Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study.

BACKGROUND: Activating KRAS and BRAF mutations predict unresponsiveness to EGFR-targeting therapies in colorectal cancer (CRC), but their prognostic value needs further validation. In this study, we investigated the impact of KRAS codons 12 and 13, and BRAF mutations on survival from CRC, overall and stratified by sex, in a large prospective cohort study. METHODS: KRAS codons 12 and 13, and BRAF mutations were analysed by pyrosequencing of tumours from 525 and 524 incident CRC cases in The Malmö Diet and Cancer Study. Associations with cancer-specific survival (CSS) were explored by Cox proportional hazards regression, unadjusted and adjusted for age, TNM stage, differentiation grade, vascular invasion and microsatellite instability (MSI) status. RESULTS: KRAS and BRAF mutations were mutually exclusive. KRAS mutations were found in 191/ 525 (36.4%) cases, 82.2% of these mutations were in codon 12, 17.3% were in codon 13, and 0.5% cases had mutations in both codons. BRAF mutations were found in 78/524 (14.9%) cases. Overall, mutation in KRAS codon 13, but not codon 12, was associated with a significantly reduced CSS in unadjusted, but not in adjusted analysis, and BRAF mutation did not significantly affect survival. However, in microsatellite stable (MSS), but not in MSI tumours, an adverse prognostic impact of BRAF mutation was observed in unadjusted, but not in adjusted analysis. While KRAS mutation status was not significantly associated with sex, BRAF mutations were more common in women. BRAF mutation was not prognostic in women; but in men, BRAF mutation was associated with a significantly reduced CSS in overall adjusted analysis (HR = 3.50; 95% CI = 1.41-8.70), but not in unadjusted analysis. In men with MSS tumours, BRAF mutation was an independent factor of poor prognosis (HR = 4.91; 95% CI = 1.99-12.12). KRAS codon 13 mutation was associated with a significantly reduced CSS in women, but not in men in unadjusted, but not in adjusted analysis. CONCLUSIONS: Results from this cohort study demonstrate sex-related differences in the prognostic value of BRAF mutations in colorectal cancer, being particularly evident in men. These findings are novel and merit further validation.

Clinicopathological correlates and prognostic significance of KRAS mutation status in a pooled prospective cohort of epithelial ovarian cancer.

BACKGROUND: Activating KRAS mutations are common in ovarian carcinomas of low histological grade, less advanced clinical stage and mucinous histological subtype, and form part of the distinct molecular alterations associated with type I tumors in the dualistic model of ovarian carcinogenesis. Here, we investigated the occurrence, clinicopathological correlates and prognostic significance of specific KRAS mutations in tumours from 153 epithelial ovarian cancer (EOC) cases from a pooled, prospective cohort. METHODS: KRAS codon 12,13 and 61 mutations were analysed by pyrosequencing in tumours from 163 incident EOC cases in the Malmö Diet and Cancer Study and Malmö Preventive Project. Associations of mutational status with clinicopathological and molecular characteristics were assessed by Pearson Chi Square test. Ovarian cancer-specific survival (OCSS) according to mutational status was explored by Kaplan-Meier analysis and Cox proportional hazards modelling. KRAS-mutation status was also analysed in 28 concomitantly sampled benign-appearing fallopian tubes. RESULTS: Seventeen (11.1%) EOC cases harboured mutations in the KRAS gene, all but one in codon 12, and one in codon 13. No KRAS mutations were found in codon 61 and all examined fallopian tubes were KRAS wild-type. KRAS mutation was significantly associated with lower grade (p = 0.001), mucinous histological subtype (p = < 0.001) and progesterone receptor expression (p = 0.035). Kaplan-Meier analysis revealed a significantly improved OCSS for patients with KRAS-mutated compared to KRAS wild-type tumours (p = 0.015). These associations were confirmed in unadjusted Cox regression analysis (HR = 2.51; 95% CI 1.17-5.42) but did not remain significant after adjustment for age, grade and clinical stage. The beneficial prognostic impact of KRAS mutation was ony evident in tumours of low-intermediate differentiation grade (p = 0.023), and in a less advanced clinical stage (p = 0.014). Moreover, KRAS mutation was associated with a significantly improved OCSS in the subgroup of endometroid carcinomas (p = 0.012). CONCLUSIONS: The results from this study confirm previously demonstrated associations of KRAS mutations with well-differentiated and mucinous ovarian carcinomas. Moreover, KRAS-mutated tumours had a significantly improved survival in unadjusted, but not adjusted, analysis. A finding that merits further study is the significant prognostic impact of KRAS mutation in endometroid carcinomas, potentially indicating that response to Ras/Raf/MEK/ERK-targeting therapies may differ by histological subtype. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1788330379100147.

Liquid-based cytology using CytoRich Red/Tripath is diagnostically equivalent to conventional smears for bronchial washings and brushings and reduces the cost.

A split sample study was performed on 109 bronchial brushings and washings and evaluated from conventional slides (CS) and CytoRich Red/Tripath preparations (CRR/Tripath). Unassessable bronchial washings were significantly more frequent in CS (5 vs. 0), but as all brushings were assessable with both methods, no overall diagnostic advantage was found. CS and CRR/Tripath gave discordant diagnoses in one case with a final benign diagnosis and in six cases with final malignant diagnoses. In the benign case, atypia was assessed in CS. In the malignant cases, suspected malignancy was found in one CRR/Tripath and one CS, atypia vs. benign assessment was also balanced, with three atypias in CRR/Tripath and two in CS. The better preserved cells in CRR/Tripath facilitated correct diagnosis in some cases, but might also lead to false positive diagnoses. In small cell carcinomas diagnostic hints such as smearing and moulding were less pronounced in CRR/Tripath but this did not affect the diagnostic accuracy. Overall, the diagnostic performance with CRR/Tripath was at least as good as with conventional slides, although statistically no difference could be seen. The number of slides and screening time, and thereby cost was significantly reduced with CRR/Tripath, thus the liquid-based method is preferred for bronchial washings and brushings.

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

BACKGROUND: Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. METHODS: DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. RESULTS: All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. CONCLUSIONS: Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society.

High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer.

PURPOSE: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. EXPERIMENTAL DESIGN: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). RESULTS: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.

Increased androgen receptor expression in serous carcinoma of the ovary is associated with an improved survival.

BACKGROUND: Altered androgen hormone homeostasis and androgen receptor (AR) activity have been implicated in ovarian carcinogenesis but the relationship between AR expression in ovarian cancer and clinical outcome remains unclear. METHODS: In this study, the prognostic impact of AR expression was investigated using immunohistochemistry in tissue microarrays from 154 incident cases of epithelial ovarian cancer (EOC) in the prospective, population-based cohorts Malmö Diet and Cancer Study and Malmö Preventive Project. A subset of corresponding fallopian tubes (n = 36) with no histopathological evidence of disease was also analysed. RESULTS: While abundantly expressed in the majority of fallopian tubes with more than 75% positive nuclei in 16/36 (44%) cases, AR was absent in 108/154 (70%) of EOC cases. AR expression was not related to prognosis in the entire cohort, but in the serous subtype (n = 90), AR positivity (> 10% positive nuclei) was associated with a prolonged disease specific survival in univariate (HR= 0.49; 95% CI 0.25-0.96; p= 0.038) and multivariate (HR= 0.46; 95% CI 0.22-0.97; p= 0.042) analysis, adjusted for age, grade and clinical stage. CONCLUSIONS: AR expression is considerably reduced in EOC as compared to fallopian tubes, and in EOC of the serous subtype, high AR expression is a favourable prognostic factor. These results indicate that assessment of AR expression might be of value for treatment stratification of EOC patients with serous ovarian carcinoma.

Telomerase activity in effusions: a comparison between telomere repeat amplification protocol in situ and conventional telomere repeat amplification protocol assay.

CONTEXT: We previously found telomere repeat amplification protocol (TRAP) in situ helpful in the diagnosis of malignancy in effusions, whereas varying sensitivities and specificities for malignancy were reported by investigators using extract-based TRAP. OBJECTIVE: To compare the 2 methods and to elucidate the discrepancies between them. DESIGN: Twenty-three effusions were analyzed. Telomerase activity of whole cell lysate was measured with a Telo TAGGG telomerase polymerase chain reaction ELISA PLUS kit with modifications to exclude polymerase chain reaction inhibitors. TRAP in situ was performed on cytospins. An estimate of total TRAP activity in the specimen was made based on the amount of positive cells, their fluorescence intensity, and the proportion of different cell types in the specimen. The estimate was compared with the level of telomerase activity in cell lysate-based TRAP. RESULTS: TRAP in situ: Thirteen of 14 malignant cases and 2 of 2 equivocal cases showed moderate/strong reactivity. Five of 7 benign effusions were negative; in 2 of 7, mesothelial cells showed weak reactivity. Cell lysate-based TRAP assay: In 4 cases no internal standard was detected, indicating the presence of polymerase chain reaction inhibitors. The relative telomerase activities were 33.1 to 72.7 with a considerable overlap between malignant (48 +/- 9, mean +/- SD) and benign (43 +/- 9) cases. CONCLUSIONS: The TRAP in situ results correlated to final diagnoses, whereas the cell lysate-based TRAP assay did not differentiate between malignant and benign cases. The varying proportions of positive cells and the variation in fluorescence intensity in the TRAP in situ slides explained some of the discrepancies. The problems encountered with TRAP performed on cell lysates are partly overcome using TRAP in situ.

Comparison of NCL-hTERT antibody reactivity and telomere repeat amplification protocol in situ in effusions.

OBJECTIVE: To compare the performances of 2 methods, telomerase repeat amplification protocol (TRAP) in situ and antibodies to the hTERT protein, in assessing telomerase activity. STUDY DESIGN: TRAP in situ and immunohistochemistry with a commercial antibody (NCL-hTERT) was performed on 54 body cavity effusions. The results were compared and correlated to diagnosis. RESULTS: Thirty-four effusions from patients with verified malignant disease contained cytologically malignant cells. Both methods were positive in 33 of the cases, whereas only hTERT was positive in 1 case. Twenty effusions, all containing mesothelial cells, came from patients with benign conditions. In 2 fluids atypical, hyperplastic mesothelial cells were both TRAP in situ and hTERT positive. All remaining 18 fluids were TRAP in situ negative, whereas 12 of 18 were hTERT positive. Thus the results of TRAP in situ and hTERT immunohistochemistry disagreed in 1 of 34 (3%) malignant and 12 of 20 (60%) benign cases. CONCLUSION: The sensitivities for malignancy were similar for TRAP in situ and hTERT immunohistochemistry. The specificity of the applied hTERT antibody was significantly lower, due to hTERT reactivity in mesothelial cells.

Lower proliferation rate in metastatic effusion mesothelial cells than in benign effusions.

OBJECTIVE: To determine the proliferation rates of mesothelial cells in metastatic and benign effusions. STUDY DESIGN: Immunohistochemistry was performed on formalin-fixed pellets from 16 malignant and 9 benign clinical effusions. Dual staining with antibodies against Ki-67 (MIB-1) and desmin was applied to all effusions to differentiate between benign mesothelial cells and malignant cells, and the proportions of desmin+/Ki-67+ and desmin+/Ki-67- cells were calculated. RESULTS: In 7 malignant effusions no proliferating mesothelial cells were found, whereas some rate of proliferation could always be demonstrated in mesothelial cells in the benign effusions. Further, the median proportions of proliferating cells, malignant 2% vs. benign 11%, differed significantly. CONCLUSIONS: To our knowledge this finding has not been previously described, and it may have implications for both cytologic diagnosis and the understanding of tumor biology and the interaction between tumor cells and mesothelial cells.

Weak telomerase activity in malignant cells in metastatic serous effusions: correlation to short survival time.

OBJECTIVE: The purpose of this study was to evaluate whether the intensity of telomerase activity measured on the cellular level in effusions correlates to survival time in cases of metastatic spread to the serous cavities. STUDY DESIGN: Telomere repeat amplification protocol (TRAP) in situ was applied to 46 effusions containing metastatic cancer cells. RESULTS: Weak telomerase activity in tumor cells was seen in 7 cases, and moderate or strong activity in 39 cases. The intensity of the enzyme activity correlated significantly to survival (Kaplan-Meier survival analysis), the median survival time being 18 days for patients with weakly positive tumors and 100 days for patients with moderately or strongly positive tumors (Kruskal-Wallis test, p = 0.021). CONCLUSION: The strong relationship between telomerase activity in tumor cells in effusions and survival time has never been described before. Determination of telomerase activity on the cellular level provides a way to identify a subgroup of patients with a high probability of short survival.

Telomere repeat amplification protocol (TRAP) in situ reveals telomerase activity in three cell types in effusions: malignant cells, proliferative mesothelial cells, and lymphocytes.

Telomerase Repeat Amplification Protocol (TRAP) in situ was performed on cytospin preparations from 65 effusions from the serous cavities (45 pleural and 19 ascitic fluids and one pericardial fluid) submitted for routine diagnosis and the results were correlated to cytological morphology. Three types of cells with nuclear fluorescence were identified: malignant cells, hyperplastic mesothelial cell and lymphocytes. Of 38 cytologically malignant effusions, 12 showed strong reactivity in all malignant cells, three strong reactivity in part of the malignant population, whereas 12 showed moderate reactivity in the whole and five in part of the malignant population, respectively. In five malignant effusions weak reactivity was found in all (one case) and in scattered (four cases) malignant cells. Two effusions contained telomerase-negative malignant cells. Two pleural and two ascitic fluids contained proliferative mesothelial cells with weak or, in one case, moderate reactivity. Lymphocytes usually showed weak telomerase activity. Telomerase was expressed in almost all malignant tumours metastatic to serous cavities. Heterogeneity in tumour populations was demonstrated, which may have diagnostic implications, especially in cytology. Weak or moderate reactivity was found in lymphocytes and in some mesothelial proliferations and may explain the low specificity for malignancy sometimes obtained with the TRAP extract method. The weak reactivity found in lymphocytes may reduce the specificity when the extract method is used but causes no diagnostic problem with the TRAP in situ method.