Mutations in PIK3CA are present in 10 to 15% of colorectal carcinomas. We aimed to examine how PIK3CA mutations relate to other molecular alterations in colorectal carcinoma, to pathologic phenotype ...and survival. PIK3CA mutation testing was carried out using direct sequencing on 757 incident tumors from the Melbourne Collaborative Cohort Study. The status of O-6-methylguanine-DNA methyltransferase (MGMT) was assessed using both immunohistochemistry and methyLight techniques. Microsatellite instability, CpG island phenotype (CIMP), KRAS and BRAF V600E mutation status, and pathology review features were derived from previous reports. PIK3CA mutation was observed in 105 of 757 (14%) of carcinomas, characterized by location in the proximal colon (54% vs. 34%; P<0.001) and an increased frequency of KRAS mutation (48% vs. 25%; P<0.001). High-levels of CIMP were more frequently found in PIK3CA-mutated tumors compared with PIK3CA wild-type tumors (22% vs. 11%; P = 0.004). There was no difference in the prevalence of BRAF V600E mutation between these two tumor groups. PIK3CA-mutated tumors were associated with loss of MGMT expression (35% vs. 20%; P = 0.001) and the presence of tumor mucinous differentiation (54% vs. 32%; P<0.001). In patients with wild-type BRAF tumors, PIK3CA mutation was associated with poor survival (HR 1.51 95% CI 1.04-2.19, P = 0.03). In summary, PIK3CA-mutated colorectal carcinomas are more likely to develop in the proximal colon, to demonstrate high levels of CIMP, KRAS mutation and loss of MGMT expression. PIK3CA mutation also contributes to significantly decreased survival for patients with wild-type BRAF tumors.
Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with ...germline mismatch repair (MMR) gene mutations.
Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS).
Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered.
Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation.
BRAFV600E mutation in microsatellite-unstable (MSI) colorectal carcinomas (CRCs) virtually excludes Lynch syndrome (LS). In microsatellite-stable (MSS) CRCs it predicts poor prognosis. We propose a ...universal CRC LS screening algorithm using concurrent reflex immunohistochemistry (IHC) for BRAFV600E and mismatch-repair (MMR) proteins. We compared BRAFV600E IHC with multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry in 216 consecutive CRCs from 2011. Discordant cases were resolved with real-time PCR. BRAFV600E IHC was performed on 51 CRCs from the Australasian Colorectal Cancer Family Registry (ACCFR), which were fully characterized for BRAF mutation by allele-specific PCR, MMR status (MMR IHC and MSI), MLH1 promoter methylation, and germline MLH1 mutation. We then assessed MMR and BRAFV600E IHC on 1403 consecutive CRCs. By matrix-assisted laser desorption/ionization-time of flight mass spectrometry 15 cases did not yield a BRAF result, whereas 38/201 (19%) were positive. By IHC 45/216 (20%) were positive. Of the 7 discordant cases, real-time PCR confirmed the IHC result in 6. In the 51 CRCs from the ACCFR, IHC was concordant with allele-specific PCR in 50 cases. BRAFV600E and MSI IHC on 1403 CRCs demonstrated the following phenotypesBRAF/MSS (1029 cases, 73%), BRAF/MSS (98, 7%), BRAF/MSI (183, 13%), and BRAF/MSI (93, 7%). All 11/1403 cancers associated with proven LS were BRAF/MSI. We conclude that BRAF IHC is highly concordant with 2 commonly used PCR-based BRAFV600E assays; it performed well in identifying MLH1 mutation carriers from the ACCFR and identified all cases of proven LS among the 1403 CRCs. Reflex BRAFV600E and MMR IHC are simple cheap tests that facilitate universal LS screening and identify the poor prognosis of the BRAFV600E-mutant MSS CRC phenotype.
Tumor testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was ...to identify MMR gene mutation carriers in two cohorts of population-based CRC utilizing a combination of tumor and germline testing approaches.
Colorectal cancers from 813 patients diagnosed with CRC < 60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry, microsatellite instability (MSI), BRAF
somatic mutation, and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and Multiplex Ligation Dependent Probe Amplification) was performed on germline DNA of patients with MMR-deficient tumors and a subset of MMR-proficient CRCs.
Of the 813 ACCFR probands, 90 probands demonstrated tumor MMR deficiency (11.1%), and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR deficiency was identified in the tumors of 103 probands (12.5%) and seven had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 25.2% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively.
Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimized by immunohistochemistry screening of CRC diagnosed before 70 years of age. A significant proportion of MMR-deficient CRCs will have unknown etiology (Lynch-like) proving problematic for clinical management.
Mucinous differentiation is associated with both CpG island methylator phenotype and microsatellite instability in colorectal cancer. The mucinous phenotype derives from abundant expression of the ...colonic goblet cell mucin, MUC2, and de novo expression of gastric foveolar mucin, MUC5AC. We, therefore, investigated the protein expression levels of MUC2 and MUC5AC, as well as MUC5B and MUC6, in molecular subtypes of colorectal cancer. Seven-hundred and twenty-two incident colorectal carcinomas occurring in 702 participants of the Melbourne Collaborative Cohort Study were characterized for methylator status, MLH1 methylation, somatic BRAF and KRAS mutations, microsatellite-instability status, MLH1, MSH2, MSH6, and PMS2 mismatch repair, and p53 protein expression, and their histopathology was reviewed. Protein expression levels of MUC2, MUC5AC, MUC5B, MUC6, and the putative mucin regulator CDX2 were compared with molecular and clinicopathological features of colorectal cancers using odds ratios and corresponding 95% confidence intervals. MUC2 overexpression (>25% positive tumor cells) was observed in 33% colorectal cancers, MUC5B expression in 53%, and de novo MUC5AC and MUC6 expression in 50% and 39%, respectively. Co-expression of two or more of the mucins was commonly observed. Expression of MUC2, MUC5AC and MUC6 was strongly associated with features associated with tumorigenesis via the serrated neoplasia pathway, including methylator positivity, somatic BRAF p.V600E mutation, and mismatch repair deficiency, as well as proximal location, poor differentiation, lymphocytic response, and increased T stage (all P<0.001). Overexpression was observed in tumors with and without mucinous differentiation. There were inverse associations between expression of all four mucins and p53 overexpression. CDX2 expression was inversely associated with MUC2, MUC5AC and MUC6 expression. Our results suggest that, in methylator-positive tumors, mucin genes on chromosome 11p15.5 region undergo increased expression via mechanisms other than direct regulation by CDX2.
KRAS-mutated carcinomas comprise 35–40% of all colorectal carcinomas but little is known about their characteristics. The aim of this study was to examine the pathological and molecular features of ...KRAS-mutated colorectal carcinomas and to compare them with other carcinoma subgroups. KRAS mutation testing was performed in 776 incident tumors from the Melbourne Collaborative Cohort Study. O6-methylguanine DNA methyltransferase (MGMT) status was assessed using both immunohistochemistry and MethyLight techniques. Microsatellite instability (MSI) phenotype and BRAF V600E mutation status were derived from earlier studies. Mutation in KRAS codon 12 or codon 13 was present in 28% of colorectal carcinomas. Compared with KRAS wild-type carcinomas, KRAS-mutated carcinomas were more frequently observed in contiguity with a residual polyp (38 vs 21%; P<0.001), demonstrated mucinous differentiation (46 vs 31%; P=0.001) and were associated with different MSI status (P<0.001) and with MGMT methylation (47 vs 21%; P=0.001). Compared with tumors demonstrating neither BRAF nor KRAS mutation, KRAS-mutated carcinomas showed more frequent location in the proximal colon (41 vs 27%; P=0.001), mucinous differentiation (46 vs 25%; P<0.001), presence of a contiguous polyp (38 vs 22%; P<0.001), MGMT methylation (47 vs 26%; P=0.01) and loss of MGMT immunohistochemical expression (27 vs 19%; P=0.02). KRAS-mutated carcinomas were distributed in a bimodal pattern along the proximal–distal axis of the colorectum. Compared with male subjects, female subjects were more likely to have KRAS-mutated carcinoma in the transverse colon and descending colon (39 vs 15%; P=0.02). No difference in overall survival was observed in patients according to their tumor KRAS mutation status. In summary, KRAS-mutated carcinomas frequently develop in contiguity with a residual polyp and show molecular features distinct from other colorectal carcinomas, in particular from tumors with neither BRAF nor KRAS mutation.
Summary Adenocarcinomas of the colon and rectum are graded using a 2-tiered system into histologic low-grade and high-grade tumors based on the proportion of gland formation. The current grading ...system does not apply to subtypes of carcinomas associated with a high frequency of microsatellite instability (MSI), such as mucinous and medullary carcinomas. We investigated the combined effect of histologic grade and MSI status on survival for 738 patients with colorectal carcinoma (48% female; mean age at diagnosis 68.2 years). The proportion of high-grade adenocarcinoma was 18%. MSI was observed in 59 adenocarcinomas (9%), with higher frequency in high-grade tumors compared with low-grade tumors (20% versus 6%; P < .001). Using Cox regression models, adjusting for sex and age at diagnosis and stratifying by the American Joint Committee on Cancer stage, microsatellite stable (MSS) high-grade tumors were associated with increased hazard of all-cause and colorectal cancer–specific mortality: hazard ratio 2.09 (95% confidence interval CI, 1.58-2.77) and 2.54 (95% CI, 1.86-3.47), respectively, both P < .001. A new grading system separating adenocarcinoma into low grade (all histologic low grade and MSI high grade) and high grade (MSS histologic high grade) gave a lower Akaike information criterion value when compared with the current grading system and thus represented a better model fit to stratify patients according to survival. We found that patients with a high-grade adenocarcinoma had significantly shorter survival than patients with low-grade adenocarcinoma only if the tumor was MSS, suggesting that the grading of colorectal adenocarcinoma with high-grade histologic features should be made according to the MSI status of the tumor.
•Endometrial cancer risk is predicted by cancer in first and second degree relatives.•The strongest predictor was a first degree relative with endometrial cancer <50y.•Risk was significantly greater ...with increasing Lynch cancers reported in relatives.•Risk associated with cancer in relatives did not differ by proband tumor MMR status.
To determine endometrial cancer (EC) risk according to family cancer history, including assessment by degree of relatedness, type of and age at cancer diagnosis of relatives.
Self-reported family cancer history was available for 1353 EC patients and 628 controls. Logistic regression was used to quantify the association between EC and cancer diagnosis in ≥1 first or second degree relative, and to assess whether level of risk differed by degree of relationship and/or relative's age at diagnosis. Risk was also evaluated for family history of up to three cancers from known familial syndromes (Lynch, Cowden, hereditary breast and ovarian cancer) overall, by histological subtype and, for a subset of 678 patients, by EC tumor mismatch repair (MMR) gene expression.
Report of EC in ≥1 first- or second-degree relative was associated with significantly increased risk of EC (P=3.8×10−7), independent of lifestyle risk factors. There was a trend in increasing EC risk with closer relatedness and younger age at EC diagnosis in relatives (PTrend=4.43×10−6), and with increasing numbers of Lynch cancers in relatives (PTrend≤0.0001). EC risk associated with family history did not differ by proband tumor MMR status, or histological subtype. Reported EC in first- or second-degree relatives remained associated with EC risk after conservative correction for potential misreported family history (OR 2.0; 95% CI, 1.24–3.37, P=0.004).
The strongest predictor of EC risk was closer relatedness and younger EC diagnosis age in ≥1 relative. Associations remained significant irrespective of proband MMR status, and after excluding MMR pathogenic variant carriers, indicating that Lynch syndrome genes do not fully explain familial EC risk.
Debate continues as to the usefulness of assessing adenomas for loss of mismatch repair protein expression to identify individuals with suspected Lynch syndrome. We tested 109 polyps from 69 proven ...mutation carriers (35 females and 34 males) belonging to 49 Lynch syndrome families. All polyps were tested by immunohistochemistry for four mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. Detailed pathology review was performed by specialist gastrointestinal pathologists. The majority of polyps (86%) were conventional adenomas (n=94), with 65 tubular and 28 tubulovillous adenomas and a single villous adenoma. The remaining 15 lesions (14%) were serrated polyps. Overall, loss of mismatch repair expression was noted for 78/109 (72%) of polyps. Loss of mismatch repair expression was seen in 74 of 94 (79%) conventional adenomas, and 4 of 15 (27%) serrated polyps from mismatch repair gene mutation carriers. In all instances, loss of expression was consistent with the underlying germline mutation. Mismatch repair protein expression was lost in 27 of 29 adenomas with a villous component compared with 47 of 65 adenomas without this feature (93 vs 73%; P=0.028). A strong trend was observed for high-grade dysplasia. Mismatch repair deficiency was observed in 12 of 12 conventional adenomas with high-grade dysplasia compared with 60 of 79 with low-grade dysplasia (100 vs 76%; P=0.065). We were unable to demonstrate a significant association between conventional adenoma size or site and mismatch repair deficiency. All (4/4 or 100%) of the serrated polyps demonstrating mismatch repair deficiency were traditional serrated adenomas from a single family. Diagnostic testing of adenomas in suspected Lynch syndrome families is a useful alternative in cases where cancers are unavailable. The overwhelming majority of conventional adenomas from mutation carriers show loss of mismatch repair protein expression concordant with the underlying germline mutation.
Summary The association between ovarian endometrioid adenocarcinoma and endometriosis is well established. However, not all endometrioid adenocarcinomas are directly related to endometriosis, and it ...has been suggested that there may be clinicopathologic differences between endometriosis-positive and endometriosis-negative tumors. Molecular alterations in endometrioid adenocarcinoma include KRAS and BRAF mutations, but the incidence of these abnormalities in previous reports has been highly variable (0%-36% and 0%-24%, respectively). This may be explained by relatively small sample sizes in earlier studies but could also reflect difficulties in accurately classifying high-grade ovarian malignancies. In the current study, we investigated KRAS and BRAF mutations in 78 low-grade (FIGO grade 1 and 2) endometrioid adenocarcinomas and compared the results with the presence of endometriosis in the tumor-associated ovary and/or in other pelvic sites. KRAS mutations were identified in 12 (29%) of 42 endometriosis-associated endometrioid adenocarcinomas with satisfactory analysis but in only 1 (3%) of 29 tumors in which endometriosis was not identified. BRAF mutation was identified only in a single endometriosis-associated case. These findings support the hypothesis that endometriosis-associated and independent endometrioid adenocarcinoma may develop via different molecular pathways and that KRAS mutations have an important role only in the former tumors. In contrast, BRAF mutations do not appear to have a significant role in either endometrioid adenocarcinoma subgroup. This may be relevant to future targeted therapies in patients with high-stage or recurrent disease and indicate that histopathologists should carefully examine endometrioid adenocarcinoma specimens, including nonneoplastic tissues, for the presence of endometriosis.
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