Lars Helgeland

Detection of clonality in follicular lymphoma using formalin-fixed, paraffin-embedded tissue samples and BIOMED-2 immunoglobulin primers

Aims The BIOMED-2 multiplex PCR protocol is a commonly used procedure for assessing B cell clonality in lymphoma diagnostics. Follicular lymphoma poses a special challenge for PCR-based analyses because of high prevalence of somatic hypermutations in the rearranged immunoglobulin (IG) domains. This study aimed to evaluate the BIOMED-2 protocol performance in detection of B cell clonality in follicular lymphoma using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods FFPE samples from 118 patients diagnosed with follicular lymphoma in the period 1998–2008 were used in the study. Clonality of IG heavy (IGH) and light chains (IGK, IGL) was assessed using a PCR procedure that was optimised for FFPE tissue. Results The highest clonal detection rates were 67.8% with the IGH Vн-FR2-Jн assay and 66.1% with the IGK Vκ-Jκ assay. Clonality was detected in 94.9% of all FFPE follicular lymphoma samples when all assays were combined. FFPE samples stored for 1–5 years did not perform significantly differently from those stored for 6–11 years. Interobserver agreement of clonality was tested for all analyses. The lowest score (Cohens κ value = 0.56) was observed for the IGK Vκ-Jκ clonality assay. Conclusions An improved PCR protocol for detection of clonality in FFPE samples using BIOMED-2 IG primers is presented. For best performance, a combination of IGH and IGK analyses is recommended.

The thymus is a source of B-cell-survival factors–APRIL and BAFF–in myasthenia gravis

The accumulation of B cells in the thymus is a common feature of myasthenia gravis (MG). To understand whether factors enhancing B-cell survival are increased in MG, we studied the expression of APRIL, BAFF and three of their receptors in the thymus. In hyperplastic thymi, macrophages expressed APRIL and BAFF, and germinal-center B cells, BAFF-R. CD138-positive plasma cells were abundant in MG thymi. By contrast, BCMA-positive plasma cells were scarce. The expression of APRIL and BAFF in MG thymi may reflect the establishment of an environment favorable to B-cell survival.

Indications of 'atopic bowel' in patients with self-reported food hypersensitivity.

Aliment Pharmacol Ther 31, 1112–1122

Low‐dose oral ferrous fumarate aggravated intestinal inflammation in rats with dss‐induced colitis

Background: Oral ferrous iron therapy may reinforce intestinal inflammation. One possible mechanism is by catalyzing the production of reactive oxygen species. We studied the effects of low‐dose oral ferrous fumarate on intestinal inflammation and plasma redox status in dextran sulfate sodium (DSS)‐induced colitis in rats. Methods: Forty male Wistar rats were divided into 5 groups: no intervention, sham gavage (distilled water), ferrous fumarate, DSS, and ferrous fumarate + DSS. Ferrous fumarate was dissolved in distilled water (0.60 mg Fe2+/kg per day) and administered by gavage on days 1 to 14. All rats were fed a standard diet. Colitis was induced by 5% DSS in drinking water on days 8 to 14. Rats were killed on day 16. Histologic colitis scores, fecal granulocyte marker protein, plasma malondialdehyde, plasma antioxidant vitamins, and plasma aminothiols were measured. Results: DSS significantly increased histologic colitis scores (P < 0.001) and fecal granulocyte marker protein (P < 0.01). Ferrous fumarate further increased histologic colitis scores (P < 0.01) in DSS‐induced colitis. DSS + ferrous fumarate decreased plasma vitamin A compared with controls (P < 0.01). Otherwise, no changes were seen in plasma malondialdehyde, plasma antioxidant vitamins, or plasma aminothiols. Conclusion: Low‐dose oral ferrous iron enhanced intestinal inflammation in DSS‐induced colitis in rats.

Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.

Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion.

Time patterns of changes in biomarkers, symptoms and histopathology during pelvic radiotherapy

Acute radiation proctitis was evaluated before, during and after radiotherapy (RT) for prostate cancer. The main aims of the study were to examine changes related to the increasing radiation dose, and identify surrogate markers of gastrointestinal (GI) reaction to radiation. Twenty consecutive prostate cancer patients scheduled for 7 weeks of conformal RT were prospectively included in a longitudinal study assessing symptoms, inflammation in rectal mucosa biopsies, and blood and stool samples at four time points (before RT and 2, 6 and 11 weeks after start of RT). Blood samples were examined for acute phase response-related markers, fatty acids (FAs), vitamin E and leukotriene B4 (LTB4). Lactoferrin, calprotectin and S100A12 were measured in stool samples and FAs in biopsies from rectal mucosa. The increase in histopathological inflammation reached a maximum 2 weeks after start of RT. Symptoms of GI toxicity increased with higher radiation dose and had not returned to pre-treatment level 4 weeks after RT. Lactoferrin concentrations in stool increased significantly at week 6. Significant decreases of vitamin E, leukocyte count, hemoglobin and some groups of FAs were discovered, while a few FAs increased significantly during the study period. Time courses vary between the selected indicators of acute radiation proctitis. The biopsy grading of inflammatory changes were most intense 2 weeks into the treatment period while symptoms continued to increase until week 6. Lactoferrin in stool samples could be a non-invasive marker of GI inflammation during RT. A transient decrease in vitamin E and some FAs during RT warrants further studies.

Prognosis in adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia evaluated by uPAR-immunohistochemistry.

Adenocarcinomas of lower oesophagus, gastro‐oesophageal junction and cardia in humans are highly invasive tumours with poor prognosis. The localisation of urokinase‐type plasminogen activator receptor (uPAR) was determined in 66 patients; 60 with adenocarcinomas and six cases with Barretts oesophagus. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts at both the invasion front and the tumour core. In areas with high‐grade dysplasia or with Barretts metaplasia adjacent to the tumour tissue, no uPAR‐immunoreactivity was found. High local expression of uPAR, therefore, appears to be a characteristic marker for invasive behaviour in this tumour, suggesting that uPARs contribution to matrix degradation during invasive growth is a late event in carcinogenesis. Using a scoring system for semiquantitative estimation of uPAR‐positivity on immmunohistochemically stained specimens, a significant association was found between poor overall survival and high uPAR‐score for cancer cells in the tumour core and for macrophages peripherally at the tumour invasion zone. In multivariate analysis, these two uPAR‐scores were confirmed as highly significant prognostic parameters independent of Tumour, Node, Metastasis (TNM)‐stage and World Health Organization (WHO) classification. The proteolytic action of these malignant and nonmalignant accessory cells thus seemed to follow two main patterns: one dominated by uPAR positive cancer cells and one by uPAR‐positive macrophages. Scoring of uPAR‐positivity might be a useful parameter for onset of invasion and prognosis in these adenocarcinomas.

Evaluation of germinal center-like structures and B cell clonality in patients with primary Sjögren syndrome with and without lymphoma.

Objective. Germinal center (GC)-like structures have previously been observed in minor salivary glands (MSG) of patients with primary Sjögren syndrome (pSS). The aim of our study was to explore the prevalence and features of GC-like structures and B cell clonality in patients with pSS with and without lymphoma. Methods. Based on a nationwide survey in Norway, we included 21 patients with pSS and with a concomitant lymphoma from whom MSG and/or lymphoma biopsies were available. Tonsil biopsies and MSG from 28 patients with pSS without lymphoma were used as controls. The presence of GC-like structures was investigated with H&E staining and double staining for CD21/IgD and CD38/IgD. B cell clonality in MSG and tumors were investigated with analysis of immunoglobulin gene rearrangements. Results. H&E labeling of MSG revealed GC-like structures in 17/40 (43%) of the patients: 4/12 (33%) with and 13/28 (46%) without lymphoma. Staining for CD21/CD38/IgD demonstrated CD21+ networks in 27/40 (68%) of the patients. CD21+/CD38– infiltrates were seen in 25/40 (63%) of the patients, and 16 of these were IgD+ within the infiltrate. Five percent (2/40) of the patients presented with CD21+/CD38+ infiltrates resembling tonsillar GC. Monoclonal B cell infiltration in MSG was present in 5/12 patients (42%) with and 5/28 patients (18%) without lymphoma (p = 0.12). In 2/10 (20%) of cases where both MSG and lymphoma biopsies were available, identical clonal rearrangements were detected. Conclusion. GC-like structures seen in H&E-stained MSG may represent various subtypes of CD21+ infiltrates. We were unable to detect a clear association between cellular infiltrates, B cell clonality, and lymphoma development.

Crohn's Disease But Not Chronic Ulcerative Colitis Induces the Expression of PAI-1 in Enteric Neurons

OBJECTIVES:Chronic inflammation of the intestinal wall is the common characteristic of Crohns disease and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in neurons after experimental peripheral nerve injury and have been linked to nerve regeneration.METHODS:The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was analyzed by immunohistochemical techniques.RESULTS:PAI-1 was found in a subset of neurons primarily located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with Crohns disease, but in none of 17 cases with chronic ulcerative colitis and other severe inflammatory conditions in the intestinal wall. The PAI-1 was seen in the perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was seen in nerves in all types of lesion varying from 21% to 88% of the cases, most frequent in colon adenocarcinomas. No uPAR-positive nerves were detected in normal colon.CONCLUSIONS:PAI-1-positive neurons in inflammatory bowel disease are linked to chronic inflammation in Crohns disease, implying PAI-1 as a potential parameter for the differential diagnosis between Crohns disease and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.