Probes for KRAS

KRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation-specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin-fixed paraffin-embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild-type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild-type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild-type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors.

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

Relevant protein expression of GATA6, CK5, vimentin, and mucins using immunohistochemistry was assessed for predicting the prognosis and chemotherapy efficacy in pancreatic cancer (PC). The protein expression was examined in 159 PCs resected after neoadjuvant chemotherapy (NAC-PCs) with 120 matched biopsy specimens taken before NAC. KRAS mutations were assessed by digital PCR. NAC-PCs were classified by GATA6 expression initially and CK5 expression subsequently into four types, i.e., classical type (n = 22) showing GATA6-high (≧ 50%)/CK5-low (< 10%), hybrid type (n = 45) showing GATA6-high/CK5-high (≧ 10%), basal-like type (n = 53) showing GATA6-low (< 50%)/CK5-high (≧ 30%), and null type (n = 39) showing GATA6-low/CK5-low (< 30%), which resulted in a well-stratification of the patients’ prognosis. The classical type showed the most favorable prognosis, while the null type showed the worst prognosis (multivariate hazard ratio 3.56, 95% confidence interval (CI) 1.63−7.77, p = 0.0015). The hybrid and basal-like types were in between. The risk for hepatic recurrence was lower in the classical type than null (multivariate odds ratio (mOR) 0.18, CI 0.04−0.96, p = 0.0449) and basal-like (mOR 0.24, CI 0.05−1.16, p = 0.0750) types. In contrast, the risk for loco-regional recurrence was higher in classical type than the basal-like type (mOR 5.03, CI 1.20−21.1, p = 0.0272). The hybrid type was subclassified into transition and co-expression patterns with different gastric mucin expression. Vimentin-high (≧ 10%, n = 30) in pre-NAC-PC tissues was associated with poor prognosis (p = 0.0256). Phenotypic transitions between pre- and post-NAC were common (73/120; 61%). PCs with NAC regression grades 2 and 3 showed a transition to poorer prognostic phenotypes (p = 0.0497). KRAS mutations were not associated with these phenotypes. In conclusion, GATA6 and CK5 immunohistochemical expression phenotypes may stratify the survival of NAC-PCs and reflect post-NAC phenotypic transitions associated with poor prognosis. Prompt evaluation of immunohistochemical phenotypes may contribute to designing a precision therapeutic strategy for PC patients.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.