05), and survival was significantly reduced ( P = 0.002, log-rank test) among patients with high COX-2 expression when compared with the COX-2 low group. Five-year survival rates were 35% (95% confidence interval [CI], 23–47) and 72% (95% CI, 53–90) in COX-2 high and COX-2 low categories, respectively. Furthermore, expression of COX-2 was recognized as an independent prognostic factor by multivariate analysis (relative risk, 3.5; 95% CI, 1.6–7.9). Conclusions: Elevated expression of COX-2 protein is associated with significantly reduced survival of patients undergoing surgery for esophageal adenocarcinoma. These findings support the effort to initiate clinical studies to investigate the effect of COX-2 inhibitors as a novel (adjuvant) chemotherapeutic modality for the treatment of adenocarcinoma arising from Barrett's esophagus. GASTROENTEROLOGY 2002;122:1800-1807

To the editor: Because of the lack of curative treatment options for most advanced cancers, innovative and experimental strategies are being developed. One promising therapeutic approach is the delivery of human sodium iodide symporter (hNIS) with oncolytic viruses.1,2,3,4,5 hNIS provides the dual utility of therapy and imaging of virus distribution with radionuclides such as iodides or technetium. Although therapy is the ultimate goal, imaging of the virus is also critical because all available biodistribution information concerning oncolytic viruses is currently based on animal models, which may not be fully representative for species-specific viruses such as human adenovirus. We and others have published preclinical data on the use of radionuclide transporters as transgenes,2,3,4,5,6,7 and hNIS has thus far been the most popular approach. In general, two approaches have been used to arm oncolytic viruses with human NIS: replication-coupled expression and replication-independent expression. The former is useful for detection of virus replication but may not reveal overall virus biodistribution, and the opposite is true for the latter. Utilizing replication-independent human NIS expression, Barton et al. recently published an elegant first-in-humans phase I trial.1 Nuclear imaging showed that seven of nine (78%) patients treated intratumorally with the higher viral doses and six of six (100%) treated with the highest dose accumulated technetium-99m (99mTc). These data, together with promising preclinical data on Ad5/3-24-hNIS (ref. 4) and previous clinical experience with different oncolytic adenoviruses,8,9,10,11 led us to treat one patient in a Finnish Medicines Agencyapproved Advanced Therapy Access Program, which allows personalized patient-by-patient therapy under the “hospital exemption.”12 The patient was a 50-year-old woman with chemotherapy-refractory cervical carcinoma metastatic to the lungs, lesser pelvis, pelvic lymph nodes, liver, and bones. She had previously been treated with cisplatin, cisplatin with topotecan, paclitaxel and gemcitabine, and radiotherapy. The patient received a total of 3 × 1011 viral particles into the clinically most relevant tumors—as evaluated by positron emission tomography–computed tomography (PET-CT) imaging on the same day—in the pelvis and liver. Twenty hours after treatment with virus, the patient received an injection of iodine-123 (123I), a low-energy γ-emitter with excellent imaging properties that is frequently used for thyroid and brain imaging. A series of scans was performed: a 30-minute dynamic-imaging series at injection time (0 hour), whole-body single-photon emission-computed tomography (SPECT)-CT scans at 4 hours, 8 hours, and 24 hours after 123I injection. These scans did not reveal any accumulation of iodine into the injected regions over baseline readings (Figure 1a). Therefore, the imaging was repeated on the third day (68 hours after virus injection), this time with 99mTcO4 (to exclude iodide-specific issues), resulting in the same negative result. To exclude the possibility of protracted amplification of signal, one more round of 123I imaging was performed on day 6 post virus. Careful examination still did not reveal any signal enhancement other than that resulting from endogenous hNIS expression in the thyroid, stomach, and salivary glands. Urine was visualized due to renal excretion of iodide. Figure 1 SPEC-CT imaging of Ad5/3-24-hNIS. (a) A whole-body scan (Siemens Symbia T2, Siemens Medical Solutions, Erlangen, Germany) showing the distribution of 123I 4 hours after 123I injection and 25 hours after intratumoral Ad5/3-24–human sodium iodide ... Blood samples were taken on days 0, 1, 2, and 6 and analyzed for adenovirus copy number with quantitative PCR.9 Before treatment, no virus was detected. However, on days 1 and 2, less than 500 virus particles per milliliter (vp/ml) and 825 vp/ml were measured, respectively, suggesting that the highest peak of virus replication occurred exactly when imaging was performed (Figure 1b). On day 6, the value had declined to 0 vp/ml. The patient reported grade 1 lower-limb edema and grade 2 dyspnea and anorexia after the treatment. No changes in laboratory values were observed. The neutralizing-antibody titer measured before treatment was 1:256 and thus only moderately elevated,8,9,10,11 and it rose to 1:16,384 a week after treatment. A pretreatment biopsy contained 60% poorly differentiated carcinoma, 35% fibrotic stroma, and 5% necrotic tissue (Figure 1c). Consequently, neither antibodies nor high stromal content seem likely reasons for low hNIS expression. Our findings suggest that constructs optimized for oncolysis and featuring replication-coupled transgene expression, such as Ad5/3-24-hNIS, might not be optimal for detection of hNIS expression in humans. By comparing differences in virus design and treatment protocol with Barton et al.,1,5 we believe that important vectorological lessons can be learned. Differences between treatment protocol, dose, and patient type may nevertheless also contribute to the findings. One difference between our construct and Ad5-yCD/mutTKSR39rep-hNIS is the promoter driving hNIS expression. Ad5/3-24-hNIS expresses hNIS from the native E3 promoter, which is activated by virus replication at about 8 hours,4 whereas in Ad5-yCD/mutTKSR39rep-hNIS the transgene is under the ubiquitously active cytomegalovirus (CMV) promoter, which allows for immediate high-level expression of transgene in normal and tumor cells. This might result in a stronger signal when the injected lesion contains both cell types, which may be relevant in that the stromal component of tumors can vary between 5 and 95%. Moreover, the capacity of Ad5-yCD/mutTKSR39rep-hNIS for expressing hNIS in normal tissues was demonstrated in dog prostates.2 Dogs are not permissive for human adenoviruses,13 and the dogs in the study did not have known prostate cancer. Also, the high early activity mediated by CMV, versus slower replication-coupled expression, might be useful in a situation where the cells have limited time to produce hNIS and concentrate radiotracers, before the cell is lysed. In this regard, the high oncolytic potency of Ad5/3-24-hNIS (ref. 4) might work against imaging. Furthermore, Ad5-yCD/mutTKSR39rep-hNIS is based on dl1520, an adenoviral construct that has been safely used in many clinical trials.14 However, some studies have suggested that the productive replication of dl1520 might be attenuated even in permissive cells.15,16 Moreover, Ad5-yCD/mutTKSR39rep-hNIS represents a complex construct with three transgenes and three CMV promoters that together might further attenuate the replication, allowing for prolonged and enhanced transgene expression. Also, the trial patients received 5-fluorocytosine and valganciclovir prodrug therapy for 3 weeks, which might further hamper viral replication (at least in vitro), thus giving time for CMV-driven hNIS expression. However, it should be noted that the first images were captured before prodrug therapy.16,17 The patients also received radiation therapy, which might influence virus replication and transgene expression. The administration technique might also contribute to the findings. Our patient received Ad5/3-24-hNIS into a bulky pelvis tumor through three needle tracts and into a liver metastasis through one needle tract, as opposed to subjects in Barton and colleagues' study2 who received the whole dose along one needle tract, resulting in a higher local virus concentration that might have facilitated imaging. In summary, we believe our experience with Ad5/3-24-hNIS in comparison with the report on Ad5-yCD/mutTKSR39rep-hNIS reveals several important aspects in virus design. First, low oncolytic potency may be useful for expression of transgene products located on the cell surface. Second, slower virus replication (e.g., in combination with replication-attenuating prodrugs) may allow for better imaging of membrane-associated proteins. In addition, high levels of early transgene expression may be preferable over protracted replication-coupled transgene expression. Also, tumor-selective transgene expression may lead to less robust imaging results than expression in all transduced cells. Finally, the sensitivity of SPECT may require relatively high levels of transgene expression. Thus, Ad5-yCD/mutTKSR39rep-hNIS may represent a useful agent for combining oncolytic virotherapy with prodrug-converting enzymes, imaging, and radionuclide therapy, whereas Ad5/3-24-hNIS type viruses are more suitable for optimal oncolysis and expressing transgenes with systemic or paracrine rather than cell membranerestricted effects.9 Therefore, we are currently not planning further treatments with Ad5/3-24-hNIS.

The cyclooxygenase (Cox) enzyme catalyzes the rate-limiting oxidative and peroxidative enzymatic steps in the biosynthesis of prostanoids. Both Cox-1 and -2 genes encode the two isoenzymes that carry out similar enzymatic steps. Enhanced Cox activity is associated with proliferative diseases such as colon cancer. To determine if a cause and effect relationship exists between Cox isoenzyme overexpression and tumorigenesis, the human Cox-1 and Cox-2 isoenzymes were transfected into ECV immortalized endothelial cells. Although numerous clones of Cox-1 expressing cells were obtained, Cox-2 overexpression resulted in growth disadvantage and increased cell death. In contrast, Cox-1 overexpressing cells expressed high levels of the functional Cox-1 polypeptide in the endoplasmic reticulum and the nucleus. In vitro proliferation of these cells was reduced compared with vector-transfected ECV cells. Cox-1 overexpression also enhanced the tumor necrosis factor-α-induced apoptosis of ECV cells 2-fold. In contrast to the in vitrobehavior, ECV-Cox-1 cells proliferated aggressively and formed tumors in athymic “nude” mice, whereas the vector-transfected counterparts did not. The growth of Cox-1-induced tumors was not inhibited by indomethacin, suggesting a nonprostanoid function of Cox-1. ECV-Cox-1-derived tumors were angiosarcoma-like and contained numerous host-derived neovessels. These data suggest that Cox-1 overexpression in immortalized ECV endothelial cells results in nuclear localization of the polypeptide and tumorigenesis.

The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.

Prostacyclin (PGI2), a powerful vasodilatory prostanoid, and endothelin-1 (ET-1), a potent vasoconstrictive peptide, are produced by vascular endothelial cells. We show that human serum (10%) caused a 3.2-fold stimulation both in PGI2 and ET-1 synthesis in human endothelial cells cultured from umbilical veins, and human plasma (10%) stimulated productions of both 1.6- and 1.7-fold, respectively. In addition, releasates from thrombin-activated platelets (20 x 10(9) platelets/l) caused a 1.9-fold increase in PGI2 and a 1.4-fold increase in the ET-1 synthesis. Releasates from frozen-thawed and sonicated platelets (20 x 10(9) platelets/l) caused a 3.6-fold increase in PGI2 release but did not affect ET-1 production. We thus conclude that, in normal situation, endothelial stimulating activity present in plasma perhaps plays a role in the regulation of endothelial function, whereas platelet-derived activity in serum may be important at site of thrombosis.

Summary Atrial natriuretic peptide (ANP) and the nitrovasodilator drugs nitroglycerine and nitroprusside were shown here to decrease both basal and thrombin stimulated production of endothelin-1 (ET-1) from cultured human endothelial cells as measured by radioimmunoassay. 8-Bromo-3′,5′-cyclic guanosine monophosphate (cGMP) and papaverine also inhibited ET-1 production. The inhibitory effect of ANP and nitrovasodilators on ET-1 production thus appears to be mediated by guanylate cyclase and cGMP. Part of the vasodilatory action of ANP, nitroprusside and nitroglycerine may be due to suppression of endothelial ET-1 production. This may be an additional mechanism whereby nitrovasodilators participate in the regulation of vascular tone.

Introduction In Finland colorectal cancer is the second most common cancer among women and the third most common cancer in men. Five-year survival rates are 61% and 60% for women and men, respectively. Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) is the most prevalent hereditary colorectal cancer consisting of 3-5% of all colorectal cancer cases. Lynch syndrome is an autosomal dominant disorder caused by mutations in the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) or germline deletion in epithelial cell adhesion molecule, which leads to MSH2 silencing in approximately 1% of the cases. MMR genes form DNA mismatch repair system responsible for correcting errors occurring during DNA replication. Failure of MMR genes to do this inflicts genomic instability and can be detected in microsatellites, short tandem repeats in the DNA, as high microsatellite instability (MSI-H). Errors in the DNA sequence can lead to nonsense and frameshift mutations, deficiency of target gene functions, and eventually, improved tumorigenesis. In 10 to 15% of sporadic colorectal cancers the disease is caused by nonfunctional MMR gene, and more accurately, approximately 90% of the cases is a consequence of MLH1 gene promoter hypermethylation leading to gene silencing and MSI-H. Recent studies suggest a favorable prognosis for patients with MSI-H (0.65% hazard ratio for overall survival, 95% confidence interval 0.59-0.71). Moreover, several studies suggest that MSI-H expressing colorectal cancer patients do not benefit from adjuvant chemotherapy, which raises the question whether or not these patients should be treated with harsh adjuvant therapies at all. However, since the results are not comprehensive no patients can be left out from the common treatment practice. The significance of this project is 1) to better understand the nature of both diseases and their association with clinicopathological parameters and survival, 2) to be able to separate these diseases from each other accurate, simple and fast way, 3) to inform the clinician of bad prognosis disease (KRAS, BRAF, MGTG alteration; MSS) and the applicability of anti-EGFR treatments (KRAS/BRAF wild type), and 4) elucidate the efficiency and significance of cancer treatments. Methods The study will be based on colorectal cancer patient materials from the Helsinki University Central Hospital, and MSI status of the tissue samples, MMR proteins' expression levels, mutation and/or promoter methylation status of MMRs, and oncogenes like KRAS, BRAF and MGTG DNA alterations will be investigated. Currently, BRAF V600E mutation status has been analyzed from the sporadic colorectal cancer specimens by immunohistochemistry, quantitative RT-PCR and sequencing. Results BRAF V600E mutation is present in 20% (27/135) of the sporadic colorectal cancer cases. Conclusion Immunohistochemistry and quantitative RT-PCR are accurate and fast methods to detect BRAF V600E mutation in colorectal cancer.

Aim: Treatment of patients with RCC has improved substantially since the introduction of targeted therapies, but no biomarkers are available for predicting efficacy of such therapies. The proto-oncogene c-Met is involved in tumour angiogenesis, development and metastasis. In RCC c-Met expression is associated with poor prognosis suggesting a therapeutic role for c-Met inhibitors. The main objective of this study was to evaluate c-Met expression in metastatic RCC (mRCC). To our knowledge, c-Met expression has not previously been evaluated as a biomarker in patients with mRCC treated with VEGFR targeted agents. Methods: Clinical data and tumour tissue were available from 137 patients with mRCC treated with sunitinib at the Helsinki University Hospital between Oct 18, 2006 and May 31, 2012. Tumour c-Met expression was analysed from formalin-fixed paraffin-embedded samples using an anti-c-Met rabbit monoclonal ready-to-use antibody (Roche) and a BenchMark XT immunostainer (Ventana Medical Systems). c-Met expression was scored independently by 2 evaluators, and the discrepant cases were re-assessed jointly. Results: 78 (57%) of the 137 RCCs had no (-) or weak (+) c-Met expression (low expression group) and 59 (43%) had moderate (++ ) or strong (+++) expression (high expression group). Patients with high expression had shorter progression-free survival (PFS) than those with low expression (median, 6.5 vs. 14.2 months, p Conclusions: c-Met expression is associated with poor survival in a RCC patient population treated with sunitinib. Tumour c-Met expression warrants further investigation as a potential biomarker particularly in patients treated with dual c-Met/VEGFR inhibitors. Disclosure: P. Bono: Pfizer, Novartis and GSK advisory board honoraria. All other authors have declared no conflicts of interest.

Background & Aims—Gastrointestinal juvenile polyps may occur in juvenile polyposis syndrome (JPS) or sporadically. JPS is an autosomal-dominant condition caused by a germline defect in SMAD4 or BMPR1A in 50% to 60% of cases, and is characterized by multiple juvenile polyps, predominantly in the colorectum. JPS has an increased risk of gastrointestinal malignancy but sporadic juvenile polyps do not. Cyclooxygenase-2 (COX-2) expression is increased in gastrointestinal tumorigenesis and familial adenomatous polyposis. Inhibition of COX-2 leads to regression of colorectal adenomas in familial adenomatous polyposis patients and inhibits gastrointestinal tumorigenesis. To investigate the role of COX-2 in juvenile polyps, we compared the expression of COX-2 in juvenile polyps from a well-defined group of juvenile polyposis patients and sporadic juvenile polyps. Methods—COX-2 expression was assessed in 24 genetically well-defined JPS patients and 26 patients with sporadic juvenile polyps using tissue microarray analysis. Two additional markers, Huantigen R, a stabilizer of messenger RNA, and CCAAT/enhancer-binding protein β, a transcription factor, both associated with increased COX-2 expression, also were investigated. Results—Increased COX-2 expression in JPS patients was noted compared with patients with sporadic juvenile polyps (P < .001). Also, JPS patients with a BMPR1A germline defect had higher COX-2 expression than did JPS patients in whom no germline mutation was detected. High COX-2 levels correlated with increased cytoplasmic Hu-antigen R expression in JPS polyps (P = .022), but not in sporadic juvenile polyps. Conclusions—Juvenile polyposis and sporadic juvenile polyps show distinctive expression profiles of COX-2 that may have clinical implications. Juvenile polyps occur in about 1% of the pediatric population and most often are sporadic, solitary lesions of the colorectum. 1 These hamartomatous polyps are characterized by distorted and dilated crypts with reactive changes of the epithelium and an abundance of stroma. In

Top of pageAbstract Oncolytic adenoviruses a.k.a. conditionally replicating adenoviruses (CRAds) represent a promising approach for treatment of cancers resistant to conventional therapies. Our hypothesis was that efficacy of CRAds might be further improved by using chemotherapeutic agents in a multimodal antitumor approach. Oncolytic tumor killing differs mechanistically from conventional therapies and therefore an additive or synergistic antitumor effect is possible. The toxicity profiles of the agents may be different and could result in enhanced efficacy without increased side effects. Finally, it may be possible to use lower treatment doses and still achieve a greater efficacy than with single agents. We evaluated Ad5/3-|[Delta]|24 in combination with deoxycytidine analogue gemcitabine against human ovarian adenocarcinoma. Ad5/3-|[Delta]|24 is a serotype 3 receptor targeted CRAd containing a 24 bp deletion in the E1A gene. As a result the E1A protein is unable to bind the retinoblastoma (Rb) protein for induction of viral replication. Therefore the virus replicates selectively in cells dysfunctional in the Rb/p16 pathway, including most if not all cancer cells. Most CRAds are based on adenovirus serotype 5 (Ad5). However, the expression of the Ad5 primary receptor, coxsackie-adenovirus receptor (CAR), is highly variable on ovarian cancer cells. The lack of CAR can be circumvented by substituting the knob domain of Ad5 with the corresponding domain of serotype 3 (Ad3). Ad3 has a distinct receptor which is expressed to high degree on ovarian cancer cells. For evaluation of the potential interaction between Ad5/3-|[Delta]|24 and gemcitabine in vitro, the cell killing effect of the combination treatment was compared to single treatments on established ovarian adenocarcinoma cell lines. The combination resulted in synergistic cell killing as determined by Chou and Talalay's synergy analysis. However, the effect was dependent on dose and sequencing of the agents. In an orthotopic murine model of aggressive peritoneally disseminated ovarian cancer, the combination of Ad5/3-|[Delta]|24 and gemcitabine increased the survival of mice over either agent alone, and almost 60 % of treated mice were cured. However, sequencing of the agents was critical for toxicity versus efficacy. Mice remained free from intraperitoneal disease, but some succumbed to treatment related toxicity. Possible reasons for the interaction between Ad5/3-|[Delta]|24 and gemcitabine include the chemosensitizing activity of E1A and/or altered replication kinetics. Our results indicate that gemcitabine reduces the initial rate of Ad5/3-|[Delta]|24 replication without affecting the total amount of virus produced.