Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors.

Inactivation of deoxyribonucleic acid (DNA) mismatch repair genes, most commonly human mutL homologue 1 (hMLH1) or human mutS homologue 2 (hMSH2), is a recently described alternate pathway in cancer development and progression. The resulting genetic instability is characterized by widespread somatic mutations in tumor DNA, and is termed high-frequency microsatellite instability (MSI-H). Although described in a variety of tumors, mismatch repair deficiency has been studied predominantly in colorectal carcinoma. Most MSI-H colorectal carcinomas are sporadic, but some occur in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and are associated with germline mutations in mismatch repair genes. Until now, the identification of MSI-H cancers has required molecular testing. To evaluate the role of immunohistochemistry as a new screening tool for mismatch repair-deficient neoplasms, the authors studied the expression of hMLH1 and hMSH2, using commercially available monoclonal antibodies, in 72 formalin-fixed, paraffin-embedded tumors that had been tested previously for microsatellite instability. They compared immunohistochemical patterns of 38 MSI-H neoplasms, including 16 cases from HNPCC patients with known germline mutations in hMLH1 or hMSH2, with 34 neoplasms that did not show microsatellite instability. Thirty-seven of 38 MSI-H neoplasms were predicted to have a mismatch repair gene defect, as demonstrated by the absence of hMLH1 and/or hMSH2 expression. This included correspondence with all 16 cases with germline mutations. All 34 microsatellite-stable cancers had intact staining with both antibodies. These findings clearly demonstrate that immunohistochemistry can discriminate accurately between MSI-H and microsatellite-stable tumors, providing a practical new technique with important clinical and research applications.

Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer.

Recent characterization of the molecular genetic basis of hereditary nonpolyposis colorectal cancer provides an important opportunity for identification of individuals and their families with germline mutations in mismatch repair genes. Cancer family history criteria that accurately define hereditary colorectal cancer are necessary for cost-effective testing for germline mutations in mismatch repair genes. The present report describes the results of analysis of 33 colorectal cancer cases/families that satisfy our modified family history criteria (Mount Sinai criteria) for colorectal cancer. Fourteen of these families met the more stringent Amsterdam criteria. Germline MSH2 and MLH1 mutations were identified by the reverse transcription-polymerase chain reaction and the protein truncation test, and confirmed by sequencing. Microsatellite instability analysis was performed on available tumors from affected patients. MSH2 or MLH1 mutations were detected in 8 of 14 Amsterdam criteria families and in 5 of the remaining 19 cases/families that only satisfied the Mount Sinai criteria. Three of the latter families had features of the Muir-Torre syndrome. A high level of microsatellite instability (MSI-H) was detected in almost all (16/18) colorectal cancers from individuals with MSH2 and MLH1 mutations, and infrequently (1/21) in colorectal cancer specimens from cases without detectable mutations. Families with germline MSH2 and MLH1 mutations tended to have individuals affected at younger ages and with multiple tumors. The Amsterdam criteria are useful, but not sufficient, for detecting hereditary colorectal cancer families with germline MSH2 and MLH1 mutations, since a proportion of cases and families with mutations in mismatch repair genes will be missed. Further development of cancer family history criteria are needed, using unbiased prospectively collected cases, to define more accurately those who will benefit from MSH2 and MLH1 mutation analysis.

Autotransplantation of epithelial cells in the pig via an aerosol vehicle.

A new method of delivery of epithelial suspensions with use of an aerosolization apparatus was examined in the pig. Full-thickness pig skin was harvested, and an epithelial suspension was created using standard techniques of dispase and trypsin. Twenty-four hours after skin harvest, four full-thickness wounds were created on the flanks of the pig. The control wound was sprayed with a solution without epithelial cells. The three experimental wounds were sprayed with epithelial cell suspensions (integral 10(6) cells/suspension). Weekly evaluation with photographs, biopsies, and tracings were done for 4 weeks. At 10 weeks, the entire process was repeated with new wounds on the pig's back. Thirty-five wounds in five pigs were evaluated: 10 control (5 flank, 5 back) and 25 experimental (15 flank, 10 back). Control wounds healed by contraction alone, with epithelium at the edges only. After 4 weeks, an open area remained. Central epithelial islands developed in experimental wounds at 2 weeks. These islands coalesced to close the wounds by 4 weeks. Histology at 1 week showed groups of epithelial cells deeply embedded in granulation tissue. These groups became immature epithelial layers on the surface by 2 weeks, and all layers of epithelium were present by 4 weeks. Overall, flank experimental wounds epithelialized sooner, but contracted at the same rate as control wounds. In conclusion, epithelial cells can be delivered by an aerosolization apparatus and remain viable and proliferative in a pig model.

Overexpression of the nonpancreatic secretory group II PLA2 messenger RNA and protein in colorectal adenomas from familial adenomatous polyposis patients.

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated in various inflammatory processes and has been shown to release arachidonic acid for prostaglandin biosynthesis. In human colorectal cancer, both arachidonic acid and eicosanoid levels are elevated. Recently, sPLA2 has been identified as a candidate gene that modifies the Apc gene in the Min mouse, a murine model for familial adenomatous polyposis (FAP). Loss of sPLA2 gene function results in susceptibility to the Min phenotype and the formation of multiple intestinal polyps, whereas mice expressing an active sPLA2 gene are resistant to polyp formation. Therefore, there are two potentially contrasting roles for sPLA2 in colon cancer; one is protection against polyp formation, and the other, the release of arachidonic acid for prostaglandin production and subsequent tumor promotion. To investigate these contrasting dual roles of sPLA2, we have examined the expression and sequence of the sPLA2 mRNA in normal mucosa and duodenal and colorectal polyps from FAP patients. In 11 of 14 patients, there was a significant increase in sPLA2 mRNA levels in the adenoma over the normal tissue. In some cases, there was over 100-fold increase in mRNA levels in the adenoma compared with normal tissue. Analysis of multiple adenomatous polyps from individual patients revealed that not all polyps contained elevated levels of sPLA2 mRNA. Immunoblot analysis also showed that sPLA2 protein expression was elevated in adenoma over normal tissue in five of six FAP patients analyzed. Furthermore, sequence analysis of sPLA2 mRNA present in these samples did not reveal mutations in the coding region. The implications of the up-regulation of sPLA2 in FAP is not clear, but unlike the Min mouse model, it does not seem to have a significant effect on polyp formation. In contrast, the high level of sPLA2 expression is more likely contributing to the elevated levels of arachidonic acid found in colorectal cancer and, in conjunction with the elevated expression of cyclooxygenase-2, could be another factor in tumor formation.

Superior mesenteric vein thrombosis after the Whipple procedure: an aggressive, combined treatment approach.

It is now recognized that occlusion of the mesenteric veins not only may complicate a number of disease processes but may occur as a life-threatening complication after abdominal surgery. A 32-year-old woman had mesenteric venous thrombosis after resection of a duodenal inflammatory pseudotumour by pancreatoduodenectomy. She recovered fully after treatment, which consisted of thrombectomy, flushing with urokinase and intravenous administration of heparin. Papaverine infused for 4 days substantially improved bowel viability. Current concepts in mesenteric vein occlusion and the principles of clinical management are reviewed.

Treatment of left-sided ulcerative colitis with butyrate enemas: a controlled trial.

BACKGROUND: The colonic mucosa is highly dependent upon the presence of luminal nutrients. This dependence is most marked in the distal colon. The major luminal nutrients are short chain fatty acids that are produced as a by-product of colonic fermentation of carbohydrates. Butyrate appears to be the short chain fatty acid most avidly metabolized by the colonic mucosa. It has been suggested that ulcerative colitis is, at least in part, related to an energy deficiency state of the colonic mucosa which may be secondary to impaired short chain fatty acid production, uptake or utilization. The objective of this study was to determine if butyrate given as enema therapy is effective in the treatment of active distal ulcerative colitis. METHODS: Thirty-eight patients with distal ulcerative colitis were randomly assigned to receive nightly butyrate (n = 19) or saline/placebo (n = 19) enemas. Butyrate enemas consisted of 60 mL of 80 mM sodium butyrate titrated to a pH of 7.0. Patients were assessed clinically and endoscopically at baseline and at 3 and 6 weeks follow-up. Pre- and post-treatment mucosal biopsies were assessed histologically. Response to therapy was determined by changes in a 12-point clinical disease activity index score based on patient symptoms, endoscopic mucosal appearance and physicians' global assessment. RESULTS: Clinical improvement was noted in seven of 19 (37%) butyrate-treated patients and nine of 19 (47%) placebo-treated patients (P = 0.51). Clinical remission was achieved in three patients in each group (16%). No toxicity was observed in either treatment arm. CONCLUSIONS: The results suggests that once nightly 60 mL butyrate enemas (80 mmol/L) are not efficacious in the treatment of distal ulcerative colitis.

A preventive registry for hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a genetic disorder characterized by a strong family history of colorectal and extracolonic cancers, usually at a young age. This article presents a new provincial service for families with HNPCC. The Steve Atanas Stavro Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital is accruing patients that meet a set of criteria establishing a putative diagnosis of HNPCC. The objectives of the Registry are to develop and assess patient pedigrees, to coordinate screening procedures for at-risk persons, to maintain a prospective database of patient information, to provide education and support for families and to contribute to research. To date, surgeons and patients are the most common referral sources, while oncologists and geneticists are the least common. The ultimate goal of the HNPCC service is the secondary prevention of cancer and a corresponding decrease in mortality for HNPCC family members.

Amnioinfusions in renal agenesis.

BACKGROUND: Renal agenesis causes severe oligohydramnios, which results in compression effects, lung hypoplasia, and rapid neonatal death. CASE: We report a case of renal agenesis identified in a fetus in which serial amnioinfusions were employed to prevent pulmonary hypoplasia and compression effects. A total of ten amnioinfusions were performed between 17-33 weeks' gestation. Chorioamnionitis led to preterm delivery at 33 weeks. The infant had no significant pulmonary hypoplasia and none of the compression effects usually associated with the oligohydramnios sequence. Peritoneal dialysis was provided for the infant with a long-term aim of eventual renal replacement therapy, but dialysis was unsuccessful and the infant died at the age of 23 days. Autopsy revealed slightly small lungs and extensive cavitating lesions in the brain, which were presumed to be of peripartum or antenatal origin. The chain of events leading to this unusual course of action is described, and the ethical aspects are outlined. CONCLUSION: At present, this type of procedure is not an appropriate intervention in cases of renal agenesis, and such management is strongly discouraged.

Acute typhlitis in an immunocompromised host. Report of an unusual case and review of the literature.

A case of acute typhlitis arising in a neutropenic male is described. Light and electron microscopy reveal findings resembling those of an early stage of malakoplakia. The coexistence of these two uncommon lesions suggests a pathogenetic role in the development of septicemia in immunocompromised hosts.

Palisading granulomas associated with lung cancer.

A granulomatous response to neoplastic structures was found in three cases of resected small-cell anaplastic carcinoma of the lung. This consisted of almost continuous rims of palisading epithelioid cells surrounding viable, necrotizing, and necrotic tumor nests. None of the patients had received chemotherapy or radiation treatment prior to surgery, and no clinical, microbiological, or histological evidence of tuberculosis, fungal infection, or rheumatoid disease was found. The granulomatous rim seems to be a response to spontaneous tumor decay.

Existence of a connecting system in the flagellar apparatus and the accessory structures of Trypanosoma evansi.

The three-dimensional architecture of a quartet of microtubules (QM) of Trypanosoma evansi and its related structures was examined with a transmission electron microscope (TEM). The initiation of the QM as a successive bundle of microtubules was confirmed at the juxtaposition of the second barren body (SBB). After connecting with the distal part of the basal body (BB), the QM ascended to the level of the terminal plate (TP) and gained access most closely to the inner membrane of the flagellar pocket (FP) and formed the special structure together with macula adherens (MA) and the electron dense linear substance (EDLS); then it follows its way around the FP from the posterior to the anterior and fills the subpellicular space at the underneath of the flagellum. This special structure disappeared when the associated-flagellum became the extracellular flagellum (EF), where the QM intervened into the row of the subpellicular microtubules (SM). This EDLS is suggested to represent a demarcating line (DL). In the deep cytoplasm of the flagellar root, connections via several microtubules between the BB and the SBB and via electron zonal substance (EDZS) between the BB and the mitochondrion (M) were observed. As a result, a connecting system among the flagellar apparatus and its accessory structures seems probable.