Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced‐stage disease identified from a nation‐wide tumour registry in Denmark. Mutational analysis for hotspots in KRAS and BRAF was successful in 55 APSTs and demonstrated KRAS mutations in 34 (61.8%) and BRAF mutations in eight (14.5%). Mutational analysis was successful in 56 peritoneal implants and revealed KRAS mutations in 34 (60.7%) and BRAF mutations in seven (12.5%). Mutational analysis could not be performed in two primary tumours and in nine implants, either because DNA amplification failed or because there was insufficient tissue for mutational analysis. For these specimens we performed VE1 immunohistochemistry, which was shown to be a specific and sensitive surrogate marker for a V600E BRAF mutation. VE1 staining was positive in one of two APSTs and seven of nine implants. Thus, among 63 implants for which mutation status was known (either by direct mutational analysis or by VE1 immunohistochemistry), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST–implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST–implant pairs. Wild‐type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11 SBT/APST–implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST–implant (non‐invasive and invasive) pairs (p < 0.00001). This study provides cogent evidence that the vast majority of peritoneal implants, non‐invasive and invasive, harbour the identical KRAS or BRAF mutations that are present in the associated SBT/APST, supporting the view that peritoneal implants are derived from the primary ovarian tumour. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

KRAS (but not BRAF) mutations in ovarian serous borderline tumour are associated with recurrent low‐grade serous carcinoma

BRAF and KRAS mutations in ovarian serous borderline tumours (OSBTs) and ovarian low‐grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or whether those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumour samples from 23 recurrent LGSC patients with a known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for five patients, and either OSBTs or LGSCs were available for another 18 patients. Tumour cells from paraffin‐embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumours that appeared to have wild‐type KRAS by conventional PCR–Sanger sequencing were further analysed by full COLD (co‐amplification at lower denaturation temperature)‐PCR and deep sequencing. Full COLD‐PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in ten patients. Full COLD‐PCR deep sequencing detected low‐abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in seven OSBT samples and six LGSC samples. Surprisingly, patients with the KRAS G12V mutation have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumour cells with or without detectable KRAS mutations. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

BRAF Mutation Is Rare in Advanced-Stage Low-Grade Ovarian Serous Carcinomas

Low-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed.Serous carcinoma is the most common histological subtype of epithelial ovarian cancer. Recent evidence strongly suggests that a two-tier system stratifying serous carcinoma into low-grade and high-grade categories is biologically and clinically relevant.^1–12 Low-grade serous carcinomas are believed to arise via an adenoma–serous borderline tumor–carcinoma sequence, whereas high-grade serous carcinomas develop from an unknown precursor.^6,7 While previous reports indicate that low-grade serous carcinomas may develop from either recurrence of a serous borderline tumor or de novo, the mechanism associated with the former type of development is not fully understood.^13–15 Almost all serous borderline tumors that recur are associated with noninvasive or invasive peritoneal implants (stages II–IV) at initial diagnosis of the serous borderline tumor.^13–17According to the World Health Organization, the pattern of serous borderline tumors may be classified as either typical or micropapillary.^15,18–21 The micropapillary pattern was initially described in 1996; accounts for 8% to 18% of serous borderline tumors; and is associated with a greater frequency of bilaterality, a greater association with peritoneal implants (particularly invasive implants), and a higher risk of relapse than the typical pattern of serous borderline tumor.^15,18–21 For all serous borderline tumors, regardless of the pattern, the risk of relapsing as low-grade ovarian serous carcinoma increases over time.²²Mutation analysis could provide insight into how low-grade serous carcinoma develops. The initial report on BRAF and KRAS mutations in low-grade serous carcinomas, which were believed to be derived from serous borderline tumors with micropapillary pattern, indicated a rate of BRAF or KRAS mutation of 68%.²³ Similarly, in serous borderline tumors, a rate of BRAF or KRAS mutation of 61% was reported.²³ However, in invasive high-grade serous carcinomas, BRAF and KRAS mutations were not found.^23–25 Conversely, TP53 mutations are quite common in high-grade serous carcinomas but exceedingly rare in serous borderline tumors and low-grade serous carcinomas.²⁶ However, the correlation of BRAF/KRAS with patient outcome in low-grade ovarian serous carcinomas has not been explored. In this study, we investigated the BRAF and KRAS mutation status of advanced-stage low-grade serous carcinomas with and without a background of serous borderline tumor at initial diagnosis.     

Lineage tracing suggests that ovarian endosalpingiosis does not result from escape of oviductal epithelium

Most high-grade serous carcinomas are thought to arise from Fallopian tube epithelium (FTE), but some likely arise outside of the tube, perhaps from ectopic tubal-type epithelium known as endosalpingiosis. Importantly, the origin of endosalpingiosis is poorly understood. The proximity of the tubal fimbriae to the ovaries has led to the proposal that disruptions in the ovarian surface that occur during ovulation may allow detached FTE to implant in the ovary and form tubal-type glands and cysts. An alternative model suggests that cells present in ectopic locations outside the Müllerian tract retain the capacity for multi-lineage differentiation and can form glands with tubal-type epithelium. We used double transgenic Ovgp1-iCreER^T2;R26R^LSL-eYFP mice, which express an eYFP reporter protein in OVGP1-positive tissues following transient tamoxifen (TAM) treatment, to track the fate of oviductal epithelial cells. Cohorts of adult mice were given TAM to activate eYFP expression in oviductal epithelium, and ovaries were examined at time points ranging from 2 days to 12 months post-TAM. To test whether superovulation might increase acquisition of endosalpingiosis, additional cohorts of TAM-treated mice underwent up to five cycles of superovulation and ovaries were examined at 1, 6, and 12 months post-TAM. Ovaries were sectioned in their entirety to identify endosalpingiosis. Immunohistochemical staining for PAX8, tubulin, OVGP1, and eYFP was employed to study endosalpingiosis lesions. Ovarian endosalpingiosis was identified in 14.2% of TAM-treated adult mice. The endosalpingiotic inclusion glands and cysts were lined by secretory and ciliated cells and expressed PAX8, tubulin, OVGP1, and eYFP. Neither age nor superovulation was associated with a significant increase in endosalpingiosis. Endosalpingiosis was also occasionally present in the ovaries of pre-pubertal mice. The findings imply that ovarian endosalpingiosis in the mouse does not likely arise as a consequence of detachment and implantation of tubal epithelium and other mechanisms may be relevant. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mutational Analysis of KRAS, BRAF, and TP53 Genes of Ovarian Serous Carcinomas in Korean Women

Purpose

To assess the prevalence of KRAS, BRAF, and TP53 mutations in cases of low-grade and high-grade serous carcinomas and to evaluate the clinical outcomes of these morphologically distinct carcinomas.

Materials and Methods

Patients with primary invasive serous carcinomas were classified according to the universal grading system. Grade 2 serous tumors were excluded. A total of 100 patients were included for clinical evaluation. Thirty-seven patients, including 20 with low-grade and 17 with high-grade carcinomas, were selected for mutational analysis.

Results

The low-grade carcinoma group was characterized by young age and premenopausal period compared with the high-grade carcinoma group, but there were no statistically significant differences in stage, metastasis of lymph node and residual disease. There were no statistically significant differences in survival rates, however, the low-grade carcinoma group showed a trend for improved progression-free survival compared with the high-grade carcinoma group of early stage (p = 0.064). Mutations in KRAS and BRAF were found in 6 (30%) and 2 (10%) patients in the low-grade carcinoma group, respectively, however, they were not found in the high-grade carcinoma group. KRAS and BRAF mutations were mutually exclusive, and both mutations were observed in 40% (8/20). The frequency of TP53 mutations in low-grade and high-grade carcinoma groups were found in 20% (4/20) and 70.6% (12/17), respectively (p = 0.009).

Conclusion

Low-grade serous carcinoma shows mutation pattern different from that with high-grade carcinoma. As there were no significant differences in stage distribution and survival, especially in advanced stage, we suggest that more studies are needed to segregate these patients into distinct disease entities.     

Distinct evolutionary trajectories of primary high‐grade serous ovarian cancers revealed through spatial mutational profiling

High‐grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment‐resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2–91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole‐genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug‐resistance mechanisms. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary

Numerous studies have tested the association between TP53 mutations in ovarian cancer and prognosis but these have been consistently confounded by limitations in study design, methodology, and/or heterogeneity in the sample cohort. High‐grade serous (HGS) carcinoma is the most clinically important histological subtype of ovarian cancer. As these tumours may arise from the ovary, Fallopian tube or peritoneum, they are collectively referred to as high‐grade pelvic serous carcinoma (HGPSC). To identify the true prevalence of TP53 mutations in HGPSC, we sequenced exons 2–11 and intron–exon boundaries in tumour DNA from 145 patients. HGPSC cases were defined as having histological grade 2 or 3 and FIGO stage III or IV. Surprisingly, pathogenic TP53 mutations were identified in 96.7% (n = 119/123) of HGPSC cases. Molecular and pathological review of mutation‐negative cases showed evidence of p53 dysfunction associated with copy number gain of MDM2 or MDM4, or indicated the exclusion of samples as being low‐grade serous tumours or carcinoma of uncertain primary site. Overall, p53 dysfunction rate approached 100% of confirmed HGPSCs. No association between TP53 mutation and progression‐free or overall survival was found. From this first comprehensive mapping of TP53 mutation rate in a homogeneous group of HGPSC patients, we conclude that mutant TP53 is a driver mutation in the pathogenesis of HGPSC cancers. Because TP53 mutation is almost invariably present in HGPSC, it is not of substantial prognostic or predictive significance. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Peritoneal epithelial lesions associated with proliferative serous tumours of ovary

The peritoneal epithelial lesions in 40 cases of proliferating ovarian serous tumour are described. The lesions were varied and of both neoplastic and non-neoplastic form. The most common was serous tumour similar to that in the associated ovarian neoplasm. Tumour of this type was present in some or all of the peritoneal lesions in 77.5% of cases. In nearly two-thirds, the tumour was superficial; in the rest it invaded omentum. Occasionally, the infiltrating tumour was poorly differentiated. Benign tubular lesions resembling endosalpingiosis occurred in 16 (40%) of the 40 cases, but in seven it was associated with serous tumour. Psammoma bodies frequently accompanied serous tumour and endosalpingiosis and in occasional cases the majority of lesions consisted of psammomatous foci. The duration of follow up is too short to adequately assess the biological significance of these findings but it is clear that the peritoneal tumour occasionally may kill the patient within a few years. 'Serous tumour of low malignant potential' is the most appropriate term to describe the general group of ovarian serous tumours of so-called 'borderline malignant' type.     

Fallopian tube precursors of ovarian low‐ and high‐grade serous neoplasms

Traditionally, it was thought that ovarian high‐grade serous carcinoma arises from the ovarian surface epithelium and epithelial inclusion glands and that the pathogenesis is de novo; nonetheless, a convincing precursor in the ovary or peritoneum has not been identified to date. During the last few years, however, there has been a dramatic shift in thinking, and a candidate precursor is now recognized in the fallopian tube, especially within the fimbriated end – serous tubal intra‐epithelial carcinoma (STIC). Accordingly, STIC is probably the earliest histologically recognizable lesion in the pathogenesis of high‐grade serous carcinoma, but steps in this pathway which precede STIC have also been proposed. With subsequent progression, STIC implants onto the ovary and then develops into an invasive high‐grade serous carcinoma with rapid tumour growth. For invasive low‐grade serous carcinoma, it is well‐established that its pathogenesis begins with serous cystadenoma/adenofibroma, which develops sequentially into atypical proliferative serous tumour (typical serous borderline tumour), non‐invasive micropapillary (low‐grade) serous carcinoma (micropapillary serous borderline tumour) and then invasive low‐grade serous carcinoma in a relatively slow stepwise process. It is presumed that cystadenoma/adenofibroma arises from epithelial inclusion glands in the ovary, but recent evidence suggests that epithelial inclusion glands originate in the fallopian tube. Alternatively, it has been proposed that the other early precursors in the low‐grade pathway, including serous borderline tumours, non‐invasive implants and endosalpingiosis, may also arise directly from tubal mucosa. Therefore, the precursor of most ovarian high‐grade serous carcinomas originates in the fallopian tube, and that for low‐grade serous tumours may also be derived from the tube. This review paper provides an overview of the fallopian tube's potential role in the pathogenesis of both types of serous neoplasms of the ovary.     

Genetic alteration and immunohistochemical staining patterns of ovarian high‐grade serous adenocarcinoma with special emphasis on p53 immnnostaining pattern

We evaluated p53, KRAS, BRAF and CTNNB1 mutation and p53, WT1, p16 and beta‐catenin expression in 31 ovarian high‐grade serous adenocarcinoma. Twenty‐five (80.6%) tumors contained functional mutations of p53; three frameshift, four nonsense and 19 missense mutations. None of the tumors showed KRAS, BRAF or CTNNB1 mutation. In all 18 tumors with missense mutations, ≥60% of tumor cells were strongly positive for p53 immunostaining whereas all tumors with frameshift or nonsense mutations were completely negative. Missense mutation was correlated with diffuse and strong imunoreaction and frameshift/nonsense mutation was correlated with completely negative immunoreaction (P = 0.000). Tumors with wild‐type p53 revealed a wide range of immunostaining patterns. In 27 (87.1%) and 18 (58.1%) tumors, ≥50% of tumor cells were moderate to strongly positive for WT1 and p16, respectively. A considerable intratumoral heterogeneity for p16 expression was present. None of the tumors demonstrated nuclear beta‐catenin expression. p53 mutations appear to be a powerful molecular marker for ovarian high‐grade serous adenocarcinoma. Using p53 with an appropriate interpretation criteria together with WT1, p16 and beta‐catenin, most of the high‐grade serous adenocarcinoma could be distinguished from other ovarian tumors.     

The Li–Fraumeni syndrome (LFS): a model for the initiation of p53 signatures in the distal Fallopian tube

A candidate early precursor to pelvic serous cancer, the ‘p53 signature’, is commonly found in the benign mucosa of the distal Fallopian tube and harbours p53 mutations and evidence of DNA damage. We examined tubes from women with pre‐existing (germ‐line) mutations in p53 [Li–Fraumeni syndrome (LFS)] for evidence of this precursor. Fallopian tubes from two cases of LFS were immunostained for p53, Ki‐67 (proliferation) and H2AX (DNA damage response) and analysed for p53 mutations by laser capture microdissection (LCM) and p53 genomic sequencing (exons 2–11). A common single nucleotide repeat (snp) in exon 3 (rs1042522) and deletion sequencing chromatograms in exon 4 were examined in combination to estimate LOH in both LFS tubes and advanced serous carcinomas from the general population. LFS tubal epithelium contained abundant (10–20 per section) p53 signatures with evidence of DNA damage and low proliferative activity. Six of 11 LFS microdissected p53 signatures (55%) and 15 of 21 serous carcinomas (71%) revealed LOH at the p53 locus, relative to background epithelium. The LFS model confirms prior observations that the distal Fallopian tube is particularly prone to focal epithelial p53 gene inactivation—p53 mutation and LOH—in the absence of malignancy or increased epithelial proliferation. The fact that the LFS is not associated with ovarian cancers is consistent with the concept that loss of p53 function must be accompanied by at least one more genotoxic event (including BRCA1/2 functional inactivation) to produce the malignant phenotype. This is in keeping with a general model of carcinogenesis, in which different and often independent risk factors operate at multiple points in the serous carcinogenic spectrum. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Secretory cell outgrowth,PAX2 and serous carcinogenesis in the Fallopian tube

The ‘p53 signature’ is a benign secretory cell outgrowth in the distal Fallopian tube that shares properties with ovarian serous cancer—including p53 mutations—and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down‐regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2 and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p < 0.001); 89% were PAX2 (A) and 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from regionally unrestricted (PAX2) to greater likelihood specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the Fallopian tube. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genomic analysis of low‐grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities

Low‐grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy‐number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin‐specific protease 9X (USP9X), which is a chromosome X‐linked substrate‐specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique ‘driver’ aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mutational status of KRAS,NRAS, and BRAF in primary clear cell ovarian carcinoma

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer with characteristic biological features and aggressive clinical behavior. OCCCs show a pattern of gene mutations different from other type I ovarian malignancies, notably a higher frequency of PIK3CA mutations. In low grade serous ovarian cancer, KRAS and BRAF mutations are frequent, but little data are available on the mutational status of these genes in OCCCs. To clarify this issue, we designed a clinicopathological study with the aim to establish the incidence of KRAS, NRAS, and BRAF hot spot mutations in OCCC. Between December 2006 and June 2012, 22 patients with a proven diagnosis of OCCC were admitted to our Institutions. In all cases, final diagnosis was established according to FIGO and WHO criteria. All women received complete surgical staging. The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions on 2.5-μm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. Pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions revealed the presence of mutations only at codon 12 in exon 2 of KRAS in 3 of 22 (14 %) cases. We found no mutations in the hot spot regions of NRAF (exons 2, 3, 4) or BRAF (exon 15). The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage IA lesions in two cases associated with foci of endometriosis. We conclude that in 14 % of OCCCs, a KRAS mutation occurs in codon 2 exon 2. NRAS and BRAF mutations were not found.     

MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma

Molecular pathogenesis of high‐grade serous ovarian carcinoma (HG‐SOC) is poorly understood. Recent recognition of HG‐SOC precursor lesions, defined as serous tubal intraepithelial carcinoma (STIC) in fimbria, provides a new venue for the study of early genetic changes in HG‐SOC. Using microRNA profiling analysis, we found that miR‐182 expression was significantly higher in STIC than in matched normal Fallopian tube. Further study revealed that miR‐182 was significantly overexpressed in most HG‐SOC cases. To test whether miR‐182 plays a major role in early tumourigenesis of HG‐SOC, we overexpressed miR‐182 in immortalized ovarian surface, Fallopian tube secretory cells and malignant ovarian cell lines, and found that miR‐182 overexpression resulted in increased tumour transformation in vitro, and enhanced tumour invasiveness in vitro and metastasis in vivo. Mechanistically, we demonstrated that the oncogenic properties of miR‐182 in ovarian cancer were mediated in part by its impaired repair of DNA double‐strand breaks and negative regulation of breast cancer 1 (BRCA1) and metastasis suppressor 1 (MTSS1) expression as well as its positive regulation of the oncogene high‐mobility group AT‐hook 2 (HMGA2). Our findings suggest that miR‐182 dysregulation confers powerful oncogenic potential in the tumourigenesis of HG‐SOC. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increased tissue immunoexpression of YKL-40 protein in high grade serous ovarian cancers

YKL-40 is a glycoprotein secreted by numerous human cells, such as cartilage, synovial, and endothelial cells. The biological role of YKL-40 has not yet been fully unveiled, however, its participation is perceived in angiogenesis, growth, proliferation, differentiation, and remodeling processes. The primary goal of our study was to evaluate possible differences in tissue immunoexpression of YKL-40, assumed between high grade and low-grade ovarian cancers and between the above-mentioned cancer types and benign lesions. Another purpose was to find out whether immunoexpression of the studied protein could correlate with the tumor proliferation process, evaluated by Ki-67 immunoexpression.The analysis comprised 45 women, diagnosed and treated for epithelial ovarian tumors at the Medical University of Lodz between 1997 and 2002. YKL-40 protein immunoexpression was semiquantitatively assessed, whereas immunoexpression of Ki-67 was evaluated using a computer image analysis system. Significantly higher immunoexpression values of both examined proteins were observed in high-grade serous ovarian cancers vs. low-grade and benign tumors. Moreover, a significant positive correlation was identified between the immunoexpressions of YKL-40 and Ki-67 proteins in the studied groups of tumors.In conclusion, the obtained data suggest an overt prominence of TKL-40 tissue immunoexpression of YKL-40 in high-grade serous ovarian tumors, which could then be approached as a helpful, additional marker to identify more aggressive ovarian cancers.     

Tracing origin of serrated adenomas with <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">KRAS</Emphasis> mutations

Serrated neoplasm of the colorectum raised many as-yet unanswered issues. To characterize serrated neoplasia pathway, we investigated BRAF and KRAS mutations in 35 traditional serrated adenomas. BRAF exons 11 and 15, and KRAS exon 2 were amplified by polymerase chain reaction and directly sequenced. BRAF V599E mutation was found in 27 serrated adenomas (77.1%), and KRAS mutations were found in 3 (8.6%) of 35 traditional serrated adenomas. In 13 cases, mixed polyps composed of traditional serrated adenomas and hyperplastic (serrated) polyps were observed, and seven of them showed the same BRAF mutations in both components. Somatic mutations of BRAF and KRAS genes were mutually exclusive. These findings suggest that BRAF mutations are early and a critical event in the serrated adenomas, and most serrated adenomas in both sides of colon may progress from microvesicular hyperplastic polyps via BRAF mutations, and some left-sided serrated adenomas develop via KRAS mutations.     

Molecular analysis of the EGFR-RAS-RAF pathway in pancreatic ductal adenocarcinomas: lack of mutations in the BRAF and EGFR genes

The vast majority of tumors of the pancreas are ductal adenocarcinomas. This cancer type has an extremely poor prognosis and in many Western countries, it represents the fifth leading cause of cancer-related death. Pancreatic ductal adenocarcinomas exhibit the highest incidence of activating KRAS (Ki-Ras) mutations observed in any human cancer. It was therefore of interest to examine how this pattern would relate to mutations in the BRAF and EGFR genes, which are involved in the same signaling pathway as KRAS. We screened a series of 43 formalin-fixed, paraffin-embedded ductal adenocarcinomas of the pancreas. When DNA was extracted from whole tissue sections, KRAS codon 12 mutations were detected in 67% of the tumors. When cancerous ducts were isolated by laser-assisted microdissection, 91% were positive for KRAS mutations. Although it did not reach statistical significance, there was a trend in our material that survival after diagnosis varied according to KRAS mutation subtype, GTT-positive patients having the best prognosis. No alterations in BRAF exons 11 and 15 or in EGFR exons 18–21 were detected in KRAS-positive or KRAS-negative cases. We therefore conclude that the BRAF and EGFR mutations commonly seen in a variety of human cancers are generally absent from pancreatic ductal adenocarcinomas. Apparently, these tumors depend on no more than one genetic hit in the EGFR-RAS-RAF signaling pathway.     

The serrated pathway to colorectal carcinoma: current concepts and challenges

Approximately 30% of colorectal carcinomas develop via a serrated neoplasia pathway, named for the pattern of crypts in the precursor polyps. Molecular abnormalities consistently involve methylation of CpG islands [CpG island methylator phenotype (CIMP)] of low degree (CIMP‐L) or high degree (CIMP‐H), and activating mutations of the mitogen‐activated protein kinase pathway components BRAF or KRAS. Microsatellite instability (MSI) of a high level (MSI‐H) is often present, allowing for a molecular classification of serrated pathway carcinoma as: (i) BRAF mutant/CIMP‐H with either a) MSI‐H or b) microsatellite stable (MSS); and (ii) KRAS mutant/CIMP‐L/MSS. Precursor polyps include sessile serrated adenoma (SSA), characterized by proximal location, crypt architectural disturbance, and BRAF mutation. Microvesicular hyperplasic polyp (MVHP) probably precedes the development of SSA, and borderline lesions between MVHP and SSA occur. Cytological dysplasia in SSA portends advanced genetic abnormality and a high risk of progression to carcinoma. The traditional serrated adenoma has a predilection for the left colon, tubulovillous architecture, eosinophilic cytoplasm, and frequent KRAS mutation. Serrated morphology carcinoma is a new World Health Organization subtype with well‐differentiated, mucinous or trabecular patterns. It has frequent KRAS or BRAF mutations and a poor prognosis. This review provides an insight into the histology and molecular mechanisms driving these serrated pathway lesions.     

The extended spectrum of RAS‐MAPK pathway mutations in colorectal cancer

Current clinical guidelines recommend mutation analysis for select codons in KRAS and NRAS exons 2, 3, and 4 and BRAF V600E to guide therapy selection and prognostic stratification in advanced colorectal cancer. This study evaluates the impact of extended molecular testing on the detection of RAS‐MAPK pathway mutations. Panel next‐generation sequencing results of colorectal cancer specimens from 5795 individuals from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) were included. Mutations in RAS‐MAPK pathway genes were analyzed and functionally annotated. Colorectal cancers had recurrent pathogenic pathway activating mutations in KRAS (44%), NRAS (4%), HRAS (<1%), BRAF (10%), MAP2K1 (1%), RAF1 (<1%), and PTPN11 (<1%). The proportion of colorectal cancers with pathogenic RAS pathway mutations was 37% when only KRAS codon 12 and 13 mutations were considered, 46% when also including select KRAS and NRAS exons 2, 3, and 4 mutations, 53% when including BRAF V600E mutations, and 56% when including all pathogenic mutations. Panel next‐generation sequencing testing identifies additional RAS‐MAPK pathway driver mutations beyond current guideline recommendations. These mutations have potential implications in treatment selection for patients with advanced colorectal cancer.