Pre-operative chemoradiotherapy with UFT and Leucovorin in patients with advanced rectal cancer: a phase II study.

PURPOSE: The aim this study was to determine the pathologic complete response (pCR) rate defined as tumor regression grade 1 (TRG1) and toxicity profile of the combination of high-dose pre-operative radiotherapy and simultaneous UFT/leucovorin (LV) in patients with locally advanced rectal cancer. MATERIALS/METHODS: Eligibility included biopsy proven rectal adenocarcinoma; T3-T4 N0-N2; performance status < 2 (ECOG) and adequate blood, hepatic and renal function. Treatment consisted of radiotherapy 54 Gy at 1.8 Gy/day and UFT 300 mg/m(2)/day and LV 60 mg/day, given simultaneously daily for 6 weeks. Surgery was performed within 4-6 weeks period after chemoradiotherapy. Patients who did not achieve TGR1 were to receive 4 cycles of adjuvant UFT/LV on days 1-28, every 5 weeks. RESULTS: Sixty-eight patients were included. All but one received full dose of radiation and 62 had the total planned pre-operative UFT/LV dose. Grade 3 toxicities were diarrhea 7% and proctitis 3%. Complete resection was achieved in 62 patients (91%). Tumor regression grade 1 (TRG1) was seen in 11 patients (16%). Forty-eight patients received adjuvant UFT/LV. Grade 3 toxicity during adjuvant UFT/LV included diarrhea 12%, asthenia 4%, neutropenia 2%, and hand-foot syndrome 2%. The 3-year disease-free survival was 71%. CONCLUSIONS: Simultaneous high-dose pre-operative localized radiation therapy concurrent with UFT/LV is feasible and has a low toxicity profile. This schedule is highly effective and merits further investigation.

Tumour selection advantage of non-dominant negative P53 mutations in homozygotic MDM2-SNP309 colorectal cancer cells.

BACKGROUND: Mdm2 is a natural inhibitor of p53 function and its overexpression impairs p53 transcriptional activity. T-->G single-nucleotide polymorphism at position 309 (SNP309) of mdm2 induces overexpression of mdm2, but inhibits p53. OBJECTIVES: To determine whether SNP309 is a risk-modifier polymorphism in colorectal cancer (CRC) and whether tumour selection of P53 mutations are influenced by SNP309. METHODS: Single-stranded conformation polymorphism and automatic sequencing were performed. RESULTS: SNP309 is not associated with the risk of CRC or recurrence of tumours. These data do not over-ride the tumour-selection capabilities of P53 mutations in CRC. However, a significant association with non-dominant-negative P53 mutations (p = 0.02) was found. CONCLUSIONS: MDM2-SNP309 favours tumour selection of non-dominant negative P53 mutations in CRC, which also show an earlier age of tumour onset.

Prognostic significance of vascular endothelial growth factor and cyclooxygenase-2 in patients with rectal cancer treated with preoperative radiotherapy.

PURPOSE: To analyze the prognostic value of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in patients with locally advanced rectal cancer treated with preoperative radiotherapy. METHODS: Eighty-one patients with locally advanced rectal cancer were studied. All patients received preoperative pelvic radiotherapy. Forty-seven patients received concomitant chemotherapy. Surgical resection was performed 4-8 weeks later in all patients. Immunohistochemical examination of COX-2 and VEGF was performed on the preirradiation diagnostic biopsies. An immunohistochemical score established from the extension and intensity of the markers was used for analysis. The log-rank test and proportional hazards regression analysis were used to calculate the probability that the biomarkers were associated with patient outcome. RESULTS: COX-2 expression was positive in 38 tumors (51%) while VEGF expression was positive in 43 (57%). The only clinicopathological parameter significantly associated with COX-2 or VEGF expression was performance status. None of the 2 markers were found to predict treatment response. There was no statistically significant correlation between COX-2 and VEGF. Univariate analysis identified pathological stage (pT, pN) as prognostic for disease-free survival. When VEGF expression was analyzed, disease-free survival was reduced among patients with VEGF-positive tumors (p = 0.047). This was specifically related to metastases-free survival (p = 0.016). These results were not observed for COX-2. After multivariate analysis, the pT and pN stage remained as independent prognostic factors. CONCLUSIONS: VEGF-positive expression is an indicator of poor disease-free survival, specifically linked to distant metastasis. More aggressive treatment strategies are warranted in pT3-4 and pN1-2 rectal cancer patients.

Mechanisms of inactivation of the receptor tyrosine kinase EPHB2 in colorectal tumors.

The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/beta-catenin. Premalignant lesions of the colon express high levels of EPHB2 but the expression of this kinase is reduced or lost in most colorectal carcinomas. In addition, inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutation in the colon and rectum. In this study, we investigated the molecular mechanisms responsible for the inactivation of EPHB2 in colorectal tumors. We show here the presence of mutations in repetitive sequences in exon 17 of EPHB2 in 6 of 29 adenomas with microsatellite instability (MSI), and 101 of 246 MSI carcinomas (21% and 41%, respectively). Moreover, we found EPHB2 promoter hypermethylation in 54 of the 101 colorectal tumors studied (53%). Importantly, EPHB2 expression was restored after treatment of EPHB2-methylated colon cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. In conclusion, in this study, we elucidate the molecular mechanisms of inactivation of EPHB2 and show for the first time the high incidence of frameshift mutations in MSI colorectal tumors and aberrant methylation of the regulatory sequences of this important tumor suppressor gene.

The expression of epidermal growth factor receptor results in a worse prognosis for patients with rectal cancer treated with preoperative radiotherapy: a multicenter, retrospective analysis.

BACKGROUND AND PURPOSE: Expression of epidermal growth factor receptor (EGFR) is observed in 50-70% of colorectal carcinoma and is associated with poor prognosis. The aim of this study was to determine the prognostic value of EGFR status before radiotherapy in a group of patients with locally advanced rectal cancer treated with preoperative radiotherapy. PATIENTS AND METHODS: Eighty-seven patients were studied retrospectively. Treatment consisted of pelvic radiotherapy, in 50 patients with concomitant chemotherapy and surgical resection. Immunohistochemistry for EGFR was determined at the preradiation biopsy and in the resected specimens. Immunohistochemical analysis for EGFR expression was evaluated according to extension and staining intensity. We defined positive staining (EGFR positive), when extension was 5% or more. RESULTS: A total of 52 of 87 tumors showed EGFR positive status at biopsy (60%) and EGFR expression was associated neither with clinical tumor stage nor with clinical nodal stage. EGFR positive expression was linked to a lack of pathologic complete response to preoperative radiotherapy (P=0.006). Disease-free survival was lower among patients with EGFR positive status before radiotherapy (P=0.003). In a multivariate analysis EGFR expression at biopsy was a statistically significant predictor of disease-free survival, RR=2.88(1.1-7.8), P=0.036. CONCLUSIONS: EGFR is expressed in a significant number of rectal tumors. EGFR-positive expression before radiotherapy is an indicator for poor response and low disease-free survival.

BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes.

Recently, it was shown that the oncogenic activation of BRAF, a member of the RAS/RAF family of kinases, by the V600E mutation is characteristic for sporadic colon tumors with microsatellite instability. Further, it was shown to associate with the silencing of the mismatch repair (MMR) gene MLH1 by hypermethylation. Moreover, BRAF mutations proved to be absent in tumors from hereditary nonpolyposis colorectal cancer syndrome (HNPCC) families with germline mutations in the MMR genes MLH1 and MSH2. These data suggest that the oncogenic activation of BRAF is involved only in sporadic colorectal tumorigenesis. In order to further support this hypothesis, we have extended the analysis of the BRAF gene to a different subset of HNPCC families without germline mutations in MLH1 and MSH2. BRAF-V600E mutations were analysed by automatic sequencing in 38 tumors from HNPCC families with germline mutations in the MSH6 gene and also in HNPCC (suspected) families that do not have mutations in the MMR genes MLH1, MSH2 and MSH6. All patients belong to different families. No mutations were detected in 14 tumors from HNPCC patients with germline mutations in MSH6. Further, no mutations of BRAF were found in tumors from 23 MMR-negative families, from which 13 fulfilled the Amsterdam criteria (HNPCC) and 10 were suspected for HNPCC as they were positive for the Bethesda criteria. Overall, our data reinforce the concept that BRAF is not involved in the colorectal tumorigenesis of HNPCC. The detection of a positive BRAF-V600E mutation in a colorectal cancer suggests a sporadic origin of the disease and the absence of germline alterations of MLH1, MSH2 and also of MSH6. These findings have a potential impact in the genetic testing for HNPCC diagnostics and suggest a potential use of BRAF as exclusion criteria for HNPCC or as a molecular marker of sporadic cancer.

Low levels of microsatellite instability characterize MLH1 and MSH2 HNPCC carriers before tumor diagnosis.

Microsatellite instability (MSI) characterizes tumors arising in patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome. HNPCC is a hereditary autosomal dominant disease caused by germline mutations in genes from the DNA (MMR) mismatch repair system. In these tumors, the loss of MMR compromises the genome integrity, allowing the progressive accumulation of mutations and the establishment of a mutator phenotype in a recessive manner. It is not clear, however, whether MSI can be detected in HNPCC carriers before tumor diagnosis. The aim of this study was to evaluate the presence of genetic instability in MMR gene carriers in peripheral blood lymphocytes of carriers and non-carriers members of two HNPCC families harboring a germline MLH1 and MSH2 mutation, respectively. An extensive analysis of the allelic distribution of single molecules of the polyA tract bat26 was performed using a highly sensitive PCR-cloning approach. In non-carriers, the allelic distribution of single bat26 molecules followed a gaussian distribution with no bat26 alleles shorter than (A)21. All mutation carriers showed unstable alleles [(A)20 or shorter] with an overall frequency of 5.6% (102/1814). We therefore suggest that low levels of genomic instability characterize MMR mutation carriers. These observations suggest that somatic mutations accumulate well before tumor diagnosis. Even though it is not clear whether this is due to the presence of a small percentage of cells with lost MMR or due to MMR haploinsufficiency, detection of these short unstable alleles might help in the identification of asymptomatic carriers belonging to families with no detectable MMR gene mutations.

Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway and signals upstream of MEK1/2 kinases. Recently, activating mutations within BRAF have been reported in a high percentage of melanomas and colorectal carcinomas and shown to have oncogenic capabilities. Further, their association to mismatch-repair-deficient tumors has suggested the involvement of the RAS/RAF pathway in the tumorigenesis of microsatellite-unstable colon cancers, and that RAS and RAF mutations are alternative genetic events. We determined whether colorectal mismatch-repair-deficient tumors with BRAF mutations show a specific genotype when compared with tumors with wild-type BRAF, and whether they can be associated with a particular clinicopathological feature. Here, we report a striking association of BRAF, but not of APC, KRAS2, AXIN2, and TP53 mutations, with proximal mismatch-repair-deficient colon tumors and MLH1 hypermethylation. Our results support the hypothesis that proximal and distal colorectal tumors with mismatch repair deficiency harbor different genetic alterations, and we suggest that the involvement of the RAS/RAF pathway in colorectal tumorigenesis is differentially modulated according to tumor location and MLH1 inactivation.

BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.

Genes from the RAF family are Ras-regulated kinases involved in growth cellular responses. Recently, a V599E hotspot mutation within the BRAF gene was reported in a high percentage of colorectal tumors and significantly associated to defective mismatch repair (MMR). Additionally, BRAF mutations were described only in K-Ras-negative colon carcinomas, suggesting that BRAF/K-Ras activating mutations might be alternative genetic events in colon cancer. We have addressed to what extent the tumorigenic-positive selection exerted by BRAF mutations seen in colorectal MMR-deficient tumors was also involved in the tumorigenesis of gastric cancer. Accordingly, BRAF mutations were detected in 34% (25/74) of colorectal MMR-deficient tumors and in 5% (7/142) of MMR-proficient colorectal cases (P=0.0001). All mutations found in the MSI cases corresponded to the previously reported hotspot V599E. Two D593K and a K600E additional mutations were also detected in three MSS cases. However, only one mutation of BRAF was found within 124 MSS gastric tumors and none in 37 MSI gastric tumors, clearly suggesting that BRAF mutations are not involved in gastric tumorigenesis. Nonetheless, a high incidence of mutations of K-Ras was found within the MSI gastric group of tumors (P=0.0005), suggesting that the activation of K-Ras-dependent pathways contributes to the tumorigenesis of gastric cancers with MMR deficiency. Accordingly, our results show evidences that BRAF mutations characterize colon but not gastric tumors with MMR deficiency and are not involved in the tumorigenesis of gastric cancer of the mutator phenotype pathway.

Molecular patterns of nuclear and mitochondrial microsatellite alterations in breast tumors.

The widespread distribution of microsatellite sequences within the human genome has allowed researchers to identify alternative patterns of microsatellite alterations in cancer cells. Among them, aneuploid patterns of nuclear microsatellites, pseudo-diploid microsatellite instability patterns, and also patterns of microsatellite instability within the mitochondrial genome. In this context, while aneuploid patterns of multiple genomic gains and losses had already been described in breast tumors, data on nuclear microsatellite instability still remain controversial and limited data on mitochondrial instability are available. In order to clarify this, we undertook an extensive analysis of nuclear and mitochondrial microsatellite alterations in breast ductal adenocarcinomas, stratified by grades. No instability was detected in any of the 40 dinucleotide microsatellites analysed nor in bat26 and APDelta3 mononucleotide repeats, clearly concluding that microsatellite instability is not a feature of ductal breast tumorigenesis. Instead, microsatellites defined a clear pattern of aneuploid genomic gains and losses among which, losses of BRCA1 at D17S855 and gains of plakoglobin at D17S846 significantly associated to grade III tumors and poor prognosis. On the other hand, mitochondrial instability at the transcription control region was also detected in 10.8% of cases. None of the new mitochondrial variants was found in the normal tissue counterparts, confirming that these new variants arise as sporadic somatic mutations in the tumor cells. Also, no association was found between heteroplasmy in the normal tissue and mitochondrial instability in the tumors. We therefore suggest that these new variants arise in tumors as a consequence of the progressive accumulation of slippage somatic mutations and the intrinsic instability of these microsatellite sequences. Finally, our results also confirm that mitochondrial instability does not associate with nuclear MSI.

Epidermal growth factor receptor is a predictor of tumor response in locally advanced rectal cancer patients treated with preoperative radiotherapy.

PURPOSE: Epidermal growth factor receptor (EGFR) expression is observed in 50%-70% of colorectal carcinomas and is associated with poor prognosis. The aim of this study was to determine the EGFR expression rate in locally advanced rectal cancer and to analyze whether EGFR expression predicts tumor response to preoperative radiotherapy. METHODS AND MATERIALS: Between December 1997 and October 2000, 45 patients were included. Treatment consisted of preoperative pelvic radiotherapy and, in 21 patients, 2 courses of 5-fluorouracil leucovorin. Surgical resection was performed 4-8 weeks later. Immunohistochemistry for EGFR was determined at the preradiation diagnostic biopsy and in the resected specimens. Immunostaining was performed using EGFR monoclonal antibody (Biogenex, MU 207-UC). Immunohistochemical staining was evaluated according to extension and intensity. We defined positive staining (EGFR+) as extension of 5% or more. RESULTS: Preoperative treatment resulted in pathologic complete remission in 7 patients (15%), downstaging in 13 patients (29%), and no response in 25 patients (56%). EGFR+ was observed in 29 of 45 tumors (64%) and was associated with neither clinical tumor stage nor clinical nodal stage. The overall response rate was 34% in EGFR+ patients vs. 62% in those who were EGFR- (p = 0.07). Only 1 of the 7 pathologic complete remission patients was EGFR+ (p = 0.003). CONCLUSIONS: EGFR is expressed in a significant number of locally advanced rectal tumors. EGFR expression is an indicator for poor response to preoperative radiotherapy in advanced rectal carcinoma.