Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma

Activating mutations in KRAS and in one of its downstream mediators, BRAF, have been identified in a variety of human cancers. To determine the role of mutations in BRAF and KRAS in ovarian carcinoma, we analyzed both genes for three common mutations (at codon 599 of BRAF and codons 12 and 13 of KRAS). Mutations in either codon 599 of BRAF or codons 12 and 13 of KRAS occurred in 15 of 22 (68%) invasive micropapillary serous carcinomas (MPSCs; low-grade tumors) and in 31 of 51 (61%) serous borderline tumors (precursor lesions to invasive MPSCs). None of the tumors contained a mutation in both BRAF and KRAS. In contrast, none of the 72 conventional aggressive high-grade serous carcinomas analyzed contained the BRAF codon 599 mutation or either of the two KRAS mutations. The apparent restriction of these BRAF and KRAS mutations to low-grade serous ovarian carcinoma and its precursors suggests that low-grade and high-grade ovarian serous carcinomas develop through independent pathways.     

Mutations of BRAF and KRAS precede the development of ovarian serous borderline tumors

Molecular genetic changes that are associated with the initiating stage of tumor development are important in tumorigenesis. Ovarian serous borderline tumors (SBTs), putative precursors of low-grade serous carcinomas, are among the few human neoplasms with a high frequency of activating mutations in BRAF and KRAS genes. However, it remains unclear as to how these mutations contribute to tumor progression. To address this issue, we compared the mutational status of BRAF and KRAS in both SBTs and the adjacent epithelium from cystadenomas, the presumed precursor of SBTs. We found that three of eight SBTs contained mutant BRAF, and four SBTs contained mutant KRAS. All specimens with mutant BRAF harbored wild-type KRAS and vice versa. Thus, seven (88%) of eight SBTs contained either BRAF or KRAS mutations. The same mutations detected in SBTs were also identified in the cystadenoma epithelium adjacent to the SBTs in six (86%) of seven informative cases. As compared to SBTs, the cystadenoma epithelium, like ovarian surface epithelium, lacks cytological atypia. Our findings provide cogent evidence that mutations of BRAF and KRAS occur in the epithelium of cystadenomas adjacent to SBTs and strongly suggest that they are very early events in tumorigenesis, preceding the development of SBT.     

Alteration of the p53 Tumor Suppressor Gene Occurs Independently of K-ras Activation and More Frequently in Serous Adenocarcinomas than in Other Common Epithelial Tumors of the Human Ovary

To clarify the role of the p53 tumor suppressor gene in the development of human ovarian epithelial tumors and to study the association of p53 alterations with K-ras activation, a series of 70 common epithelial ovarian tumors from Japanese patients was studied. These included 31 serous adenocarcinomas, 12 mucinous adenocarcinomas, 5 mutinous tumors of borderline malignancy, 13 endometrioid adenocarcinomas, and 9 clear cell carcinomas. Allelic loss, recognized at the polymorphic site in codon 72 of the p53 gene, was detected in 14 of 36 (39%) informative cases by restriction fragment length polymorphism analysis and by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction(PCR)-amplified DNA fragments. Mutations in the highly conserved regions of the p53 gene were detected by SSCP analysis of PCR-amplifled fragments. Mutations were found in 22 of 70 (31%) ovarian tumors, including 1 of 5 mucinous tumors of borderline malignancy. Mutations were subsequently characterized by direct sequencing. Single missense base substitutions were detected in 13 ovarian carcinomas and in one case of mucinous tumor of borderline malignancy. Short (1-8 bp) deletions and insertions were found in 8 cases. Mutations in the p53 gene occurred more frequently in serous adenocarcinomas (14/31, 45%) than in all nonserous types of malignant epithelial tumors combined (7/34, 21%;P=0.032). Point mutations in K-ras were identified by dot blot hybridization analysis of PCR-amplified fragments with mutation-specific oligonucleotides and by direct sequencing. The overall frequency of K-ras mutations was 19/70 (27%).K-ras mutations were found in 12 of 17 (71%) mucinous tumors (8/12 mucinous carcinomas [67%] and 4/5 mucinous tumors of borderline malignancy [80%]), and occurred more frequently than in serous carcinomas (4/31, 13%;P=0.00009) or in all nonmucinous types of ovarian epithelial tumors combined (7/53, 13%; p=0.00002). These data suggest that different combinations of oncogenes and/or tumor suppressor genes may be involved in the genesis and development of histologically distinct categories of common epithelial tumors of the human ovary.     

p16、p53、BRAF、Bcl-2在卵巢浆液性肿瘤组织中的表达及临床意义

目的:探讨卵巢浆液性肿瘤组织中p16、p53、BRAF、Bcl-2的表达及临床意义。方法:收集宁夏医科大学总医院病理科2017年至2018年确诊的卵巢浆液性肿瘤136例,其中浆液性囊腺瘤52例,交界性囊腺瘤22例,低级别浆液性癌18例,高级别浆液性癌44例;另收集卵巢良性肿瘤和卵巢癌手术切除标本各30例。分别采用免疫组织化学SP法检测p16、p53、BRAF、Bcl-2的表达,实时定量PCR法检测p16、p53在卵巢良恶性肿瘤组织中的表达。结果:卵巢浆液性囊腺瘤、交界性囊腺瘤、低/高级别浆液性癌组织中p16的阳性率分别为3.8%、45.5%、88.9%、81.8%,p53为0、9.1%、55.6%、45.5%,BRAF为46.2%、45.5%、22.2%、31.8%,Bcl-2为46.2%、45.5%、38.9%、47.7%。不同类型浆液性肿瘤组织中p16、p53表达均有显著性差异（P＜0.001）,但BRAF、Bcl-2表达未见明显差异。与卵巢良性肿瘤相比,p16在交界性肿瘤、卵巢癌中的阳性表达明显升高,差异有显著统计学意义（P＜0.012 5）;p53在卵巢癌中的阳性表达明显高于良性肿瘤（P＜0.001）;p16和p53的表达呈正相关（P＜0.05）。p53、Bcl-2与卵巢癌淋巴结转移有相关性（P＜0.001）,p16、p53、Bcl-2与盆腔侵犯有关（P＜0.05）,p53、BRAF、Bcl-2与CA125表达有不同程度相关性（P＜0.05）。p16、p53联合检测对卵巢癌诊断的敏感性和特异性为90.0%、76.7%。结论:p16、p53、BRAF、Bcl-2参与卵巢癌的发生发展,p16和p53基因突变可能在卵巢浆液性肿瘤的恶性进展中发挥作用,联合检测p16、p53对卵巢癌诊断有指示意义。     

p53 mutations in ovarian tumors,detected by temperature-gradient gel electrophoresis,direct sequencing and immunohistochemistry

Samples from 94 ovarian tumors, comprising 24 cystadenomas/adenofibromas, among them 6 benign and 18 borderline tumors, one benign Brenner tumor, 39 carcinomas, 17 sex-cord stromal tumors, 5 germ-cell tumors and 8 metastatic or recurrent neoplasms were screened for p53 aberrations by polymerase chain reaction (PCR), temperature-gradient gel electrophoresis (TGGE), direct sequencing and immunohistochemistry. All sex-cord stromal and germ-cell tumors showed wild-type p53, except for a heterozygous silent germ-line mutation in one androblastoma. Somatic p53 mutations were detected in only one tumor of the cystadenoma/adenofibroma series (4.2%), in contrast to 38.5% of the carcinomas, among them 57.1% of serous papillary carcinomas, and 12.5 to 22.2% of endometrioid and mucinous carcinomas. By direct sequencing, the mutations of 13 cases were qualified as mis-sense mutations (n = 10), or I to 2-bp deletions (n = 3). Only 2 cases were immunohistochemically positive in the absence of detectable p53-gene abnormalities. The presence of p53 aberrations was significantly correlated with high grade, but not with stage of disease. For 21 bilateral tumors and/or tumors spread to the peritoneum, samples from both ovaries and/or ascites were analyzed. Among these, 16 cases were identical as to the p53 genotype, 5 cases showed discordant p53 states in ovary and/or in ascites DNA. We conclude that somatic p53 mutations are very frequent in serous papillary carcinomas, particularly in tumors of high grade, bilaterality, and peritoneal spread, less frequent in other carcinoma types and extremely rare in borderline and benign tumors of the ovary. © 1995 Wiley-Liss, Inc.     

BRAF Mutation is associated with early stage disease and improved outcome in patients with low‐grade serous ovarian cancer

BACKGROUND:

Low‐grade serous (LGS) ovarian cancer is a chemoresistant disease that accounts for 10% of serous ovarian cancers. Prior studies have reported that 28% to 35% of serous borderline (SB)/LGS ovarian tumors harbor a BRAF mutation, suggesting that BRAF inhibitors may be a rational therapeutic approach for this disease. In the current study, the authors sought to determine whether BRAF or KRAS mutation status was associated with disease stage and/or histology in patients with SB and LGS ovarian cancer.

METHODS:

Genetic profiles were constructed for 75 SB and LGS ovarian tumors to determine BRAF and KRAS mutation status. The incidence and identity of BRAF and KRAS mutations were defined, and the results were correlated with disease stage, response to treatment, and overall survival.

RESULTS:

Of 75 samples examined, 56 tumors (75%) had SB histology, and 19 tumors (25%) had LGS histology. Fifty‐seven percent of tumors harbored either a KRAS mutation (n = 17) or a BRAF mutation (a valine‐to‐glutamate substitution at residue 600 [V600E]; n = 26). The BRAF V600E mutation was associated significantly with early disease stage (stage I/II; P < .001) and SB histology (P = .002). KRAS mutations were not associated significantly with disease stage or histology. Of the 22 patients (29%) who required chemotherapy, 20 had tumors with wild‐type KRAS/BRAF, 2 had KRAS mutant tumors, and none had tumors that harbored a BRAF mutation. All patients with BRAF tumors remained alive at a median follow‐up of 3.6 years (range, 1.9–129.3 months).

CONCLUSIONS:

V600E BRAF mutations were present in 35% of patients who had SB/LGS ovarian cancers. The presence of the BRAF V600E mutation in SB/LGS ovarian cancer was associated with early stage disease and improved prognosis. The authors concluded that patients with SB/LGS ovarian cancer who require systemic therapy are unlikely to have BRAF mutant tumors. Cancer 2013. © 2012 American Cancer Society.     

Characterization of active mitogen-activated protein kinase in ovarian serous carcinomas.

PURPOSE: Mitogen-activated protein kinase (MAPK) plays a pivotal role in signal transduction. Activation of MAPK is regulated by upstream kinases including KRAS and BRAF, which are frequently mutated in low-grade ovarian serous carcinoma. This study evaluates the expression of active MAPK in ovarian serous carcinomas, with response to treatment and survival. EXPERIMENTAL DESIGN: Expression of active MAPK was assessed by immunohistochemistry in 207 cases of ovarian serous tumors. Immunoreactivity was correlated with tumor grade, mutational status of KRAS and BRAF, in vitro drug resistance, and clinical outcome. RESULT: There was a lower frequency of expression of active MAPK in high-grade ovarian serous carcinomas as compared with low-grade serous tumors, including borderline tumors and low-grade serous carcinoma (P < 0.001). Active MAPK was present in all of the 19 low-grade tumors with either KRAS or BRAF mutations as well as in 14 (41%) of 34 tumors with wild-type KRAS and BRAF in both low- and high-grade carcinomas. Expression of active MAPK alone served as a good survival indicator in the 2-year follow-up (P = 0.037) but not in the 5-year follow-up (P = 0.145). However, a combination of expression of active MAPK and in vitro sensitivity of paclitaxel significantly correlated with a better prognosis in 5-year survival rate (P = 0.048) in patients with advanced-stage high-grade serous carcinoma. CONCLUSIONS: Active MAPK is more frequently expressed in low-grade than in high-grade ovarian serous carcinoma. Active MAPK serves as a good prognostic marker in patients with high-grade serous carcinomas.     

Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms

Sequence mutations and gene amplifications lead to activation of the PIK3CA-AKT2 signaling pathway and have been reported in several types of neoplasms including ovarian cancer. Analysis of such genetic alterations, however, is usually complicated by contamination of normal cell DNA, artifacts associated with formalin-fixed tissues and the sensitivity of the techniques employed. In this study, we analyzed the sequence mutations in PIK3CA and AKT2 genes using purified tumor cells that were isolated from high-grade ovarian serous carcinomas and serous borderline tumors (SBTs) and assessed gene amplification using a dual-color FISH on tissue microarrays. Somatic sequence mutations in the kinase domain of AKT2 were not detected in any of the 65 ovarian tumors analyzed. Mutations of PIK3CA were rare, occurring only in one (2.3%) of 44 high-grade serous carcinomas and in only one (4.8%) of 21 SBTs. Dual-color FISH demonstrated that PIK3CA and AKT2 were not amplified in SBTs but amplified in 13.3% and 18.2% high-grade carcinomas, respectively. High-level amplification (>3 fold) was more frequently observed in AKT2 than in PIK3CA. Unlike mutations in ERBB2, KRAS and BRAF which are mutually exclusive in SBTs, coamplification of PIK3CA and AKT2 was present in five high-grade carcinomas including the OVCAR3 cells. Amplification in either of the genes occurred in 27% high-grade serous carcinomas. In conclusion, the methods we employed provide unambiguous evidence that somatic sequence mutations of PIK3CA and ATK2 are rare in ovarian serous tumors but amplification of both genes may play an important role in the development of high-grade ovarian serous carcinoma.     

上皮性卵巢癌起源二元论及分子生物学基础

上皮性卵巢癌是卵巢恶性肿瘤中最常见的类型,也是恶性程度最高的妇科肿瘤,基于近年来一系列的形态学和分子遗传学研究可将上皮性卵巢癌分为Ⅰ型和Ⅱ型。卵巢低级别浆液性腺癌属Ⅰ型来自交界性肿瘤,主要表现为微乳头形态伴KRAS和BRAF基因的突变,肿瘤呈渐进性发展,惰性病程,诊断时多数处于临床早期,预后较好。高级别浆液性腺癌属Ⅱ型,细胞异型性显著,多伴TP53突变,卵巢自身见不到前驱病变,呈高度侵袭性病程,多数来自输卵管伞端上皮的浆液性病变,确诊时常已处于临床晚期,发病快,生长迅速,侵袭性强,预后差。I型及II型卵巢癌表现出的生物学特性不同,在临床表现、预后等方面的明显差异提示了二者可能具有不同的起源及遗传学改变。本文将从分子遗传学角度对近期研究理论并从“二元”角度来探讨上皮性卵巢癌的起源进行综述。      

Molecular profiling of low grade serous ovarian tumours identifies novel candidate driver genes

Low grade serous ovarian tumours are a rare and under-characterised histological subtype of epithelial ovarian tumours, with little known of the molecular drivers and facilitators of tumorigenesis beyond classic oncogenic RAS/RAF mutations. With a move towards targeted therapies due to the chemoresistant nature of this subtype, it is pertinent to more fully characterise the genetic events driving this tumour type, some of which may influence response to therapy and/or development of drug resistance. We performed genome-wide high-resolution genomic copy number analysis (Affymetrix SNP6.0) and mutation hotspot screening (KRAS, BRAF, NRAS, HRAS, ERBB2 and TP53) to compare a large cohort of ovarian serous borderline tumours (SBTs, n = 57) with low grade serous carcinomas (LGSCs, n = 19). Whole exome sequencing was performed for 13 SBTs, nine LGSCs and one mixed low/high grade carcinoma. Copy number aberrations were detected in 61% (35/57) of SBTs, compared to 100% (19/19) of LGSCs. Oncogenic RAS/RAF/ERBB2 mutations were detected in 82.5% (47/57) of SBTs compared to 63% (12/19) of LGSCs, with NRAS mutations detected only in LGSC. Some copy number aberrations appeared to be enriched in LGSC, most significantly loss of 9p and homozygous deletions of the CDKN2A/2B locus. Exome sequencing identified BRAF, KRAS, NRAS, USP9X and EIF1AX as the most frequently mutated genes. We have identified markers of progression from borderline to LGSC and novel drivers of LGSC. USP9X and EIF1AX have both been linked to regulation of mTOR, suggesting that mTOR inhibitors may be a key companion treatment for targeted therapy trials of MEK and RAF inhibitors.     

High Incidence of p53 Gene Mutation in Human Ovarian Cancer and Its Association with Nuclear Accumulation of p53 Protein and Tumor DNA Aneuploidy

Using the poylmerase chain reaction and single-strand conformation polymorphism analysis, p53 gene mutations were examined in 24 cases of ovarian tumor including 14 ovarian carcinomas and 2 borderline cases of common epithelial type, 7 germ cell tumors, and one stromal tumor. Abnormal bands indicating mutations were detected in 12 (50%) of the cases examined, being present most frequently in common "epithelial" ovarian carcinoma (71%, 10/14). One case each of squamous cell carcinoma originating in a dermoid cyst and anaplastic dysgerminoma were positive for mutation. Direct sequencing confirmed 12 mutations and revealed G→A and G→C nucleotide changes in 5 and 3 cases (42% and 25%), respectively. The mutation was localized at the CpG site of the gene in 3 cases. Immunohistochemical examination of p53 protein in 21 cases and DNA flow-cytometrical analysis in 17 cases were also performed. Nuclear accumulation of the p53 protein and DNA aneuploidy pattern were detected in 11 (52%) and 9 (53%) cases, respectively. These were significantly correlated with p53 gene mutation ( P <0.01 and P <0.05, respectively; Fisher's exact test). Neither mutation of the p53 gene, nuclear accumulation of p53 protein nor DNA aneuploidy was detected in borderline cases of common "epithelial" type, typical dysgerminoma and immature teratoma. These results suggest that p53 gene mutation, nuclear accumulation of the protein and the DNA aneuploidy pattern are events occurring almost simultaneously in the progression of ovarian tumors, and that p53 abnormalities seem to be correlated with a high grade of malignancy.     

High incidence of p53 gene mutation in human ovarian cancer and its association with nuclear accumulation of p53 protein and tumor DNA aneuploidy.

Using the polymerase chain reaction and single-strand conformation polymorphism analysis, p53 gene mutations were examined in 24 cases of ovarian tumor including 14 ovarian carcinomas and 2 borderline cases of common epithelial type, 7 germ cell tumors, and one stromal tumor. Abnormal bands indicating mutations were detected in 12 (50%) of the cases examined, being present most frequently in common "epithelial" ovarian carcinoma (71%, 10/14). One case each of squamous cell carcinoma originating in a dermoid cyst and anaplastic dysgerminoma were positive for mutation. Direct sequencing confirmed 12 mutations and revealed G-->A and G-->C nucleotide changes in 5 and 3 cases (42% and 25%), respectively. The mutation was localized at the CpG site of the gene in 3 cases. Immunohistochemical examination of p53 protein in 21 cases and DNA flow-cytometrical analysis in 17 cases were also performed. Nuclear accumulation of the p53 protein and DNA aneuploidy pattern were detected in 11 (52%) and 9 (53%) cases, respectively. These were significantly correlated with p53 gene mutation (P < 0.01 and P < 0.05, respectively; Fisher's exact test). Neither mutation of the p53 gene, nuclear accumulation of p53 protein nor DNA aneuploidy was detected in borderline cases of common "epithelial" type, typical dysgerminoma and immature teratoma. These results suggest that p53 gene mutation, nuclear accumulation of the protein and the DNA aneuploidy pattern are events occurring almost simultaneously in the progression of ovarian tumors, and that p53 abnormalities seem to be correlated with a high grade of malignancy.     

The mutational profile of sporadic epithelial ovarian carcinoma

Mutations occurring in sporadic epithelial ovarian carcinomas are reviewed and their functional significance in terms of prognosis and prediction of anticancer drug activity are discussed. Alterations in the BRCA1/2 genes, TP53, PTEN, PI3Kinase, KRAS/BRAF and CTNNB1 are described. TP53 is likely to be a driver in high grade serous tumours, but is less useful than BRCA status in prediction of response to the platinum or PARPi agents. It is expected that mutation profiling will become integrated into current morphological/immunohistochemical primary diagnostic assessment of tumours once the cost and quality control issues of the technology are addressed.     

Second primary or recurrence? Comparative patterns of p53 and K-ras mutations suggest that serous borderline ovarian tumors and subsequent serous carcinomas are unrelated tumors

The role of serous borderline ovarian tumors (BOTs) in the pathogenesis of serous ovarian carcinomas is unclear. Some authors have compared mutations in serous BOTs to those in serous ovarian carcinomas, but the data on two common oncogenes, p53 and K-ras, remain inconclusive. To further clarify the relationship between the two tumors, we performed mutational analysis on tumors from a set of eight patients who first presented with advanced-stage serous BOTs and later developed grade 1 serous carcinomas. Epithelium from eight advanced-stage serous BOTs and subsequent grade 1 papillary serous carcinomas was microdissected and retrieved using a PixCell laser-capture microscope. Stroma was dissected as an internal control. The DNA was extracted with proteinase K and analyzed by single-strand conformational polymorphism-PCR for p53 and K-ras mutations. Bands with altered motility were analyzed by direct cycle sequencing. Seven of eight patients demonstrated different mutations in the secondary tumor compared with the primary tumor. For three patients, p53 mutations were identified in the BOTs that were absent from the carcinomas, suggesting a nonclonal origin for the carcinomas. These findings are consistent with the hypothesis that advanced-stage serous BOTs represent a distinct pathological entity compared with grade 1 serous epithelial ovarian carcinoma.     

Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer

The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal carcinomas. To clarify the extent of BRAF mutations in HNPCC colorectal carcinomas, which are typical mismatch repair deficient carcinomas, we compared the frequency of BRAF mutations between HNPCC, familial adenomatous polyposis (FAP) and sporadic cases. The frequency of KRAS mutations was also compared between these three syndromes. No BRAF mutations were detected in 33 HNPCC colorectal carcinomas, while they were detected in 3 of 26 (12%) FAP carcinomas and 2 of 53 (4%) microsatellite stable sporadic carcinomas. KRAS mutations were detected in 2 of 33 (6%) HNPCC, 9 of 26 (35%) FAP and 18 of 53 (34%) sporadic carcinomas. Such extremely low frequencies of BRAF and KRAS mutations in HNPCC colorectal carcinomas suggest that the participation of RAS-RAF signaling is minor in HNPCC, and that the previously suggested high frequency of BRAF mutations in mismatch repair deficient colorectal carcinomas is not due to mutations of mismatch repair genes.     

High Frequency of Genes’ Promoter Methylation, but Lack of BRAF V600E Mutation among Iranian Colorectal Cancer Patients

Gene silencing due to DNA hypermethylation is a major mechanism for loss of tumor suppressor genes function in colorectal cancer. Activating V600E mutation in BRAF gene has been linked with widespread methylation of CpG islands in sporadic colorectal cancers. The aim of the present study was to evaluate the methylation status of three cancer-related genes, APC2, p14ARF, and ECAD in colorectal carcinogenesis and their association with the mutational status of BRAF and KRAS among Iranian colorectal cancer patients. DNA from 110 unselected series of sporadic colorectal cancer patients was examined for BRAF V600E mutation by PCR-RFLP. Promoter methylation of genes in tumors was determined by methylation specific PCR. The frequency of APC2, E-CAD, and p14 methylation was 92.6%, 40.4% and 16.7%, respectively. But, no V600E mutation was identified in the BRAF gene in any sample. No association was found in cases showing epigenetic APC, ECAD, and p14 abnormality with the clinicopathological parameters under study. The association between KRAS mutations and the so called methylator phenotype was previously reported. Therefore, we also analyzed the association between the hot spot KRAS gene mutations in codons of 12 and 13 with genes' promoter hypermethylation in a subset of this group of patients. Out of 86 tumors, KRAS was mutated in 24 (28%) of tumors, the majority occurring in codon 12. KRAS mutations were not associated with genes' methylation in this tumor series. These findings suggest a distinct molecular pathway for methylation of APC2, p14, and ECAD genes from those previously described for colorectal cancers with BRAF or KRAS mutations.     

BRAF mutation, CpG island methylator phenotype and microsatellite instability occur more frequently and concordantly in mucinous than non-mucinous colorectal cancer

Mucinous colorectal cancer (CRC) has been reported to have distinct clinicopathological and genetic characteristics. However, the incidence and the relationship among microsatellite instability (MSI), CpG island methylator phenotype (CIMP) and BRAF and KRAS mutations in mucinous and non-mucinous CRC are not known. Activating mutations of BRAF and KRAS and their relationship with MSI and CIMP were examined in 83 sporadic CRC specimens (26 mucinous and 57 non-mucinous CRC). MSI, CIMP, BRAF and KRAS mutation were observed in 17, 24, 25 and 36% of the tumors, respectively. BRAF mutation was highly correlated with MSI (p < 0.001) and CIMP (p < 0.001). A higher incidence of MSI (27% vs. 12%), CIMP (38% vs. 18%, p < 0.05) and BRAF mutation (46% vs. 16%, p < 0.01) was observed in mucinous CRC. KRAS mutation (27% vs. 40%) was observed more frequently in non-mucinous CRC. Significantly higher percentages of mucinous CRC (54%, p < 0.05) had MSI or CIMP or BRAF mutations. Concordant occurrence of 2 or more of these alterations was observed in 39% of mucinous CRC and only 11% of non-mucinous CRC (p < 0.01). The more frequent occurrence and closer association among MSI, CIMP and BRAF mutation in mucinous CRC observed in our study further supports the idea that its pathogenesis may involve distinct genetic and epigenetic changes.     

Inactivation of the mitogen-activated protein kinase pathway as a potential target-based therapy in ovarian serous tumors with KRAS or BRAF mutations

Activation of mitogen-activated protein kinase (MAPK) occurs in response to various growth stimulating signals and as a result of activating mutations of the upstream regulators, KRAS and BRAF, which can be found in many types of human cancer. To investigate the roles of MAPK activation in tumors harboring KRAS or BRAF mutations, we inactivated MAPK in ovarian tumor cells using CI-1040, a compound that selectively inhibits MAPK kinase, an upstream regulator of MAPK and thus prevents MAPK activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated tumor cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. Long serial analysis of gene expression identified several differentially expressed genes in CI-1040-treated MPSC1 cells harboring an activating mutation in BRAF (V599L). The most striking changes were down-regulation of cyclin D1, COBRA1, and transglutaminase-2 and up-regulation of tumor necrosis factor-related apoptosis-induced ligand, thrombospondin-1, optineurin, and palladin. These patterns of gene expression were validated in other CI-1040-treated tumor cells based on quantitative PCR. Constitutive expression of cyclin D1 partially reversed the growth inhibitory effect of CI-1040 in MPSC1 cells. Our findings indicate that an activated MAPK pathway is critical in tumor growth and survival of ovarian tumors with KRAS or BRAF mutations and suggest that the CI-1040 induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian tumors.     

Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas

Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1-2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.     

Expression of pro-apoptotic (p53, p21, bax, bak and fas) and anti-apoptotic (bcl-2 and bcl-x) proteins in serous versus mucinous borderline ovarian tumours

BACKGROUND: We examined the expression of apoptosis-related proteins in serous versus mucinous borderline ovarian tumours, in comparison with benign and malignant ovarian tumours. MATERIALS AND METHODS: Immunohistochemical expression of pro-apoptotic (p53, p21, bax, bak, fas) and anti-apoptotic proteins (bcl-2, bcl-x) was determined in 34 borderline (19 mucinous, 15 serous), 20 benign (10 mucinous, 10 serous) and 28 malignant ovarian tumours (9 mucinous, 19 serous). RESULTS: A difference in semi-quantitative p53 expression was found between benign and borderline tumours (P = 0.01), but not between borderline and malignant tumours. Increased p21 expression was found in borderline versus benign tumours (P = 0.004). Bcl-2 expression was lower in borderline than in benign (P = 0.01) and malignant tumours (P = 0.02). No difference in bax, bak, fas or bcl-x expression was observed among the three tumour types. Higher percentage of p21 positive cells was found in serous than in mucinous borderline tumours (P < 0.001). Bcl-2 expression was higher in serous than in mucinous forms of benign (P < 0.001), borderline (P < 0.001), and malignant tumours (P < 0.003). No difference in p53, bax, bak, fas or bcl-x expression was observed between serous and mucinous borderline ovarian tumours. CONCLUSION: Although p53 overexpression was a common feature of both mucinous and serous borderline tumours, p21 and bcl-2 overexpression appeared specific to serous tumours.