Xiuli Liu

Implementation of Universal Microsatellite Instability and Immunohistochemistry Screening for Diagnosing Lynch Syndrome in a Large Academic Medical Center

PURPOSE In 2009, the Evaluation of Genomic Applications in Practice and Prevention recommended that all colorectal cancers (CRCs) be screened for Lynch syndrome (LS) through microsatellite instability (MSI) or immunohistochemistry (IHC). No studies report how this process is implemented on a health system-wide basis. METHODS Since 2004, Cleveland Clinic has screened CRC specimens with MSI/IHC. Between January 2004 and July 2007, MSI/IHC results went only to the colorectal surgeon (approach 1). Between August 2007 and June 2008, colorectal surgeons and a genetic counselor received the MSI/IHC results, and the counselor e-mailed the colorectal surgeon regarding appropriate patients for genetic counseling (GC) referral (approach 2). After July 2008, the colorectal surgeon and counselor received MSI/IHC results, but the counselor contacted the patient to facilitate referral (approach 3). In approaches 2 and 3, patients were presumed to have sporadic CRC if the tumor lacked MLH1 expression and was also BRAF mutated or if the patient was diagnosed at age greater than 72 years and had no cancer family history. RESULTS Abnormal MSI/IHC results occurred in 178 (16%) of 1,108 patients. In approach 1, 21 (55%) of 38 patients with abnormal MSI/IHC were referred for GC, 12 (32%) of 38 underwent GC, and 10 (26%) of 38 underwent genetic testing (GT). In approach 2, nine (82%) of 11 patients were referred for GC, seven (64%) of 11 underwent GC, and five (45%) of 11 underwent GT. In approach 3, 56 (100%) of 56 patients were referred for GC, 40 (71%) of 56 underwent GC, and 37 (66%) of 56 underwent GT. Time from referral to GC was 10-fold quicker in approach 3 than approach 1. CONCLUSION Implementation of universal MSI/IHC with GC/GT, along with effective multidisciplinary communication and plans of responsibility for patient contact, resulted in increased identification of patients with LS.

BRAF mutations in colorectal cancer are associated with distinct clinical characteristics and worse prognosis.

BACKGROUND: Colorectal cancer is a heterogeneous disease with multiple underlying genetic mutations causing different clinical phenotypes. Mutation in the BRAF oncogene is a key step in malignant transformation within the methylator pathway to colorectal cancer. However, there is a paucity of information about BRAF mutant colorectal tumors. OBJECTIVE: This study defines the clinical characteristics and oncologic outcome associated with colorectal cancer BRAF mutations. DESIGN: Colorectal adenocarcinomas from a single-institution frozen-tumor biobank were studied. Genomic DNA was isolated and analyzed for mutations in the BRAF oncogene by polymerase chain reaction amplification followed by direct sequencing. A sample was classified as mutant if any of the tested loci were mutated. Patient and tumor characteristics were recorded including patient age, sex, tumor location, tumor differentiation, and microsatellite instability. MAIN OUTCOME MEASURES: Statistical associations with BRAF mutant tumors were determined by the Fisher exact probability test, &khgr;2 test, or Wilcoxon analysis. Kaplan-Meier estimates and multivariate Cox regression analysis were performed for overall survival. RESULTS: Four hundred seventy-five colorectal adenocarcinomas were included in the study population; 56 samples harbored a BRAF mutation (12%). There were significant differences between BRAF wild-type and mutant tumors in age (66 vs 75 years, p = 0.004), female sex (44% vs 71%, p < 0.001), proximal tumor location (44% vs 95%, p < 0.001), and frequency of microsatellite instability (16% vs 76%, p < 0.001). There was no difference in cancer stage between BRAF mutant and wild-type populations. Survival data were analyzed for 322 patients with stage I to III disease, and patients with a BRAF mutation had decreased overall survival than those without a mutation (p = 0.018). With the use of Cox regression analysis, BRAF mutation conferred a worse overall survival (HR 1.79, CI 1.05–3.05, p = 0.03) independent of microsatellite instability status. CONCLUSIONS: BRAF mutations in colorectal cancers are associated with distinct clinical characteristics and worse prognosis.

POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMAS OF THE PANCREAS: A CLINICOPATHOLOGIC ANALYSIS OF 44 CASES

Background:In the pancreas, poorly differentiated neuroendocrine carcinomas include small cell carcinoma and large cell neuroendocrine carcinoma and are rare; data regarding their pathologic and clinical features are very limited. Design:A total of 107 pancreatic resections originally diagnosed as poorly differentiated neuroendocrine carcinomas were reassessed using the classification and grading (mitotic rate/Ki67 index) criteria put forth by the World Health Organization in 2010 for the gastroenteropancreatic system. Immunohistochemical labeling for neuroendocrine and acinar differentiation markers was performed. Sixty-three cases were reclassified, mostly as well-differentiated neuroendocrine tumor (NET) or acinar cell carcinoma, and eliminated. The clinicopathologic features and survival of the remaining 44 poorly differentiated neuroendocrine carcinomas were further assessed. Results:The mean patient age was 59 years (range, 21 to 82 y), and the male/female ratio was 1.4. Twenty-seven tumors were located in the head of the pancreas, 3 in the body, and 11 in the tail. The median tumor size was 4 cm (range, 2 to 18 cm). Twenty-seven tumors were large cell neuroendocrine carcinomas, and 17 were small cell carcinomas (mean mitotic rate, 37/10 and 51/10 HPF; mean Ki67 index, 66% and 75%, respectively). Eight tumors had combined components, mostly adenocarcinomas. In addition, 2 tumors had components of well-differentiated NET. Eighty-eight percent of the patients had nodal or distant metastatic disease at presentation, and an additional 7% developed metastases subsequently. Follow-up information was available for 43 patients; 33 died of disease, with a median survival of 11 months (range, 0 to 104 mo); 8 were alive with disease, with a median follow-up of 19.5 months (range, 0 to 71 mo). The 2- and 5-year survival rates were 22.5% and 16.1%, respectively. Conclusions:Poorly differentiated neuroendocrine carcinoma of the pancreas is a highly aggressive neoplasm, with frequent metastases and poor survival. Most patients die within less than a year. Most (61%) are large cell neuroendocrine carcinomas. Well-differentiated NET and acinar cell carcinoma are often misdiagnosed as poorly differentiated neuroendocrine carcinoma, emphasizing that diagnostic criteria need to be clearly followed to ensure accurate diagnosis.

Histological evaluation in ulcerative colitis

This review summarizes diagnostic problems, challenges and advances in ulcerative colitis (UC). It emphasizes that, although histopathological examination plays a major role in the diagnosis and management of UC, it should always be interpreted in the context of clinical, endoscopic, and radiological findings. Accurate diagnosis requires knowledge of the classic morphological features of UC, as well as a number of atypical pathological presentations that may cause mis-classification of the disease process, either in resection or biopsy specimens. These atypical pathological presentations include rectal sparing and patchiness of disease at initial presentation of UC in pediatric patients or in the setting of medically treated UC, cecal or ascending colon inflammation in left-sided UC, and backwash ileitis in patients with severe ulcerative pancolitis. Loosely formed microgranulomas, with pale foamy histiocytes adjacent to a damaged crypt or eroded surface, should not be interpreted as evidence of Crohn’s disease. Indeterminate colitis should only be used in colectomy specimens as a provisional pathological diagnosis. Patients with UC are at risk for the development of dysplasia and carcinoma; optimal outcomes in UC surveillance programs require familiarity with the diagnostic criteria and challenges relating to UC-associated dysplasia and malignancy. Colon biopsy from UC patients should always be evaluated for dysplasia based on cytological and architectural abnormalities. Accurate interpretation and classification of dysplasia in colon biopsy from UC patients as sporadic adenoma or UC-related dysplasia [flat, adenoma-like, or dysplasia-associated lesion or mass (DALM)] requires clinical and endoscopic correlation. Isolated polypoid dysplastic lesions are considered to be sporadic adenoma if occurring outside areas of histologically proven colitis, or adenoma-like dysplasia if occurring in the diseased segment. Recent data suggest that such lesions may be treated adequately by polypectomy in the absence of flat dysplasia in the patient. UC patients with DALM or flat high-grade dysplasia should be treated by colectomy because of the high probability of adenocarcinoma. The natural history of low-grade dysplasia (LGD) is more controversial: while multifocal LGD, particularly if detected at the time of initial endoscopic examination, is treated with colectomy, unifocal flat LGD detected during surveillance may be managed by close follow-up with increased surveillance. The surveillance interval and treatment options for UC patients with dysplasia are reviewed in detail.

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.

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. Copyright

Lynch syndrome screening in newly diagnosed colorectal cancer in general pathology practice: from the revised Bethesda guidelines to a universal approach.

Abstract Objective. Lynch syndrome (LS) is the most common hereditary form of colorectal cancer (CRC). The revised 2004 Bethesda guidelines were developed to identify potential LS patients. This study aimed to retrospectively evaluate utilization and adequacy of the guidelines in general pathology practice and to determine if a universal LS screening approach increased the potential LS detection rate in newly diagnosed CRCs. Material and Methods. Included were 445 primary CRCs surgically resected from November 2006 to March 2009, when reflex microsatellite instability (MSI) testing was based on histomorphology and age as well as 145 CRCs resected from July 2009 to July 2010 when a universal LS testing paradigm was used. Reflex MSI testing rates and MSI testing results were determined. Results. The overall LS screening rate from November 2006 to March 2009 was 34.8%, and the extrapolated microsatellite instability-high (MSI-H) rate was 8.5% (38/445). Strict adherence to the revised Bethesda guidelines, that is, without testing CRC diagnosed in patients ‡60 years, would have missed 26 (68.4%) MSI-H CRCs. The overall LS screening rate from July 2009 to July 2010 was 76.3% and the MSI-H rate was 20.6% (30/145). Compared with the MSI tested group, the untested group had more CRCs removed by local excision (22.2% vs. 4.8%, p = 0.00035). Conclusion. The revised Bethesda guidelines are inadequate for LS screening when personal and family cancer history is not available to the pathologist, a universal screening paradigm greatly increased the rate of MSI testing and MSI-H CRC detection and CRCs less likely to be screened for LS were those diagnosed in locally excised specimens.

Prevalence of germline PTEN, BMPR1A, SMAD4, STK11, and ENG mutations in patients with moderate-load colorectal polyps

BACKGROUND & AIMS Gastrointestinal polyposis is a common clinical problem, yet there is no consensus on how to best manage patients with moderate-load polyposis. Identifying genetic features of this disorder could improve management and especially surveillance of these patients. We sought to determine the prevalence of hamartomatous polyposis-associated mutations in the susceptibility genes PTEN, BMPR1A, SMAD4, ENG, and STK11 in individuals with ≥5 gastrointestinal polyps, including at least 1 hamartomatous or hyperplastic/serrated polyp. METHODS We performed a prospective, referral-based study of 603 patients (median age: 51 years; range, 2-89 years) enrolled from June 2006 through January 2012. Genomic DNA was extracted from peripheral lymphocytes and analyzed for specific mutations and large rearrangements in PTEN, BMPR1A, SMAD4, and STK11, as well as mutations in ENG. Recursive partitioning analysis was used to determine cutoffs for continuous variables. The prevalence of mutations was compared using Fishers exact test. Logistic regression analyses were used to determine univariate and multivariate risk factors. RESULTS Of 603 patients, 119 (20%) had a personal history of colorectal cancer and most (n = 461 [76%]) had <30 polyps. Seventy-seven patients (13%) were found to have polyposis-associated mutations, including 11 in ENG (1.8%), 13 in PTEN (2.2%), 13 in STK11 (2.2%), 20 in BMPR1A (3.3%), and 21 in SMAD4 (3.5%). Univariate clinical predictors for risk of having these mutations included age at presentation younger than 40 years (19% vs 10%; P = .008), a polyp burden of ≥30 (19% vs 11%; P = .014), and male sex (16% vs 10%; P = .03). Patients who had ≥1 ganglioneuroma (29% vs 2%; P < .001) or presented with polyps of ≥3 histologic types (20% vs 2%; P = .003) were more likely to have germline mutations in PTEN. CONCLUSIONS Age younger than 40 years, male sex, and specific polyp histologies are significantly associated with risk of germline mutations in hamartomatous-polyposis associated genes. These associations could guide clinical decision making and further investigations.

Distinct clinicohistologic features of inflammatory bowel disease-associated colorectal adenocarcinoma: in comparison with sporadic microsatellite-stable and Lynch syndrome-related colorectal adenocarcinoma.

Long-standing inflammatory bowel disease (IBD), either ulcerative colitis or Crohn disease, is associated with a high risk of developing colorectal adenocarcinoma (CAC). However, histomorphology of IBD-associated CAC has not been thoroughly examined, and it is unclear whether and how these patients should be screened for Lynch syndrome (LS). We evaluated the demographic and morphologic features of 108 IBD-associated CACs, including ulcerative colitis-associated (n=95) and Crohn disease-associated CACs (n=13), against 93 control cases of sporadic microsatellite-stable (MSS) CAC, 20 cases of sporadic microsatellite instability high (MSI-H) CAC, and 23 CAC cases of LS. The mean age of patients with IBD-associated CAC was 50 years, which was lower compared with the mean age of 63.7 years of the sporadic MSS controls and 76.5 years of the sporadic MSI-H group but not statistically different from that of the LS patients. Synchronous CACs were noted in 20.4% of the IBD patients and 13% of LS patients but in only 2.1% of the sporadic MSS controls and in none of the MSI-H patients. Right-sided CACs were significantly less frequent in the IBD group than in sporadic MSS controls, MSI-H group, and LS patients (P<0.05 for all). In contrast to sporadic MSS CAC, IBD-associated CACs are characterized by lack of tumor necrosis, Crohn-like reaction, tumor histologic heterogeneity, the presence of mucin, and signet ring cell differentiation and tumor well differentiation. The histomorphologic similarity among IBD-associated and MSI-H tumors, either sporadic MSI-H or LS-related, is independent of MSI status. The young age of patients with IBD-associated CAC and the morphological similarities among IBD-associated, sporadic MSI-H, and LS-related CAC suggest that an age-based and morphology-based strategy before the screening test for LS may be less effective in IBD patients than in the non-IBD population.

Predictive and prognostic roles of ribonucleotide reductase M1 in resectable pancreatic adenocarcinoma

Ribonucleotide reductase M1 (RRM1) is an important molecule in different types of cancer. The objective of this study was to evaluate the predictive roles of RRM1 in the survival of patients with resectable pancreatic adenocarcinoma who received treatment with gemcitabine or nongemcitabine adjuvant therapy.