Deepa T. Patil

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.

Gastrointestinal Stromal Tumor: Advances in Diagnosis and Management

CONTEXT Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and should be differentiated from other mesenchymal tumors. They harbor specific activating mutations in the KIT or platelet-derived growth factor receptor α ( PDGFRA ) receptor tyrosine kinases, which makes them responsive to pharmacologic inhibitors, such as imatinib mesylate and sunitinib malate. OBJECTIVES To provide a comprehensive review of the pathogenesis of GIST and the underlying principles of targeted therapy, to review the salient histologic and immunohistochemical features that facilitate the distinction of GIST from other mesenchymal neoplasms of the gastrointestinal tract, and to present the prognostic parameters for risk stratification that guide clinical management. DATA SOURCES Review of the English literature through PubMed as well as personal experience. Photographs were taken from cases encountered at the Cleveland Clinic. CONCLUSIONS The discovery of the KIT -GIST connection has not only improved the diagnostic accuracy of GISTs but also provided us with a better understanding of the histogenesis and molecular pathogenesis of these neoplasms.

Delay to formalin fixation ‘cold ischemia time’: effect on ERBB2 detection by in-situ hybridization and immunohistochemistry

The American Society of Clinical Oncology/College of American Pathologists ERBB2 testing guidelines address several pre-analytical variables known to affect ERBB2 testing accuracy. According to 2010 updated guidelines, the pre-analytical variable of time to tissue fixation (cold ischemia time) should be kept to <1 h, however, little has been published about cold ischemia time and its significance in ERBB2 testing. To that end, this study evaluated ERBB2 status using two different FDA-approved in-situ hybridization methods and an FDA-approved immunohistochemistry (IHC) assay in the largest cohort to date (n=84) of invasive breast carcinomas with tracked cold ischemia time. Cold ischemia time was stratified into four groups (<1 h (n=45), 1–2 h (n=27), 2–3 h (n=6), and >3 h (n=6)) and ERBB2 status was evaluated in each group by IHC (4B5) and by in-situ hybridization methodologies (PathVysion® fluorescence in situ hybridization and the INFORM HER2® dual in situ DNA probe assay). Both in-situ hybridization methods were evaluated using three ERBB2 scoring criteria (dual-probe guidelines, single-probe guidelines, and the FDA package insert scoring instructions). Fluorescence in-situ hybridization (FISH) and INFORM HER2® demonstrated 100% concordance in the detection of ERBB2 amplification by all three scoring guidelines at all cold ischemia time points. Agreement between in-situ hybridization methodologies and IHC was superior using single-probe guidelines compared with dual probe or FDA scoring instructions. In addition, Inform HER2® in-situ hybridization signals were significantly more intense than FISH at all cold ischemia time points, however, no significant loss of either chromosome 17 or ERBB2 signal was detected by FISH or Inform HER2® in-situ hybridization in cold ischemia times up to 3 h. On the basis of our findings, cold ischemia time up to 3 h has no deleterious effect on the detection of ERBB2 via in-situ hybridization or IHC.

Evolution of nonalcoholic fatty liver disease recurrence after liver transplantation

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is the fourth most common indication for liver transplantation. Risk factors for NAFLD can persist and even worsen after liver transplantation. However, the risk and significance of NAFLD recurrence remain unclear. Reported posttransplant NAFLD and NASH recurrence rates vary widely across studies. There is little information detailing the histological evolution of NAFLD recurrence, and the long‐term natural history of NAFLD recurrence is unclear. In this review, we summarize the findings of studies on the prevalence of recurrent NAFLD and its risk factors in the posttransplant setting, and we explore reasons for the discrepant reported recurrence rates. On the basis of currently available data, the relatively low rates of advanced fibrosis and NAFLD‐associated graft loss and the comparability of the survival rates for these patients and patients undergoing transplantation for other diseases suggest that although NAFLD or NASH can recur, the clinical significance of disease recurrence for graft or patient survival may be small. Liver Transpl 18:1147–1153, 2012.

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

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.

A five-marker panel in a multiplex PCR accurately detects microsatellite instability-high colorectal tumors without control DNA.

Microsatellite instability (MSI) testing is used to screen for Lynch syndrome. The current technique for MSI determination requires DNA from normal and neoplastic tissue and is expensive and laborious. Five quasi-monomorphic markers (NR-21, BAT-25, MONO-27, NR-24, and BAT-26) are included in the Promega MSI analysis kit. With the working hypothesis that this 5-marker panel can accurately determine the MSI status of colorectal tumors without using paired control DNA, we evaluated 478 colorectal tumors and divided them into a test group (N=172, colorectal adenocarcinomas) and a validation group (N=306 including 179 colorectal adenocarcinomas and 127 adenomas). The quasi-monomorphic variation range of each marker was generated from the test group (172 normal samples) and used as a reference value in the subsequent interpretation of MSI status in the test and validation groups. Considering the MSI result using a 5-marker panel with paired control DNA as the gold standard, we identified 136 microsatellite stable (MSS) and 36 microsatellite instability-high (MSI-H) colorectal tumors in the test group and 259 MSS and 47 MSI-H colorectal tumors in the validation group. Using the quasi-monomorphic variation range of each marker rather than paired normal DNA, the 5-marker panel identified all MSI-H colorectal tumors in the test and validation groups, when MSI-H was defined as ≥2 unstable markers. Our study demonstrates that the 5-marker panel within a multiplex polymerase chain reaction of the Promega MSI analysis kit accurately identifies all MSI-H and 95.2% MSS colorectal tumors without using paired normal DNA.

Risk for esophageal neoplasia in Barrett's esophagus patients with mucosal changes indefinite for dysplasia.

Patients with Barretts esophagus (BE) are at increased risk for esophageal adenocarcinoma (EAC) and therefore require surveillance. Biopsies are classified as indefinite for dysplasia (IND) when the significance of epithelial abnormalities is uncertain due to inflammation or sampling. Our aim was to characterize the neoplastic risk of IND in BE patients and to identify predictors of neoplastic risk.

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.