Sanjay Kakar

SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Protocol for the Examination of Specimens From Patients With Primary Carcinoma of the Colon and Rectum

The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the “Surgical Pathology Cancer Case Summary (Checklist)” portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice. The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with these documents. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of this document.

Significance of intraepithelial lymphocytosis in small bowel biopsy samples with normal mucosal architecture

OBJECTIVES:The aim of this study was to determine the specificity of increase in intraepithelial lymphocytes (IELs) with normal villous architecture in small bowel biopsy samples for diagnosis of gluten sensitivity (GS) and its significance in the absence of GS.METHODS:Small bowel biopsy samples from 43 patients with increased IELs and no other pathology were reviewed. Patients with prior diagnosis of GS were excluded. A group of 46 patients with normal duodenal biopsy during the same period served as controls. The clinical records of patients and controls were examined for presenting symptoms, laboratory tests, and final clinicopathological diagnosis. Immunohistochemical characterization of IELs was performed in 13 cases.RESULTS:Four (9.3%) patients had GS based on positive IgA antiendomysial antibodies (n = 3) and favorable response to gluten-free diet (n = 4). One patient (2.2%) had partially treated tropical sprue; six patients (14%) had disorders of immune regulation including Hashimotos thyroiditis (n = 2) and one case each of Graves’ disease, rheumatoid arthritis, psoriasis, and multiple sclerosis; and six patients (14%) were on nonsteroidal anti-inflammatory drugs (NSAIDs). In contrast, none of the control subjects had GS (p = 0.05), tropical sprue, or immunoregulatory disorders (p = 0.011), and one (2.2%) was on NSAIDs (p = 0.04). Increased IELs were also observed in Crohns disease, lymphocytic/collagenous colitis, and bacterial overgrowth, but the association did not reach statistical significance. Histological features (number and distribution of IELs, crypt mitoses) and immunophenotypic analysis of IELs did not reliably distinguish GS-related from non–GS-related causes of increased IELs.CONCLUSION:Intraepithelial lymphocytosis in an otherwise normal small bowel biopsy is somewhat nonspecific, but in nearly 10% of cases can be the initial presentation of GS. Therefore all patients with this finding should be investigated for GS. Increased IELs may also be associated with autoimmune disorders and NSAIDs.

Histological patterns in drug-induced liver disease

The diagnosis of drug-induced liver injury (DILI) is a challenging problem, often confounded by incomplete clinical information and the difficulty of eliciting exposure to herbal products, over-the-counter agents and toxins. The task is further rendered difficult on biopsy, as drugs can mimic all the patterns found in primary liver disease. Acute hepatitis, with or without cholestasis, is the most common histological pattern of DILI, and drugs such as acetaminophen are the leading causes of acute liver failure. Most cases of DILI resolve on discontinuation of the drug, but recovery can take months or rarely the disease can progress despite drug withdrawal. Drugs such as methotrexate can lead to chronic hepatitis and cirrhosis, while others such as minocycline, nitrofurantoin and methyldopa are implicated in autoimmune hepatitis. Prolonged cholestasis and ductopenia resembling primary chronic biliary disease can occur. Drug-induced steatohepatitis is also an uncommon pattern, but is well described with drugs such as amiodarone and irinotecan. In the presence of risk factors such as obesity and diabetes, some drugs such as tamoxifen, oestrogens and nifedipine can precipitate or exacerbate steatohepatitis. Other observed patterns include granulomatous hepatitis, vascular injury (eg, sinusoidal obstruction syndrome), Ito cell lipidosis and neoplasms (eg, adenomas).

Utility and limitations of glypican-3 expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation spectrum

Glypican-3 is a heparin sulfate proteoglycan normally expressed in fetal liver and placenta, but not in normal adult liver. Preliminary studies have shown that glypican-3 can be useful for the diagnosis of hepatocellular carcinoma. We performed immunohistochemistry for glypican-3 on 80 resection cases of hepatocellular lesions to examine the utility of glypican-3 immunohistochemistry in hepatocellular carcinoma at two ends of the differentiation spectrum. Staining was compared to Hep Par 1 in poorly differentiated cases. Glypican-3 was expressed in 46 (79%) hepatocellular carcinomas (56, 83 and 89% of well, moderately and poorly differentiated respectively) and seven (64%) fibrolamellar carcinomas. Of the 16 well differentiated cases, 10 closely resembled adenoma and were diagnosed due to focal abnormalities and/or loss of reticulin. Glypican-3 expression was seen in 50% in this group. Hepatocellular carcinomas arising in cirrhotic liver were more likely to be glypican-3 positive (91 vs 57%, P=0.004). All hepatic adenomas and macroregenerative nodules were negative, and three (43%) high grade dysplastic nodules were positive. Focal staining was seen in regenerative nodules in four (11%) cirrhosis cases. Glypican-3 was significantly more sensitive than Hep Par 1 for diagnosis of poorly differentiated hepatocellular carcinomas (89 vs 63%, P=0.02). The difference was more significant when only cases with diffuse positive staining were considered (83 vs 21%, P<0.001). In conclusion, glypican-3 has high sensitivity for the diagnosis of hepatocellular carcinoma, but is less sensitive in the extremely well differentiated hepatocellular carcinoma and fibrolamellar variant of hepatocellular carcinoma. Caution should be exercised in using glypican-3 in biopsy specimens as cirrhotic nodules can show strong expression. Glypican-3 can be especially useful in the identification of poorly differentiated hepatocellular carcinoma as it has higher sensitivity compared to Hep Par 1.

Association of Large Serrated Polyps With Synchronous Advanced Colorectal Neoplasia

OBJECTIVES:Serrated polyps of the colorectum are a histologically and genetically heterogeneous group of lesions, which include classic hyperplasic polyps, sessile serrated adenomas (SSAs), and traditional serrated adenomas. Accumulating evidence suggests that they may have different malignancy potentials. This study sought to determine the association between the presence of large serrated colorectal polyps and synchronous advanced colorectal neoplasia.METHODS:Among 4,714 asymptomatic subjects who underwent screening colonoscopy, cases of advanced colorectal neoplasia (tubular adenoma ≥1 cm, adenoma with any villous histology, adenoma with carcinoma in situ / high-grade dysplasia, or invasive adenocarcinoma) were compared with controls without advanced neoplasia with respect to candidate predictors, including age, sex, family history of colorectal cancer, body mass index, the presence and number of small tubular adenomas (<1 cm), the presence of multiple small serrated polyps (<1 cm), and the presence of large serrated polyps (≥1 cm). Independent predictors of advanced neoplasia were determined by multivariate logistic regression analysis.RESULTS:Among 467 cases and 4,247 controls, independent predictors of advanced colorectal neoplasia were increasing age (odds ratio (OR)=4.51; 95% confidence interval (CI), 1.43–14.3; P=0.01 for subjects ≥80 years vs. 50–54 years of age); non-advanced tubular adenomas (OR=2.33; 95% CI 1.37–3.96, P=0.0017 for 3 or more); and large serrated polyps (OR=3.24; 95% CI 2.05–5.13, P<0.0001). In total, 109 subjects (2.3% of the study population) had large serrated polyps. Right- and left-sided large serrated polyps had a similar association with advanced colorectal neoplasia (OR=3.38 vs. 2.66, P=0.62).CONCLUSIONS:Large serrated polyps are strongly and independently associated with synchronous advanced colorectal neoplasia. Our results suggest that large serrated polyps may be a marker for advanced colorectal neoplasia. Further studies are needed to determine whether the association with advanced neoplasia differs among subsets of serrated polyps, particularly SSAs and classic hyperplastic polyps.

The fate of low grade dysplasia in ulcerative colitis

Abstract OBJECTIVE: The optimal strategy for the management of definite low grade dysplasia (LGD) detected in surveillance in ulcerative colitis (UC) is unknown, because the natural history of LGD has not been well described. METHODS: We reviewed the Mayo Clinic records of patients with UC found to have flat LGD between 1990 and 1993 in whom a nonoperative strategy was pursued for 2 months or more. RESULTS: Eighteen patients with UC and LGD were observed for a median of 32 months. Nine of 18 patients identified with UC and LGD developed advanced neoplastic lesions during follow-up, which were defined as adenocarcinoma, raised dysplasia, or high grade dysplasia. The cumulative incidence of progression to an advanced lesion was 33% at 5 yr (95% CI = 9–56%). Only one patient developed adenocarcinoma, diagnosed 74 months after his initial finding of LGD and 20 months after his last surveillance exam. Besides this patient, adenocarcinoma was not detected in the colectomy specimens of 13 other patients who underwent surgery. Colectomies were performed for dysplasia or cancer in seven patients, active colitis in five patients, and unknown reasons in two patients. Four patients did not have colectomies. CONCLUSIONS: Neoplastic progression in patients with UC and LGD is common. Total proctocolectomy should be offered to all patients with flat LGD. Our study illustrates numerous pitfalls in the practice of surveillance.

Clinicopathologic features and survival in fibrolamellar carcinoma: comparison with conventional hepatocellular carcinoma with and without cirrhosis.

Fibrolamellar carcinoma arises in noncirrhotic livers of young individuals and has been considered to be less aggressive than conventional hepatocellular carcinoma. This study compares survival and clinicopathologic features of fibrolamellar carcinoma with hepatocellular carcinoma arising in noncirrhotic and cirrhotic livers. Clinical and pathologic features including age, gender, tumor size, stage and survival were recorded in 20 resected cases of fibrolamellar carcinoma. Survival was compared with resected hepatocellular carcinoma without (n=32) and with cirrhosis (n=30). Proliferative activity was determined by immunohistochemistry for Ki-67. In all, 12 (60%) patients with fibrolamellar carcinoma died during follow-up; the 5-year survival was 45%. Mortality in fibrolamellar carcinoma was higher with metastatic disease at presentation (6/7, 86% vs 5/13, 39%, P=0.06). Age, gender and tumor size did not correlate with survival. The 5-year (45 vs 56%, P=0.4) as well as overall survival (40 vs 56.3%, P=0.3) was similar in fibrolamellar carcinoma and hepatocellular carcinoma without cirrhosis. The 5-year and overall survival in hepatocellular carcinoma with cirrhosis was 27 and 23.3%, respectively, which was not significantly different compared to fibrolamellar carcinoma (P=0.2). Among the cases without metastases at presentation, 5-year survival in fibrolamellar carcinoma (62%) and hepatocellular carcinoma without cirrhosis (57%) was significantly better (P=0.03) than hepatocellular carcinoma with cirrhosis (27%). The mean Ki-67 index was similar in all three groups (P=0.1). In conclusion, fibrolamellar carcinoma is an aggressive neoplasm with 45% 5-year survival and overall mortality of 60%. Nearly half the patients develop lymph node or distant metastasis. The prognosis of fibrolamellar carcinoma is similar to conventional hepatocellular carcinoma. Among nonmetastatic cases, the prognosis is better in fibrolamellar carcinoma and hepatocellular carcinoma without cirrhosis compared to hepatocellular carcinoma with cirrhosis. The better outcome in fibrolamellar carcinoma appears to be due to the absence of cirrhosis rather than its distinct clinicopathologic features.

The Relationship between Global Methylation Level, Loss of Heterozygosity, and Microsatellite Instability in Sporadic Colorectal Cancer

Purpose: The relationship between global hypomethylation, chromosomal instability (CIN), and microsatellite instability (MSI) remains unclear in colorectal cancer. The aim of this study was to investigate the relationship between global methylation status, loss of heterozygosity (LOH), and MSI in sporadic colorectal cancer. Experimental Design: We determined global methylation levels in 80 sporadic colorectal cancers, 51 adjacent normal tissues, and 20 normal tissues using the long interspersed nucleotide elements–combined bisulfite restriction analysis method. We also analyzed 80 colorectal cancers for MSI status and LOH at chromosomes 5q21, 8p12-22, 17p13, and 18q21. Results: We identified 14 cases of MSI (17.5%) and 58 cases of LOH (72.5%). LOH was observed more frequently in microsatellite stable (MSS) cancers than in MSI cancers at all loci. Colorectal cancers showed significantly lower global methylation levels than did normal tissues (41.0 ± 9.7% versus 54.3 ± 6.5%; P < 0.001). MSS cancers showed significantly lower global methylation levels when compared with MSI cancers (39.5 ± 9.4% versus 48.2 ± 8.2%; P = 0.003). Tumors with global hypomethylation (with ≤40% of methylation levels) had a significantly increased number of chromosomal loci with LOH than did tumors without global hypomethylation (1.9 versus 0.9; P < 0.001); 11 tumors (13.9%) lacked both MSI and LOH. This subgroup had significantly higher global methylation levels (46.8 ± 8.7%) than did MSS cancers with LOH (38.0 ± 9.0%; P = 0.006). Conclusions: These data showed a significant association between global hypomethylation and chromosomal instability in sporadic colorectal cancer. This suggests that global hypomethylation plays an important role in inducing genomic instability in colorectal carcinogenesis.

Mucinous carcinoma of the colon: correlation of loss of mismatch repair enzymes with clinicopathologic features and survival

Colorectal carcinoma with microsatellite instability (MSI-H) has a characteristic clinicopathologic profile, typically forming right-sided, lymphocyte-rich tumors that are often mucinous. Mucinous histology in general has been linked to adverse prognosis in some studies, but not in others. MSI-H carcinoma, in contrast, has a better prognosis than microsatellite stable carcinoma in most studies. We assessed the relationship between MSI status, clinicopathologic features and outcome for 248 consecutive patients with resected mucinous carcinoma. All cases were reviewed to confirm mucinous histology. Immunohistochemical stains for DNA mismatch repair enzymes hMLH1, hMSH2 and hMSH6 were performed on a representative block from each case. Tumors lacking expression of a mismatch repair enzyme were designated MSI-H; all others were classified as microsatellite stable. Age, sex, tumor size, site, grade, stage, growth pattern, Crohns-like reaction, vascular invasion and number of tumor-infiltrating lymphocytes were evaluated without knowledge of MSI status or patient outcome. 72 (29.3%) mucinous carcinomas were MSI-H. Compared to microsatellite stable mucinous cancers, they were more likely to be right-sided (83.3 vs 48.6%, P<0.001), have a Crohns -like reaction (65.7 vs 29.8%, P<0.001) and have many tumor infiltrating lymphocytes (72.2 vs 20.8%, P<0.001). MSI-H mucinous cancers presented more often as localized disease (66.7 vs 38.1%, P<0.001) and less often with lymph node (26.4 vs 44.9%) or distant (4.2 vs 16.5%) metastases. In univariate analysis, MSI had a favorable effect on age-adjusted survival (hazard ratio 0.597, P=0.02). In multivariate analysis, age, grade, Crohns-like reaction and stage were independent predictors of survival, but MSI status was not. In conclusion, MSI-H mucinous carcinomas are right-sided, low-stage tumors with Crohns-like reaction and tumor-infiltrating lymphocytes. The outcome for MSI-H mucinous carcinoma is better than that of microsatellite-stable mucinous carcinoma, but MSI status is not an independent predictor of survival.