How autoinflammation may turn into autoimmune inflammation: Insights from monogenetic and complex IL-1 mediated auto-inflammatory diseases.

IL-1 mediated auto-inflammatory diseases are characterised by episodes of unexplained fever, generalized and localized inflammation. The characteristic symptoms predominantly result from exaggerated activation of innate immune pathways. However, in some patients with typical IL-1 mediated diseases, chronic disease manifestations develop in the absence of acute inflammation, suggesting the involvement of adaptive immune pathways. We discuss clinical observations as well as novel insights in how chronic activation of innate immune pathways can lead to auto-immune disease features in patients with auto-inflammatory diseases and how we need to better understand these sequelae in order to improve treatment strategies.

Alterations in classical and nonclassical HLA expression in recurrent and progressive HPV-induced usual vulvar intraepithelial neoplasia and implications for immunotherapy.

Immunotherapy of usual vulvar intraepithelial neoplasia (uVIN) is promising; however, many patients still fail to show clinical responses, which could be explained by an immune escape through alterations in human leukocyte antigen (HLA) expression. Therefore, we analyzed a cohort of patients with a primary (n = 43) and subsequent recurrent uVIN lesion (n = 20), vaccine-treated uVIN patients (n = 12), patients with human papillomavirus (HPV)-induced vulvar carcinoma (n = 21) and healthy controls (n = 26) for the expression of classical HLA-class I/II and nonclassical HLA-E/-G and MHC class I chain-related molecule A (MICA). HLA-class I was downregulated in 70% of uVIN patients, including patients with a clinical response to immunotherapy. Downregulation of HLA-class I is probably reversible, as only 15% of the uVIN cases displayed loss of heterozygosity (LOH) and HLA-class I could be upregulated in uVIN keratinocyte cultures by interferon γ. HLA-class I downregulation is more frequently associated with LOH in vulvar carcinomas (25-55.5%). HLA-class II was found to be focally expressed in 65% of uVIN patients. Of the nonclassical molecules, MICA was downregulated in 80% of uVIN whereas HLA-E and -G were expressed in a minority of cases. Their expression was more prominent in vulvar carcinoma. No differences were found between the alterations observed in paired primary and recurrent uVIN. Importantly, downregulation of HLA-B/C in primary uVIN lesions was associated with the development of recurrences and progression to cancer. We conclude that downregulation of HLA is frequently observed in premalignant HPV-induced lesions, including clinical responders to immunotherapy, and is associated with worse clinical outcome. However, in the majority of cases downregulation may still be reversible.

Spindle cell morphology is related to poor prognosis in vulvar squamous cell carcinoma.

BACKGROUND: Vulvar cancer is the fourth most common gynaecological malignancy, with an annual incidence of 2 out of 100,000 women. Although most cases of early stage vulvar cancer have a good prognosis, recurrence and rapid tumour progression can occur. We investigated the prevalence of spindle cell morphology in vulvar cancer and its association with survival. METHODS: This retrospective cohort study included 108 patients with primary vulvar squamous cell carcinoma who were treated at the Leiden University Medical Center during 2000-2009. Paraffin-embedded tissue was examined for the presence of spindle cell morphology. Survival and histology data were compared between cases with spindle and without spindle cell morphology. RESULTS: Twenty-two (20%) tumours showed spindle cells infiltrating the stromal tissue. All spindle cell tumours were human papillomavirus (HPV) negative. Spindle cell morphology was strongly associated with poor prognosis and with a high risk of lymph node involvement at the time of diagnosis (relative risk 2.26 (95% CI 1.47-3.47)). Five-year disease-specific survival was lower in patients with vs without spindle cell morphology (45.2% vs 79.7%, respectively; P=0.00057). CONCLUSION: Vulvar spindle cell morphology occurs frequently and seems to develop through the non-HPV pathway. It is associated with a worse prognosis than conventional vulvar squamous cell carcinoma.

Multiparameter DNA content analysis identifies distinct groups in primary breast cancer.

BACKGROUND: Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique. METHODS: The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis. RESULTS: Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and 'multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (10%) independently associated with poor overall survival (P=0.027, CR). CONCLUSION: Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

CXCR7 expression is associated with disease-free and disease-specific survival in cervical cancer patients.

BACKGROUND: The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients. METHODS: CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed. RESULTS: CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7-11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5-10.2, P=0.005). CONCLUSION: CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.

High frequency of copy-neutral LOH in MUTYH-associated polyposis carcinomas.

Genetic instability is known to drive colorectal carcinogenesis. Generally, a distinction is made between two types of genetic instability: chromosomal instability (CIN) and microsatellite instability (MIN or MSI). Most CIN tumours are aneuploid, whereas MSI tumours are considered near-diploid. However, for MUTYH-associated polyposis (MAP) the genetic instability involved in the carcinogenesis remains unclear, as near-diploid adenomas, aneuploid adenomas and near-diploid carcinomas have been reported. Remarkably, our analysis of 26 MAP carcinomas, using SNP arrays and flow sorting, showed that these tumours are often near-diploid (52%) and mainly contain chromosomal regions of copy-neutral loss of heterozygosity (LOH) (71%). This is in contrast to sporadic colon cancer, where physical loss is the main characteristic. The percentage of chromosomal gains (24%) is comparable to sporadic colorectal cancers with CIN. Furthermore, we verified our scoring of copy-neutral LOH versus physical loss in MAP carcinomas by two methods: fluorescence in situ hybridization, and LOH analysis using polymorphic markers on carcinoma fractions purified by flow sorting. The results presented in this study suggest that copy-neutral LOH is an important mechanism in the tumorigenesis of MAP.

Elevated expression of SerpinA1 and SerpinA3 in HLA-positive cervical carcinoma.

In cervical cancer, an important mechanism by which tumour cells escape immune surveillance is loss of HLA class I, enabling tumours to evade recognition and lysis by cytotoxic T lymphocytes. Some tumours, however, escape from immune surveillance without accumulating defects in antigen presentation. We hypothesized that tumours with no or partial loss of HLA class I develop alternative mechanisms to prevent immune elimination. To investigate this hypothesis, genome-wide expression profiling using Illumina arrays was performed on cervical squamous cell carcinomas showing overall loss of HLA class I, partial, and normal HLA class I protein expression. Statistical analyses revealed no significant differences in gene expression between tumours with partial (n = 11) and normal HLA class I expression (n = 10). Comparison of tumours with normal/partial HLA class I expression (n = 21) with those with overall loss of HLA class I expression (n = 11) identified 150 differentially expressed genes. Most of these genes were involved in the defence response (n = 27) and, in particular, inflammatory and acute phase responses. Especially SerpinA1 and SerpinA3 were found to be up-regulated in HLA-positive tumours (3.6- and 8.2-fold, respectively), and this was confirmed by real-time PCR and immunohistochemistry. In a group of 117 tumours, high SerpinA1 and SerpinA3 expression in association with normal/partial HLA expression correlated significantly with poor overall survival (p = 0.035 and p = 0.05, respectively). Thus, HLA-positive tumours are characterized by higher expression of genes associated with an inflammatory profile. In addition, expression of the acute phase proteins SerpinA1 and SerpinA3 in HLA-positive tumours is associated with worse prognosis.

PCR artifacts in LOH and MSI analysis of microdissected tumor cells.

Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.

Genetic alterations on chromosome 16 and 17 are important features of ductal carcinoma in situ of the breast and are associated with histologic type.

We analysed the involvement of known and putative tumour suppressor- and oncogene loci in ductal carcinoma in situ (DCIS) by microsatellite analysis (LOH), Southern blotting and comparative genomic hybridization (CGH). A total of 78 pure DCIS cases, classified histologically as well, intermediately and poorly differentiated, were examined for LOH with 76 markers dispersed along all chromosome arms. LOH on chromosome 17 was more frequent in poorly differentiated DCIS (70%) Compared to well-differentiated DCIS (17%), whereas loss on chromosome 16 was associated with well- and intermediately differentiated DCIS (66%). For a subset we have done Southern blot-and CGH analysis. C-erbB2/neu was amplified in 30% of poorly differentiated DCIS. No amplification was found of c-myc, mdm2, bek, flg and the epidermal growth factor (EGF)-receptor. By CGH, most frequent alterations in poorly differentiated DCIS were gains on 8q and 17q22-24 and deletion on 17p, whereas in well-differentiated DCIS amplification on chromosome 1q and deletion on 16q were found. In conclusion, our data indicates that inactivation of a yet unknown tumour suppressor gene on chromosome 16q is implicated in the development of most well and intermediately differentiated DCIS whereas amplification and inactivation of various genes on chromosome 17 are implicated in the development of poorly differentiated DCIS. Furthermore these data show that there is a genetic basis for the classification of DCIS in a well and poorly differentiated type and support the evidence of different genetic routes to develop a specific type of carcinoma in situ of the breast.

Cyclin D1 gene amplification and overexpression are present in ductal carcinoma in situ of the breast.

Cyclin D1 (CCND1) amplification is found in 10-15 per cent of invasive breast carcinomas, but it is not well established whether this gene alteration also occurs in the precursor of invasive breast carcinoma, ductal carcinoma in situ (DCIS). By Southern blot analysis, cyclin D1 gene amplification was detected in 10 per cent (3/32) of DCIS cases. In addition, 15 cases of DCIS were analysed using bright field in situ hybridization (BRISH), of which 11 had already been analysed by Southern blotting. One additional case with gene amplification was found by BRISH. The use of BRISH for the detection of gene amplification is shown to be a novel and reliable in situ method on paraffin-embedded tissue sections. By immunohistochemistry, 147 cases of DCIS were analysed for the expression of cyclin D1. Cyclin D1 overexpression was found in 9 per cent of well-differentiated, 29 per cent of intermediately differentiated, and 19 per cent of poorly differentiated DCIS. No statistically significant association was found between cyclin D1 overexpression and the differentiation grade of DCIS, although 90 per cent of the cases that show overexpression are classified as intermediately and poorly differentiated. An association was found between cyclin D1 overexpression and oestrogen receptor positivity. Cyclin D1 overexpression was found in all four cases with cyclin D1 gene amplification, but was also found in 30 per cent (8/27) of cases without detectable gene amplification. It is concluded that cyclin D1 gene amplification is an early event in the development of breast carcinoma and occurs in poorly differentiated DCIS. Cyclin D1 protein overexpression is also present in tumours without cyclin D1 gene amplification and is seen predominantly in DCIS of intermediately and poorly differentiated histological type and oestrogen receptor positivity.

Distribution of T cells and signs of T-cell activation in the rheumatoid joint: implications for semiquantitative comparative histology.

A prerequisite for comparative histology of synovial tissue by means of biopsies is insight into the distribution of a marker under study. This investigation focuses on the variation in the presence of T cells and signs of T-cell activation within the rheumatoid joint. For this purpose, multiple slides from several pieces of synovial tissue from different parts of a joint were stained and scored for the expression of CD3, CD25, HLA-DR, Ki67 and interferon-gamma. The variation in scores for the presence of T cells and markers of activation was more pronounced in slides prepared from different pieces of tissue than in slides from one piece of tissue. Based on multiple analysis of variance, methods are suggested to establish a reliable overall score for the expression of a certain marker within a joint. Following validation, such methods may prove to be useful by allowing semiquantitative histology of synovial tissue for studies on arthritis.

E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis.

In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS without adjacent invasive carcinoma also lacks E-cadherin expression and whether the gene mutations present in ILC are already present in LCIS is not known. We report here that E-cadherin expression is absent in six cases of LCIS and present in 150 cases of ductal carcinoma in situ (DCIS), both without an adjacent invasive component. Furthermore, using mutation analysis, we could demonstrate the presence of the same truncating mutations and loss of heterozygosity (LOH) of the wild-type E-cadherin in the LCIS component and in the adjacent ILC. Our results indicate that E-cadherin is a very early target gene in lobular breast carcinogenesis and plays a tumour-suppressive role, additional to the previously suggested invasion-suppressive role.

Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patients with rheumatoid arthritis.

OBJECTIVE: To investigate whether T cells in the inflamed joints of patients with rheumatoid arthritis (RA) preferentially produce the T helper 1 (Th1) cytokines, interferon-gamma (IFN gamma) and interleukin-2 (IL-2), or the Th2 cytokine, IL-4, when compared with corresponding peripheral blood-derived T cells. METHODS: Synovial fluid mononuclear cells (SFMC) and corresponding peripheral blood mononuclear cells (PBMC) from 10 patients with RA were analyzed, either directly or after in vitro stimulation, for the intracellular presence of Th1 and Th2 cytokines. The amount of secreted cytokine in the cell culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: IFN gamma-containing cells were detected in the unstimulated SFMC, but not in the PBMC, of 3 patients with RA. Cells positive for IL-2 or IL-4 were not detected in the unstimulated samples. Following stimulation, the mean percentage of cells containing Th1 cytokines was significantly increased in the SFMC compared with the PBMC; no differences were found in the mean percentage of IL-4-containing cells. A comparable shift toward Th1 cytokines was observed when the amount of secreted cytokine was determined by ELISA. CONCLUSION: A shift toward T cells with a Th1 cytokine profile was observed in the joints of patients with RA. Since an imbalance between Th1 and Th2 cells is thought to be of pathogenic significance, this finding might have implications for the development of new therapies for RA.

Increased expression of interferon (IFN)-gamma together with IFN-gamma receptor in the rheumatoid synovial membrane compared with synovium of patients with osteoarthritis.

Data concerning the presence of T-cell-derived cytokines in the rheumatic joint are conflicting, challenging the hypothesis that rheumatoid arthritis (RA) is a T-cell-mediated disease. In this study synovial tissue specimens of 11 patients with RA and eight patients with osteoarthritis (OA) were stained for interferon-gamma (IFN-gamma) and its receptor. The level of expression of IFN-gamma was compared with that in tissue specimens of delayed-type hypersensitivity (DTH) reactions of the skin and of chronic tonsillitis. Furthermore, the percentage of T-lymphocytes which stained positive for IFN-gamma was determined using double staining techniques. IFN-gamma and its receptor were detected in all patients with RA and in 7/8 and 3/8, respectively, of patients with OA. Expression of IFN-gamma (P<0.02) and IFN-gamma receptor (P<0.01) in synovial tissue of patients with RA was more abundant compared with that in patients with OA. Although IFN-gamma could be detected in RA synovial tissue, the level of expression was less when compared with DTH reactions of the skin and tonsillitis. The percentage of CD3+ cells being positive for IFN-gamma was approximately 1% in RA, whereas in DTH reactions of the skin it was >90% and in tonsillitis approximately 30%. We conclude that the presence of IFN-gamma and its receptor in RA synovial tissue suggests a role for this cytokine in the ongoing immunological reaction of the inflamed joint.

Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.

Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.