Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and ...24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.
To identify markers that are relevant as predictors of lymph node metastasis in head and neck squamous cell cancer.
Expression of p53, Rb, cyclin D1, E-cadherin, and epithelial cell adhesion molecule ...was examined using immunohistochemical analysis and traditional histological parameters, and the correlation of these markers with the histologically verified presence of regional metastases was determined.
The study sample comprised 121 patients with head and neck squamous cell cancer from whom paraffin-embedded material of primary tumors was used.
Lymph node metastasis was correlated with the loss of expression of Rb (P =.04) and marginally correlated with the loss of expression of E-cadherin (P =.06). If the results are broken down to subsites, loss of E-cadherin expression in oral cancer (P =.04) and absence of eosinophilic infiltration in laryngeal cancer (P =.003) correlated with nodal metastasis. None of the other markers correlated. A combination of relevant parameters did not result in a much stronger correlation.
The expression of the investigated genetic markers and histopathological features of primary tumors can supply limited information on the metastatic behavior of tumors. Although the use of markers for regional metastasis would be a welcome additional tool, these results do not warrant the use of these parameters for clinical decision making concerning the treatment of the neck in patients with head and neck squamous cell cancer.
Dectin-1 is a pattern recognition receptor (PRR) expressed by myeloid cells that specifically recognizes beta-1,3 glucan, a polysaccharide and major component of the fungal cell wall. Upon ...activation, dectin-1 signaling converges, similar to NOD2, on the adaptor molecule CARD9 which is associated with inflammatory bowel disease (IBD). An early stop codon polymorphism (c.714T>G) in DECTIN-1 results in a loss-of-function (p.Y238X) and impaired cytokine responses, including TNF-alpha, interleukin (IL)-1beta and IL-17 upon in vitro stimulation with Candida albicans or beta-glucan. The aim of the present study was to test the hypothesis that the DECTIN-1 c.714T>G (p.Y238X) polymorphism is associated with lower disease susceptibility or severity in IBD and to investigate the level of dectin-1 expression in inflamed and non-inflamed colon tissue of IBD patients. Paraffin embedded tissue samples from non-inflamed and inflamed colon of IBD patients and from diverticulitis patients were immunohistochemically stained for dectin-1 and related to CD68 macrophage staining. Genomic DNA of IBD patients (778 patients with Crohn's disease and 759 patients with ulcerative colitis) and healthy controls (n = 772) was genotyped for the c.714T>G polymorphism and genotype-phenotype interactions were investigated. Our data demonstrate that dectin-1 expression is elevated on macrophages, neutrophils, and other immune cells involved in the inflammatory reaction in IBD. The DECTIN-1 c.714T>G polymorphism however, is not a major susceptibility factor for developing IBD.
Regional metastasis is an important factor in the treatment and prognosis of patients with head and neck squamous cell carcinoma. Although in recent years imaging techniques have improved, it is ...still impossible to detect small metastatic deposits. Metastasis is mainly determined by properties of the primary tumor and its interaction with surrounding structures.
To identify markers that predict the presence of metastasis based on the features of the primary tumor.
Correlation of the results of histological, immunohistochemical, and molecular biological analysis with clinical and histopathological data.
Several histological features and biological markers were examined in 31 laryngeal carcinomas. The following markers were selected on their putative role in the process of metastasis and were studied using immunohistochemical and/or Southern blot techniques: proliferating cell nuclear antigen (PCNA), p53, retinoblastoma tumor-suppressor gene (Rb), myc, bcl-2 (inhibitor of apoptosis), epidermal growth factor (EGF), EGF-receptor (EGFR), neu, nm23 (also known as NME1, putative metastasis suppressor), desmoplakin, neuron cell-adhesion molecule (N-CAM), epithelial cell-adhesion molecule (Ep-CAM), E-cadherin, cyclin D1 (CCND1), and EMS1.
The presence of an inflammatory reaction surrounding the tumor (P = .07), eosinophilic infiltration (P = .16), positive immunostaining for Rb (P = .02), negative immunostaining for Ep-CAM (P = .13), and amplification of CCND1 and EMS1 (P = .05) correlated with nodal metastasis. The combination of an inflammatory reaction, eosinophilic infiltration, and staining for Rb and Ep-CAM resulted in a superior accuracy in assessing nodal metastasis.
These results indicate that it is possible to predict and exclude lymph node metastasis by studying the features of the primary tumor only. When these results are confirmed in a larger series, biological markers may be powerful diagnostic tools with great impact on clinical decision making.
Background. Transformation to diffuse large cell lymphoma (DLCL) is a frequent event in patients with follicular lymphoma (FL), occurring in approximately 30–50% of patients. Upon transformation the ...prognosis of these patients is dismal, with a median overall survival of 1–2 years. However, a subset of patients does respond to aggressive chemotherapy regimens, including autologous stem cell transplantation and can achieve long-term disease-free survival. Currently, there are no prognostic factors reliably predicting response to treatment. We used gene expression profiling to identify a profile which can predict responsiveness to chemotherapy and long-term survival at transformation.
Patients and methods. From the pathology archives of the 4 participating institutions, 46 cases with transformation of FL were identified for whom frozen tissue was available. 11 cases were excluded from the analysis: 4 because of insufficient clinical information and 7 because the patients were not treated with aggressive chemotherapy regimens (defined as containing at least CHOP-like chemotherapy). RNA was extracted from frozen tissue. All samples were hybridized to cDNA microarray slides prepared at the Central Microarray Facility at the Netherlands Cancer Institute. The arrays contain 18336 biological transcripts as well as 864 control probes. Samples were cohybridized with a tonsil reference. Data analysis was performed in the statistical package R as well as with BrB array tools.
Results. Of the 35 patients, 62% was male. The median interval between the initial diagnosis of FL and transformation was 27 months (range 0–252; 4 patients had transformed FL at initial diagnosis). Median age at transformation was 52 years (range 32–78). Using hierarchical clustering, no clear separation of the cases was obtained. Of the 35 cases, 13 reached a CR or Cru and had an overall survival of >24 months (range 37–134+, median 60 months). 6 patients achieved a partial remission and had an overall survival of 12–24 months, and 16 patients had stable or progressive disease and died within 13 months (range 3–13, median 5.5 months). Using supervised analysis on patients grouped based on survival, a classifier with an accuracy of up to 88% accuracy could be reached in an LOOCV procedure.
An analysis of the most differentially expressed genes related to survival identified mostly genes involved in cell cycle, metabolism and immune related genes.
Conclusions. Using gene expression profiling a classifier can be constructed that can separate transformed FL with a long survival from transformed FL with a short survival. This classifier can help to identify patients who benefit from intensive chemotherapy. Functional annotation of the differentially expressed genes identified mostly genes involved in cell cycle control and metabolism, and immune related genes. We plan to validate this approach using immunohistochemistry.
Lynch syndrome is the most common cause of hereditary intestinal cancer, with a 30-70% risk of colorectal cancer (CRC). Prevention of CRC by colonoscopy in family members with Lynch syndrome is ...highly effective; therefore, it is important to trace as many people with this syndrome as possible. Criteria have been developed in the Netherlands to increase detection of hereditary colorectal cancer in a practically feasible and cost-effective way. Based on these criteria, the pathologist can perform microsatellite instability testing in patients recently diagnosed with CRC. The criteria are: CRC under the age of 50, second CRC under the age of 70, or CRC under the age of 70 with a concurrent or previous malignancy associated with Lynch syndrome. For family members and patients diagnosed with CRC more than a year ago, a digital test can be used to determine whether genetic counselling by a geneticist is indicated (www.umcn.nl/verwijzers).
Background: Invasion and metastasis is a complex process governed by the interaction of genetically altered tumor cells and the immunological and inflammatory host reponse. Specific T-cells directed ...against tumor cells and the nonspecific inflammatory reaction due to tissue damage, cooperate against invasive tumor cells in order to prevent recurrences. Data concerning involvement of individual cell types are readily available but little is known about the coordinate interactions between both forms of immune response. Patients and methods: The presence of inflammatory infiltrate and eosinophils was determined in 1530 patients with rectal adenocarcinoma from a multicenter trial. We selected 160 patients to analyze this inflammatory infiltrate in more detail using immunohistochemistry. The association with the development of local and distant relapses was determined using univariate and multivariate log rank testing. Results: Patients with an extensive inflammatory infiltrate around the tumor had lower recurrence rates (3.4% versus 6.9%, p = 0.03), showing the importance of host response against tumor cells. In particular, peritumoral mast cells prevent local and distant recurrence (44% versus 15%, p = 0.007 and 86% versus 21%, p < 0.0001, respectively), with improved survival as a consequence. The presence of intratumoral T-cells had independent prognostic value for the occurrence of distant metastases (32% versus 76%, p < 0.0001). Conclusions: We showed that next to properties of tumor cells, the amount and type of inflammation is also relevant in the control of rectal cancer. Knowledge of the factors involved may lead to new approaches in the management of rectal cancer.
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