Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model.

Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

Animal immunization merges with innovative technologies: A new paradigm shift in antibody discovery.

Animal-derived antibody sources, particularly, transgenic mice that are engineered with human immunoglobulin loci, along with advanced antibody generation technology platforms have facilitated the discoveries of human antibody therapeutics. For example, isolation of antigen-specific B cells, microfluidics, and next-generation sequencing have emerged as powerful tools for identifying and developing monoclonal antibodies (mAbs). These technologies enable not only antibody drug discovery but also lead to the understanding of B cell biology, immune mechanisms and immunogenetics of antibodies. In this perspective article, we discuss the scientific merits of animal immunization combined with advanced methods for antibody generation as compared to animal-free alternatives through in-vitro-generated antibody libraries. The knowledge gained from animal-derived antibodies concerning the recombinational diversity, somatic hypermutation patterns, and physiochemical properties is found more valuable and prerequisite for developing in vitro libraries, as well as artificial intelligence/machine learning methods to discover safe and effective mAbs.

A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired VH and VL sequences from 79% to 96% of antigen-specific B cells. All cognate VH and VL transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired VH and VL transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

Pathology in Patients with Chronic Liver Disease: A Practical Approach to Liver Biopsy Interpretation in Patients with Acute and Chronic Liver Diseases.

Liver biopsy and histologic examination are the mainstay for diagnosing liver diseases, despite advances in imaging and molecular procedures. Liver biopsy can provide useful information regarding the structural integrity and type and degree of injury, disease activity, response to treatment, progression of disease and degree/staging of fibrosis. Liver biopsies evaluate acute and chronic liver diseases, and mass-forming lesions. The role of the pathologist is to integrate clinical, serologic, and biochemical data with morphologic changes and provide a comprehensive diagnosis. This review focuses on basic principles necessary for proper interpretation of liver biopsy specimens in patients with chronic liver disease.

Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway.

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Oncocytic papillary cystadenoma with prominent mucinous differentiation of parotid gland: A case report.

We describe the case of an oncocytic papillary cystadenoma with mucinous differentiation of the parotid gland in a 64-year-old male. Histologically, the tumor exhibited distinctive areas of intracystic papillary growth pattern with microcystic and macrocystic spaces containing mucinous secretions and small crystals. The cyst wall and papillary fronds were lined by oncocytic admixed with numerous mucocytes. Lymphoid tissue and invasive features were not identified. The tumor showed strong expression of CK7 and mammaglobin in oncocytes, and BRST-2 and MUC4 in mucocytes. p63, ER, PR, SOX10, DOG-1, and S100 stains were negative. No rearrangement of the MAML2 gene region or ETV6-NTRK3 fusion transcript was detected. The diagnosis of oncocytic papillary tumor with prominent mucinous differentiation is particularly problematic owing to the large number of potential mimics and should prompt consideration of appropriate molecular studies.

Rhabdoid carcinoma of anal canal: role of electron microscopy and immunohistochemistry in establishing lineage.

ABSTRACT Rhabdoid carcinoma is a high-grade carcinoma with rhabdoid features and it is different from rhabdoid tumors that are broadly defined as malignant neoplasms with rhabdoid cellular appearance found primarily in the pediatric population, but adult cases have been reported in many anatomic locations. To date, no cases of anal canal rhabdoid carcinoma have been reported in the adult or pediatric population. We are reporting the first case of anal canal rhabdoid carcinoma, found in a 75-year-old male. We utilized ultrastructural as well as immunohistochemical studies to arrive at our diagnosis. Ultrastructural studies demonstrated the intermediate filament congregating to impart a rhabdoid appearance to tumor cells, and cytokeratin intermediate filaments and short microvilli indicating nature of tumor as carcinoma. Immunohistochemical phenotype showed neoplastic cells were positive for vimentin, pan-cytokeratin AE1/3, p63 and D2-40, which supports the genesis of tumor from skin adnexa. Even in the modern era of surgical pathology that routinely utilizes immunohistochemistry and molecular studies, adequate use of electron microscopy to help pinpoint the diagnosis in challenging cases is important.

Aldoketoreductase family 1B10 (AKR1B10) as a biomarker to distinguish hepatocellular carcinoma from benign liver lesions.

Hepatocellular carcinoma (HCC) is one of the most common highly aggressive malignant tumors worldwide. Aldoketoreductase 1B10 (AKR1B10) was first isolated from HCC and further identified to be over-expressed in many cancers from various organs. AKR1B10 contributes to detoxification of xenobiotics by lipid peroxidation and metabolizes physiological substrates such as farnesal, retinal, and carbonyls. Metabolizing these lipid substrates plays a crucial role in promoting carcinogenesis. In the present study, immunohistochemical analysis was performed to determine the prevalence/pattern of AKR1B10 expression in HCC and its usefulness to differentiate benign liver lesions from HCC. Oncogenic function of AKR1B10 was examined in hepatocellular carcinoma cells in vitro using Western blotting and shRNA knockdown approaches, with emphasis on cell apoptosis and response to chemotherapy. Immunohistochemistry analysis revealed AKR1B10 was overexpressed in 97% (86/89) of hepatocellular carcinomas, with minimal to no expression in adjacent hepatic tissue, while hepatic adenomas and focal nodular hyperplasia did not exhibit expression of AKR1B10. shRNA-mediated silencing of AKR1B10 expression in hepatocellular carcinoma cells resulted in (1) increased cell apoptosis, (2) decreased colony formation and size, and (3) enhanced cytoreductive response following exposure to doxorubicin chemotherapy. Our findings provide first time evidence that AKR1B10 is a unique biomarker involved in hepatocellular carcinogenesis via modulation of proliferation, cell apoptosis and chemoresistance and is a potential promising biomarker to differentiate HCCs from benign hepatic lesions.

Human peripheral blood mononuclear cells exhibit heterogeneous CD52 expression levels and show differential sensitivity to alemtuzumab mediated cytolysis.

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. METHODS: In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. RESULTS: EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). CONCLUSIONS: These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

Magnetic resonance elastography and biomarkers to assess fibrosis from recurrent hepatitis C in liver transplant recipients.

BACKGROUND: Imaging techniques evaluating liver stiffness (magnetic resonance elastography [MRE]) and biomarkers may be useful indicators of fibrosis stage in hepatitis C virus (HCV)+patients. Our aim was to compare the accuracy of MRE and biomarkers in staging fibrosis because of recurrent HCV in liver transplant (LT) recipients with hepatocellular carcinoma. METHODS: Liver magnetic resonance imaging and MRE, FIBROSpectII, aspartate aminotransferase-to-platelet ratio index (aspartate aminotransferase [AST]: platelet index), AST:alanine aminotransferase ratio, and magnetic resonance imaging/MRE-guided biopsies targeting the stiffest regions (right and left lobes) were performed in HCV+LT recipients. Sensitivity, specificity, positive predictive value (PPV)/negative predictive value (NPV), and likelihood ratios were calculated for the best cutoff by receiver operating characteristic analysis. RESULTS: Thirty-two recipients were included: 28 men, age 60 (±6.4) years, and time since LT 3.25 (±1.68) years. Both MRE (P=0.0001) and FIBROSpectII (P=0.009) were significantly different between no fibrosis and more than or equal to stage 1 groups, whereas aspartate aminotransferase-to-platelet ratio index and AST:alanine aminotransferase ratio were not different. Areas under the receiver operating characteristic curve were 0.87 for MRE and 0.84 for FIBROSpectII. MRE cutoff of 3.81 kPa had 87.5% sensitivity, 79.2% specificity, 58.3% PPV, and 95.0% NPV; FIBROSpectII cutoff of 42 had 87.5% sensitivity, 70.0% specificity, 53.8% PPV, and 93.3% NPV for detection of more than or equal to stage 1 fibrosis. Two patients had high MRE values because of unexpected acute rejection and portal vein thrombosis. CONCLUSIONS: MRE and FIBROSpectII are highly sensitive in detecting fibrosis due to recurrent HCV. Both are limited by the low specificity/PPV and confounding because of other graft complications. Values below the MRE and FIBROSpectII cutoffs, however, strongly suggest the absence of fibrosis and may avert the need for protocol biopsy staging.

Aberrant expression of functional BAFF-system receptors by malignant B-cell precursors impacts leukemia cell survival.

Despite exhibiting oncogenic events, patient's leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM) microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies.We observed that primary leukemia B-cell precursors aberrantly express receptors of the BAFF-system, BAFF-R, BCMA, and TACI. These receptors are functional as their ligation triggers activation of NF-κB, MAPK/JNK, and Akt signaling. Leukemia cells express surface BAFF and APRIL ligands, and soluble BAFF is significantly higher in leukemia patients in comparison to age-matched controls. Interestingly, leukemia cells also express surface APRIL, which seems to be encoded by APRIL-δ, a novel isoform that lacks the furin convertase domain. Importantly, we observed BM microenvironmental cells express the ligands BAFF and APRIL, including surface and secreted BAFF by BM endothelial cells. Functional studies showed that signals through BAFF-system receptors impact the survival and basal proliferation of leukemia B-cell precursors, and support the involvement of both homotypic and heterotypic mechanisms.This study shows an unforeseen role for the BAFF-system in the biology of precursor B-cell leukemia, and suggests that the target disruption of BAFF signals may constitute a valid strategy for the treatment of this cancer.

A high omega-3 fatty acid diet mitigates murine pancreatic precancer development.

BACKGROUND: Diets containing omega-3 (ω-3) fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in ω-3 fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro. MATERIALS AND METHODS: Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 mo and compared with age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay; cell cycle analysis was performed by flow cytometry; and apoptosis was assessed with annexin/PI staining. RESULTS: The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared with mice fed a standard chow. In vitro, DHA treatment resulted in a concentration-dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis. CONCLUSIONS: A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.

Alteration of strain background and a high omega-6 fat diet induces earlier onset of pancreatic neoplasia in EL-Kras transgenic mice.

Diets containing omega-6 (ω-6) fat have been associated with increased tumor development in carcinogen-induced pancreatic cancer models. However, the effects of ω-6 fatty acids and background strain on the development of genetically-induced pancreatic neoplasia is unknown. We assessed the effects of a diet rich in ω-6 fat on the development of pancreatic neoplasia in elastase (EL)-Kras(G12D) (EL-Kras) mice in two different backgrounds. EL-Kras FVB mice were crossed to C57BL/6 (B6) mice to produce EL-Kras FVB6 F1 (or EL-Kras F1) and EL-Kras B6 congenic mice. Age-matched EL-Kras mice from each strain were compared to one another on a standard chow. Two cohorts of EL-Kras FVB and EL-Kras F1 mice were fed a 23% corn oil diet and compared to age-matched mice fed a standard chow. Pancreata were scored for incidence, frequency, and size of neoplastic lesions, and stained for the presence of mast cells to evaluate changes in the inflammatory milieu secondary to a high fat diet. EL-Kras F1 mice had increased incidence, frequency, and size of pancreatic neoplasia compared to EL-Kras FVB mice. The frequency and size of neoplastic lesions and the weight and pancreatic mast cell densities in EL-Kras F1 mice were increased in mice fed a high ω-6 fatty acid diet compared to mice fed a standard chow. We herein introduce the EL-Kras B6 mouse model which presents with increased frequency of pancreatic neoplasia compared to EL-Kras F1 mice. The phenotype in EL-Kras F1 and FVB mice is promoted by a diet rich in ω-6 fatty acid.

Serum B12 concentration is elevated in patients receiving chronic parenteral nutrition, but is not a marker of intestinal failure-associated liver disease.

BACKGROUND: Cobalamin is released during hepatic cytolysis associated with liver injury. Serum B12 concentration is frequently elevated in patients that receive long-term parenteral nutrition (PN). We hypothesized that serum B12 concentration would become elevated in intestinal failure-associated liver disease and would reflect in disease severity. METHODS: We retrospectively evaluated 13 patients with short bowel syndrome (<200 cm residual small intestine) that included complete terminal ileum resection (3 male and 10 female, aged 42 to 78 y) that had received parenteral nutrition (PN) 6.1+/-3 years. All 13 patients had received at least 1 liver biopsy for presumed intestinal failure-associated liver disease. At the time of biopsy, patients had received PN between 2 and 7 days a week (4.7+/-1.9 d). The liver biopsies were evaluated and prospectively scored for pathology using 3 independent scoring systems validated for nonalcoholic steatohepatitis and nonalcoholic fatty liver disease [Brunt, NAFLD activity score (NAS) and Dixon methods], whereby numeric values were assigned to degrees of steatosis, inflammation, and fibrosis. Serum B12 concentration and hepatic chemistries (aspartate transaminase, alanine transaminase, alkaline phosphatase, and bilirubin) were recorded within 1 week of the biopsies. RESULTS: Thirteen biopsies were available for analysis. Serum B12 concentration and hepatic chemistries were available for all biopsy times. The mean serum B12 concentration was 619+/-222 pg/mL. The mean daily parenteral B12 dose was 3.3+/-1.3 mcg. Mean NAS, Brunt, and Dixon scores were 2, 1, and 1, respectively. The Spearman correlation coefficients between serum B12 concentration and liver biopsy scores were 0.15, 0.1, and 0.1 for the NAS, Brunt, and Dixon scores, respectively, indicating that there was no correlation between serum B12 concentration and liver pathology. The Spearman correlation coefficient between the NAS inflammation subscore and serum B12 concentration was 0.02. B12 concentration also failed to correlate with hepatic chemistries. There was surprisingly little correlation between serum B12 concentration and exogenous B12 daily dose through PN (r=0.19, P=0.45). CONCLUSIONS: Elevated serum B12 concentration is commonly encountered in patients who receive long-term parenteral nutrition. This does not seem to be an indicator of hepatic pathology; rather it may reflect the provision of excessive intravenous vitamin B12 and other as yet unknown factors.

Mucinous nonneoplastic cyst of the pancreas: apomucin phenotype distinguishes this entity from intraductal papillary mucinous neoplasm.

Mucinous nonneoplastic cyst of the pancreas is a newly described and rare cystic lesion with unknown histogenesis. It is defined as a cystic lesion lined with mucinous epithelium, supported by hypocellular stroma and not communicating with the pancreatic ducts. It is very challenging to differentiate this lesion from other cystic mucinous neoplasms of the pancreas such as branch-duct intraductal papillary mucinous neoplasm by morphology. In this study, a total of 436 pancreatic specimens resected between 2002 and 2007 in our institution were reviewed. Fifteen (3.4%, 15/436) mucinous nonneoplastic cysts were identified. They included 3 males and 12 females, with a median age of 60 years. Forty-six percent of cases (7/15) occurred in pancreatic head, 27% (4/15) in neck, 7% (1/15) in body, and 20% (3/15) in tail. The size of lesions ranged from 0.5 to 3.5 cm in greatest dimension. In most cases (12/15, 80%), mucinous nonneoplastic cyst was associated or adjacent to acinar-ductal mucinous metaplasia. These morphologic data indicate that mucinous nonneoplastic cyst is not really a rare disease and may originate from acinar-duct mucinous metaplasia histogenestically. Furthermore, apomucin immunostains of mucinous nonneoplastic cyst showed MUC1 expressed in 27% (4/15) cases, MUC5AC in 67% (10/15 cases), and MUC2 was were negative in all cases, whereas intraductal papillary mucinous neoplasm (n = 17; 5 main duct type, 12 branch-duct type) showed focal and weak MUC1 positivity in 18% (3/17) cases, MUC2 positivity in 71% (12/17) cases, and all intraductal papillary mucinous neoplasm (17/17) were MUC5AC positive. The clonality assay with the HUMARA gene revealed that the mucinous nonneoplastic cysts were of polyclonal origin. For the first time, using HUMARA assay, we demonstrate the nonneoplastic nature of these cysts and further characterize morphologic and immunophenotypic properties that allow differentiation from intraductal papillary mucinous neooplasm.

Overexpression of 5-lipoxygenase in colon polyps and cancer and the effect of 5-LOX inhibitors in vitro and in a murine model.

PURPOSE: Arachidonic acid metabolism via the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways modulates cell growth and apoptosis. Many studies have examined the effects of COX inhibitors on human colorectal cancer, but the role of 5-LOX in colonic cancer development has not been well studied. The purpose of this study was to evaluate the expression of 5-LOX in colonic polyps and cancer and the effect of 5-LOX inhibition on colon cancer cell proliferation. EXPERIMENTAL DESIGN: Colonic polyps, cancer, and normal mucosa were evaluated for 5-LOX expression by immunohistochemistry. Reverse transcription-PCR was used to establish 5-LOX expression in colon cancer cells. Thymidine incorporation and cell counts were used to determine the effect of the nonspecific LOX inhibitor Nordihydroguaiaretic Acid and the 5-LOX inhibitor Rev5901 on DNA synthesis. A heterotopic xenograft model in athymic mice using HT29 and LoVo human colon cancer cells was used to evaluate the effect of the 5-LOX inhibitor zileuton on tumor growth. RESULTS: 5-LOX is overexpressed in adenomatous polyps and cancer compared with that of normal colonic mucosa. LOX inhibition and 5-LOX inhibition decreased DNA synthesis in a concentration- and time-dependent manner in the Lovo cell line (P < 0.05). Inhibition of 5-LOX in an in vivo colon cancer xenograft model inhibited tumor growth compared with that of controls (P < 0.05). CONCLUSIONS: This study showed that 5-LOX is up-regulated in adenomatous colon polyps and cancer compared with normal colonic mucosa. The blockade of 5-LOX inhibits colon cancer cell proliferation both in vitro and in vivo and may prove a beneficial chemopreventive therapy in colon cancer.

Clear cell carcinoma of the pancreas: histopathologic features and a unique biomarker: hepatocyte nuclear factor-1beta.

Clear cell carcinoma as a variant of ductal carcinoma of the pancreas is not well recognized. Hepatocyte nuclear factor-1beta as a transcription factor has been identified as a specific biomarker of clear cell tumor of the female genital tract. The aim of this study was to systematically analyze clear cell carcinoma of the pancreas and its unique biomarker hepatocyte nuclear factor-1beta. A total of 84 pancreatic adenocarcinomas were analyzed pathologically and with an immunohistochemical approach with hepatocyte nuclear factor-1beta antibody. The identified clear cell carcinomas were further studied by PAS, DPAS, and mucicarmine stains. Pathologic features and clinical follow-up were documented. Of them, 20 (24%) pancreatic adenocarcinomas were identified with clear cell features, including 12 clear cell carcinomas and 8 ductal adenocarcinomas with clear cell component (defined as less than 75% of tumor with clear cells). Cytologically, the clear cell carcinomas exhibited clear cytoplasm with centrally located, atypical nuclei. PAS, DPAS, and mucicarmine stains confirmed that the clear cytoplasm was not due to accumulation of glycogen or mucin. The results of immunostaining showed that hepatocyte nuclear factor-1beta is overexpressed in all clear cell carcinomas and in the clear cell components of eight ductal carcinomas with clear cell features. In contrast, in usual ductal adenocarcinoma, hepatocyte nuclear factor-1beta exhibited overall weak or focally moderate staining; only eight cases were strongly positive (15%) of which 38% were high grade and 63% were moderate grade. However, when included with the strong staining cases in mixed and clear cell carcinoma, this group regardless of morphology appeared to correlate with worse survival compared to the group with weak hepatocyte nuclear factor-1beta staining across morphologies (P<0.01). Thus, clear cell carcinoma of the pancreas is not an uncommon variant of pancreatic ductal adenocarcinoma. Hepatocyte nuclear factor-1beta is a useful marker to identify these clear cell carcinomas, and its overexpression may aid in stratifying survival rate.