Colorectal carcinomas with submucosal invasion (pT1): analysis of histopathological and molecular factors predicting lymph node metastasis.

Submucosally invasive colorectal carcinoma (pT1) has the potential to be cured by local excision. In US surgical intervention is reserved for tumors with high-grade morphology, lymphvascular invasion, and close/positive margin. In other countries, particularly Japan, surgical therapy is also recommended for mucinous tumors, tumors with >1000 µm of submucosal invasion, and those with high tumor budding. These histological features have not been well evaluated in a western cohort of pT1 carcinomas. In a cohort of 116 surgically resected pT1 colorectal carcinomas, high tumor budding (P<0.001), lymphatic invasion (P=0.003), depth of submucosal invasion >1000 µm (P=0.04), and high-grade morphology (P=0.04) were significantly associated with lymph node metastasis on univariate analysis. Mucinous differentiation, tumor location, tumor growth pattern, and size of invasive component were not significant. On multivariate analysis, only high tumor budding was associated with lymph node metastasis with an odds ratio of 4.3 (P=0.004). A subset of 48 tumors (22 node-positive and 26 node-negative) was analyzed for mutations in 50 oncogenes and tumor suppressors. No statistically significant molecular alterations in these 50 genes were associated with lymph node status. However, lymphatic invasion was associated with BRAF mutations (P=0.01). Furthermore, high tumor budding was associated with mutations in TP53 (P=0.03) and inversely associated with mutations in the mTOR pathway (PIK3CA and AKT, P=0.02). In conclusion, this study demonstrates the importance of identifying high tumor budding in pT1 carcinomas when considering additional surgical resection. Molecular alterations associated with adverse histological features are identified.

Diagnostic utility of TP53 and cytokeratin 7 immunohistochemistry in idiopathic inflammatory bowel disease-associated neoplasia.

Long-standing inflammatory bowel disease is associated with increased risk of developing colorectal adenocarcinoma. Significant intra- and inter-observers' variability exists in histologic interpretation of dysplasia in surveillance biopsies. In this study, we evaluated the utility of a panel of immunohistochemical markers in diagnosing inflammatory bowel disease-associated neoplasia. We reviewed 39 colectomy specimens with inflammatory bowel disease-associated neoplasia. In these 39 cases, we identified 172 foci of interest (5 normal, 58 negative for dysplasia, 15 indefinite for dysplasia, 59 low-grade dysplasia, 18 high-grade dysplasia, and 17 invasive adenocarcinoma). They were subjected to immunohistochemistry for TP53 and CK7. Logistic regression was used to evaluate their association with the presence of dysplasia. Receiver operating characteristic curves were used to determine the optimal cutoffs and assess the diagnostic performance of TP53 and CK7. Both TP53 nuclear staining and CK7 immunoreactivity gradually increased in the progression of inflammatory bowel disease-associated neoplasia (P<0.0001). CK7 immunoreactivity increased along with the increase of inflammation severity (P=0.0002) as well as reactive changes (P=0.04) in the colonic mucosa. But TP53 nuclear staining was independent of either feature. When both TP53>8% and CK7>30% as identified from logistic regression and receiver operating characteristic curves were used to diagnose dysplasia, the specificity achieved as high as 95%. When either TP53>8% or CK7>30% was used to diagnose dysplasia, the sensitivity achieved was 82%. Our results suggested that a combination of CK7 and TP53 immunohistochemistry may be helpful in diagnosing inflammatory bowel disease-associated dysplasia in difficult cases.

Gene expression profiling of serrated polyps identifies annexin A10 as a marker of a sessile serrated adenoma/polyp.

Sessile serrated adenomas/polyps (SSA/Ps) are precursors of colon cancer, particularly those that exhibit microsatellite instability. Distinguishing SSA/Ps from the related, but innocuous, microvesicular hyperplastic polyp (MVHP) can be challenging. In this study seven gastrointestinal pathologists reviewed 109 serrated polyps and identified 60 polyps with histological consensus. Microarray analysis was performed on six distal consensus MVHPs < 9 mm, six proximal consensus SSA/Ps > 9 mm, and six normal colon biopsies (three proximal, three distal). Comparative gene expression analysis confirmed the close relationship between SSA/Ps and MVHPs as there was overlapping expression of many genes. However, the gene expression profile in SSA/Ps had stronger and more numerous associations with cancer-related genes compared with MVHPs. Three genes (TFF2, FABP6, and ANXA10) were identified as candidates whose expression can differentiate SSA/Ps from MVHPs, and the differences in expression were confirmed by quantitative RT-PCR. As ANXA10 showed the most promise in differentiating these polyps, the expression of ANXA10 was evaluated by immunohistochemistry in consensus SSA/Ps (n = 26), MVHPs (n = 21), and normal colon (n = 9). Immunohistochemical expression of ANXA10 was not identified in separate samples of normal colon or in the normal colonic epithelium adjacent to the serrated polyps. Consistent with the microarray and quantitative RT-PCR experiments, immunohistochemical expression of ANXA10 was markedly increased in SSA/Ps compared to MVHPs (p < 0.0001). An ANXA10 score ≥ 3 has a sensitivity of 73% and a specificity of 95% in the diagnosis of an SSA/P. In conclusion, we show that SSA/Ps and MVHPs have significant overlap in gene expression, but also important differences, particularly in cancer-related pathways. Expression of ANXA10 may be a potential marker of the serrated pathway to colon cancer.

Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status.

Although the serrated neoplasia pathway is thought to give rise to the majority of sporadic microsatellite instability-high (MSI-H) colon cancer, the exact proportion of these tumors that arise from serrated precursors has not been fully studied. Tubular and tubulovillous adenomas with features of the serrated neoplasia pathway have been described, and unlike sessile serrated adenomas, these lesions lack BRAF mutations. The contribution of these adenomas to sporadic MSI-H colon cancer is unclear. To this end, we conducted an analysis of right-sided sporadic MSI-H and microsatellite stable (MSS) colon cancer, with emphasis on precursor lesions. Overall 25% (19/75) of MSI-H colon cancer had a precursor, of which only 4 were recognized histologically as arising from a sessile serrated adenoma, and the remaining were best classified as adenomas. Of the 31 (of 89) MSS colon cancers with a precursor, only 1 was a sessile serrated adenoma (P=0.06). Histological analysis of the precursor adenomas to sporadic MSI-H colon cancer demonstrated a high frequency of crypt serrations compared with MSS colon cancer (93 vs 36%, P<0.001). BRAF mutations were found in 57/75 (76%) sporadic MSI-H and 10/89 (11%) MSS colon cancers (P<0.001). Molecular analysis demonstrated BRAF mutations in 11/12 adenoma and 3/3 sessile serrated adenoma precursors adjacent to BRAF-mutated MSI-H colon cancer. Similarly, all 4 precursors to BRAF-mutated MSS colon cancer were also BRAF mutated. The presence of BRAF mutations in these adenomatous precursors suggests that they represent sessile serrated adenomas with complete cytologic dysplasia. Finally, patients with sporadic MSI-H colon cancer were more likely to harbour synchronous sessile serrated adenomas (20 vs 8%; P=0.023). This is the largest study to rigorously evaluate the precursor and synchronous lesions in patients with right-sided colon cancer. Detailed molecular and histological analysis of these lesions confirms the importance of serrated precursors in the development of sporadic MSI-H colon cancer.

Gene Expression Changes Accompanying the Duodenal Adenoma-Carcinoma Sequence in Familial Adenomatous Polyposis.

OBJECTIVES: Duodenal cancer in familial adenomatous polyposis (FAP) arises from adenomas. Differentially expressed genes (DEGs) in the duodenal adenoma-carcinoma pathway have been identified in murine FAP models, but similar data in patients with FAP are limited. Identifying such changes may have significance in understanding duodenal polyposis therapies and identifying cancer biomarkers. We performed a genome-wide transcriptional analysis to describe the duodenal adenoma-carcinoma sequence and determine changes distinguishing patients with FAP with and without duodenal cancer. METHODS: Transcriptional profiling was performed with the Affymetrix Human Transcriptome Array 2.0 on duodenal biopsies from 12 FAP patients with duodenal cancer (FAP cases) and 12 FAP patients without cancer (FAP controls). DEGs were compared between cancer-normal, adenoma-normal, and cancer-adenoma in FAP cases and between adenomas from FAP cases and FAP controls. Significant results at P < 0.05 were filtered using fold change > 2. RESULTS: Two hundred twenty-four DEGs were identified at an absolute fold change > 2. In adenoma-normal, downregulation of DEGs involved in metabolism of brush border proteins (LCT), lipids (APOB/A4), reactive oxygen species (GSTA2), and retinol (RBP2) was observed. In the cancer-adenoma comparison, upregulation of DEGs involved in cell invasion/migration (POSTN, SPP1) and downregulation of DEGs involved in Paneth differentiation (DEFA5/6) were observed. In the adenoma-adenoma comparison, downregulation of several DEGs (CLCA1, ADH1C, ANXA10) in FAP case adenomas was observed. DEGs with therapeutic potential include SPP1, which is involved in both cyclooxygenase and epidermal growth factor receptor pathways targeted by the sulindac/erlotinib combination for duodenal polyposis. DISCUSSION: We describe DEGs in the human duodenal adenoma-carcinoma sequence in FAP, which may have prognostic and therapeutic significance. Validation studies are needed to confirm these findings.

Abrupt loss of MLH1 and PMS2 expression in endometrial carcinoma: molecular and morphologic analysis of 6 cases.

Given that endometrial cancer (EC) is often the sentinel cancer for female Lynch syndrome patients, we have successfully implemented universal screening of ECs and have previously shown that this is the preferred method to identify these patients. However, during the course of universal screening of EC, we encountered 6 cases with an unusual pattern of mismatch-repair protein immunohistochemistry that has not been previously described in this setting. In these 6 cases, there was an abrupt loss of MLH1 and PMS2 expression in a portion of the tumor. In 3 cases, marked histologic differences were identified between the areas of the tumor with retained expression and areas with loss of expression. In 2 cases, the areas with loss of expression were of higher grade (1 demonstrated solid growth and the other demonstrated increased nuclear atypia with diffuse p53 expression). In 4 tumors, histologic features associated with microsatellite instability (MSI) were present, including increased intraepithelial lymphocytes. The areas with loss of and retained MLH1/PMS2 expression were separately microdissected and assessed for MSI and MLH1 promoter methylation. The areas with loss of MLH1 and PMS2 more commonly demonstrated MSI compared with the areas with intact expression (83% vs. 33%). MLH1 promoter methylation analysis demonstrated heterogenous hypermethylation, as all areas with loss of MLH1/PMS2 expression had more extensive methylation of MLH1 compared with those areas with retained expression. In summary, we describe the histologic and molecular features of 6 cases of EC with abrupt loss of MLH1 and PMS2 expression and demonstrate that heterogenous methylation of the MLH1 promoter results in this distinct and unusual pattern of immunohistochemical expression.

A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification.

PCR amplification of part of the X-Y homologous amelogenin gene with a single primer pair has been used as a sex identification test because it generates different length products from the X and Y chromosomes. Using a commercially available kit that contains amelogenin primers, we report a single phenotypically normal Caucasian male out of 327 males tested to date that failed to show an X chromosome-specific PCR product. Using alternative amelogenin primers external to but encompassing the initial amplicon, an X chromosome-specific product was seen. Sequence analysis of this X-specific PCR product revealed a C to G mutation at the most 3' base of the initial reverse amelogenin PCR primer. An alternative reverse PCR primer with this most 3' base deleted showed X- and Y-specific products from the case study male. Rare mutations that result in a failure to amplify sex chromosome-specific products can result in incorrect gender identification.

A comparison of multiplex and monoplex T-cell receptor gamma PCR.

T-cell receptor gamma (TCRgamma) PCR is often used to detect clonal T-cell populations. Because TCRgamma contains a limited number of variable (Vgamma) and joining (Jgamma) regions, a small number of PCR primers can be used to assess T-cell clonality. The seven primers used in the current study were described previously and were split into 2 or 3 multiplex primer sets. In this study, a single 7-primer multiplex (7-plex) PCR reaction was compared with all 12 possible monoplex primer combinations on 18 samples previously analyzed for T-cell receptor rearrangements by TCRbeta Southern blot and/or TCRgamma PCR followed by temporal temperature gradient gel electrophoresis. Using fluorescent Vgamma-region primers, unlabeled Jgamma-region primers, and capillary electrophoresis, we show all TCRgamma rearrangements seen by 7-plex PCR on known positive samples were seen following monoplex PCR. However, additional TCRgamma gene rearrangements were seen in monoplex PCR reactions that were not seen in the 7-plex PCR reactions. Monoplex but not 7-plex PCR of known negative samples occasionally showed TCRgamma gene rearrangements, often with less frequently used Vgamma and Jgamma-region primers, and may have represented false positive results. In summary, the single 7-plex PCR reaction correctly identified specimens with TCRgamma clonal populations and represents an improvement over existing assays that use these same primers split into several smaller multiplex reactions. Monoplex PCR has no advantage over multiplex PCR and has the potential to lead to false positive results.

Detection of a novel point mutation of the prothrombin gene at position 20209.

Detection of the prothrombin G20210A mutation was performed on the LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using commercially available primers and hybridization probes. Herein we report four cases from unrelated African American individuals where LightCycler analysis showed atypical melting curves. Sequence analysis of the four samples showed heterozygosity for a C to T mutation 1 bp upstream from the known 20210 mutation at position 20209. The clinical significance of this mutation is not known, but three patients in whom it was detected had a history of venous thrombosis or stroke. The fourth patient had severe liver disease, which may have masked thrombotic predisposition. Since most assays used to detect the G20210A mutation use a PCR/restriction digestion assay, which would not detect the C20209T mutation, this new mutation may be underrecognized when prothrombin gene mutation testing is performed by PCR/restriction digestion assay.

Morphologic and molecular characterization of traditional serrated adenomas of the distal colon and rectum.

Of the serrated polyps, the origin, morphologic features, molecular alterations, and natural history of traditional serrated adenomas (TSAs) are the least understood. Recent studies suggest that these polyps may arise from precursor lesions. The frequencies of KRAS and BRAF mutations vary between these studies, and only 1 small study has measured CpG island methylation using current markers of methylation. Mutations in GNAS, a gene commonly mutated in colorectal villous adenomas, have not been fully evaluated in TSAs. Finally, the expression of annexin A10 (ANXA10), a recently discovered marker of sessile serrated adenomas/polyps, has not been studied in these polyps. To further characterize these polyps, 5 gastrointestinal pathologists reviewed 55 left-sided polyps diagnosed as TSA at a single institution. Pathologists assessed various histologic features including cytoplasmic eosinophilia, ectopic crypt foci, presence of conventional dysplasia, and presence of precursor serrated lesions. KRAS, BRAF, and GNAS mutational analysis was performed, as well as CpG island methylation and ANXA10 immunohistochemistry. Ectopic crypt foci were seen in 62% of TSAs. Precursor lesions were seen in 24% of the study polyps, most of which were hyperplastic polyps. KRAS and BRAF mutations were common and were present in 42% and 48% of polyps, respectively. GNAS mutations occurred in 8% of polyps, often in conjunction with a BRAF mutation. Unlike sessile serrated adenomas/polyps, TSAs rarely had diffuse expression of ANXA10. Importantly, BRAF-mutated TSAs had more widespread methylation of a 5-marker CpG island panel compared with KRAS-mutated polyps. However, ectopic crypt foci, a proposed defining feature of TSA, were not associated with any specific molecular alteration.

Immunohistochemistry for annexin A10 can distinguish sporadic from Lynch syndrome-associated microsatellite-unstable colorectal carcinoma.

Differentiating sporadic microsatellite-unstable colorectal carcinoma due to MLH1 promoter hypermethylation from Lynch syndrome (LS)-associated tumors due to mutations in mismatch-repair proteins is time consuming, cost intensive, and requires advanced laboratory testing. A mutation in BRAF has been shown to be highly specific for sporadic tumors; however, a significant proportion of sporadic microsatellite-unstable tumors lack BRAF mutations. MLH1 promoter methylation analysis is subsequently used to differentiate LS and sporadic tumors, but both tests require specialized laboratories and are costly. Through previous gene expression profiling of serrated polyps, we identified annexin A10 as a protein highly expressed in sessile serrated adenomas/polyps. As these polyps give rise to the majority of sporadic microsatellite-unstable tumors, we evaluated the ability of annexin A10 expression to discriminate between LS and sporadic tumors. A marked increase in annexin A10 mRNA was observed in sporadic microsatellite-unstable tumors compared with LS tumors (378-fold increase, P<0.001). Using immunohistochemistry, annexin A10 was expressed in 23/53 (43%) BRAF-mutated and 9/22 (41%) BRAF wild-type sporadic tumors. In contrast, only 3/56 (5%) LS tumors were positive for annexin A10 (P<0.0001). One patient had a deleterious MSH2 mutation, and another had a variant of uncertain significance in MSH6. These 2 tumors could be easily distinguished from sporadic tumors using mismatch-repair protein immunohistochemistry. Only 1/28 (4%) LS tumors with loss of MLH1 was positive for annexin A10. This patient did not have a deleterious MLH1 mutation but rather germline promoter hypermethylation of MLH1. On the basis of these results, immunohistochemistry for annexin A10 may be a useful marker to distinguish sporadic from LS-associated microsatellite-unstable colon cancer.

Serrated lesions of the appendix frequently harbor KRAS mutations and not BRAF mutations indicating a distinctly different serrated neoplastic pathway in the appendix.

Appendiceal serrated polyps often morphologically resemble their colorectal counterparts and most pathologists employ colorectal diagnostic terminology when evaluating appendiceal serrated lesions. We analyzed 132 appendiceal lesions for mutations in the RAS/RAF/MAPK pathway in an attempt to (1) determine the frequency of these mutations in appendiceal serrated lesions and (2) correlate the histopathologic features with molecular alterations. The study group of appendiceal serrated lesions (n = 46) was divided into a non-dysplastic group (28/46, subclassified as 7 hyperplastic polyps and 21 sessile serrated adenoma/polyps (SSA/P) using colorectal diagnostic terminology) and dysplastic group (18/46, subclassified as 9 SSA/Ps with cytological dysplasia, 7 traditional serrated adenomas, and 2 adenomas with prominent serrations). Appendiceal non-serrated dysplastic lesions (n = 86) comprised the control group. Of the 123 lesions analyzed, KRAS mutations were identified in 64 (52%) appendiceal lesions. No significant difference in the presence of KRAS mutations were identified between serrated non-dysplastic lesions (13/25, 52%), serrated dysplastic lesions (7/14, 50%) and the control group of non-serrated dysplastic lesions (44/84, 52%) (P = 1.0). Importantly, KRAS mutations were identified in lesions that were histologically identical to colorectal hyperplastic polyps (2/6, 33%), SSA/Ps (11/19, 58%), and SSA/Ps with cytological dysplasia (4/7, 57%). Of the 126 lesions tested, BRAF V600E mutations were identified in only 5 (4%) appendiceal lesions. Our results indicate that serrated lesions of the appendix often harbor KRAS mutations rather than BRAF mutations and suggest that the serrated pathway in the appendix is likely different than in the colon and rectum.

Histomorphologic and molecular features of pouch and peripouch adenocarcinoma: a comparison with ulcerative colitis-associated adenocarcinoma.

The occurrence of adenocarcinoma after ileal pouch-anal anastomosis for ulcerative colitis (UC) is an infrequent but potentially lethal complication. Neither histomorphologic nor molecular features of pouch adenocarcinoma after ileal pouch-anal anastomosis have been fully investigated. We report the largest series of 12 pouch and peripouch adenocarcinomas and compared them with 58 randomly selected UC-associated adenocarcinomas. The mean age of patients with pouch/peripouch adenocarcinoma was 55.2 years (SD 14.8), which was not significantly different from that of controls (P=0.52). Pouch/peripouch adenocarcinoma and UC-associated adenocarcinoma had a comparable frequency of tumor-infiltrating lymphocytes, lack of dirty necrosis, mucin differentiation, signet ring cell differentiation, heterogeneity, and well differentiation (P>0.05 for all). Pouch/peripouch adenocarcinoma was more likely to show Crohn-like reaction compared with UC-associated adenocarcinoma (P=0.047). Loss of at least 1 mismatch repair protein was noted in 9% of pouch/peripouch adenocarcinomas and 9.6% of UC-related adenocarcinomas (P=1.0). There was no significant difference in the frequency of p53 overexpression (36.4% vs. 61.1%, P=0.184) or nuclear immunoreactivity for ß-catenin (9% vs. 7.4%, P=0.99) in pouch/peripouch versus UC-associated adenocarcinomas, respectively. Pouch/peripouch and UC-associated adenocarcinoma had a comparable positive rate for CK7 (54.5% vs. 55.5%, P=0.99), CK20 (100% vs. 98.1%, P=0.99), and CDX2 (72.8% vs. 72.2%, P=0.99) by immunohistochemistry. In summary, pouch and peripouch adenocarcinoma can occur in patients without colorectal neoplasia and in those with idiopathic inflammatory bowel disease, can be potentially lethal, and has histomorphologic and molecular features similar to those of UC-associated adenocarcinoma.

BRAF-mutated, microsatellite-stable adenocarcinoma of the proximal colon: an aggressive adenocarcinoma with poor survival, mucinous differentiation, and adverse morphologic features.

The association of BRAF V600E mutation and the presence of the CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) often confound analysis of BRAF mutation status and survival in colorectal carcinoma. We evaluated a consecutive series of proximal colonic adenocarcinomas for mismatch repair protein abnormalities/MSI, BRAF V600E mutation, and KRAS mutations in an attempt to determine the prognostic significance of these abnormalities and to correlate histopathologic features with molecular alterations. Of the 259 proximal colon adenocarcinomas analyzed for mismatch repair protein abnormalities and/or MSI, 181 proximal colonic adenocarcinomas demonstrated proficient DNA mismatch repair using either MSI PCR (n=78), mismatch repair protein immunohistochemistry (n=91), or both MSI PCR and mismatch repair immunohistochemistry (n=12); these were tested for the BRAF V600E mutation and KRAS mutations. Compared with BRAF wild-type adenocarcinomas, BRAF-mutated adenocarcinomas more frequently demonstrated adverse histologic features such as lymphatic invasion (16/20, 80% vs. 75/161, 47%; P=0.008), mean number of lymph node metastases (4.5 vs. 2.2; P=0.01), perineural invasion (8/20, 40% vs. 13/161, 8%; P=0.0004), and high tumor budding (16/20, 80% vs. 83/161, 52%; P=0.02). BRAF-mutated adenocarcinomas frequently contained areas with mucinous histology (P=0.0002) and signet ring histology (P=0.03), compared with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas. Clinical follow-up data were available for 173 proximal colonic adenocarcinomas with proficient DNA mismatch repair. Patients with BRAF-mutated adenocarcinomas had a median survival of 12.3 months with a 1-year probability of survival of 54% and a 1-year disease-free survival of 56%. Patients with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas had significantly improved overall survival (unadjusted log-rank P=0.03 and unadjusted log-rank P=0.0002, respectively) and disease-free survival (unadjusted log-rank P=0.02 and unadjusted log-rank P=0.02, respectively) compared with patients with BRAF-mutated adenocarcinomas. When adjusting for tumor stage, survival analysis demonstrated that patients with BRAF-mutated adenocarcinoma had a significantly poor overall survival and disease-free survival (hazard ratios 6.63, 95% CI, 2.60-16.94; and 6.08, 95% CI, 2.11-17.56, respectively) compared with patients with KRAS/BRAF wild-type adenocarcinomas. No significant difference in overall or disease-free survival was identified between patients with KRAS-mutated and KRAS/BRAF wild-type adenocarcinomas. Our results demonstrate that BRAF-mutated proximal colon adenocarcinomas with proficient DNA mismatch repair have a dismal prognosis with an aggressive clinical course and often display mucinous differentiation, focal signet ring histology, and other adverse histologic features such as lymphatic and perineural invasion and high tumor budding.