Nancy Van Damme

Immunomodulatory effects of anti-tumor necrosis factor alpha therapy on synovium in spondylarthropathy: histologic findings in eight patients from an open-label pilot study.

OBJECTIVE To assess the effects of treatment with anti-tumor necrosis factor alpha (anti-TNFalpha) on synovial histology in patients with spondylarthropathy (SpA) in order to confirm the effect on peripheral synovitis and to investigate the immunologic mechanisms involved in anti-TNFalpha therapy. METHODS Patients with treatment-resistant SpA were treated with infliximab (5 mg/kg) at weeks 0, 2, and 6 in an open-label pilot study. In 8 patients, synovial biopsy tissues obtained at baseline, week 2, and week 12 were used for histologic and immunohistochemical evaluation. RESULTS In all 8 patients (3 with ankylosing spondylitis, 1 with undifferentiated SpA, and 4 with psoriatic arthritis), there was a clear clinical improvement in the peripheral arthritis after anti-TNFalpha therapy. Histologic analysis of the synovial biopsy tissues indicated that the synovial lining layer thickness tended to decrease, with a significant reduction of CD55+ synoviocytes, at week 12. In the sublining layer, vascularity was reduced at week 12, with a decreased endothelial expression of vascular cell adhesion molecule 1 but not intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, and E-selectin. Although at week 2 and week 12, the number of neutrophils and CD68+ macrophages in the sublining layer was decreased, the overall degree of inflammatory infiltration remained unchanged. This could be related to the lymphocyte infiltration since at week 12, only CD4+ cells (but not CD3+, CD45RO+, and CD8+ cells) tended to decrease, while CD20+ lymphocytes and plasma cells were clearly increased. CONCLUSION The reduction in lining layer thickness, vascularity, and infiltration with neutrophils and macrophages paralleled the beneficial effect of anti-TNFalpha therapy on peripheral synovitis in 8 patients with different subtypes of SpA. The adhesion molecule expression, T cell infiltration, and, most important, B cell infiltration seemed to contrast with previous observations in RA. Although these preliminary data need to be confirmed in a larger cohort, they suggest distinct immunomodulatory mechanisms of anti-TNFalpha in SpA.

Macrophages expressing the scavenger receptor CD163: a link between immune alterations of the gut and synovial inflammation in spondyloarthropathy.

The objective of this study was to investigate CD163+ macrophages in the synovial membrane of patients with spondyloarthropathy (SpA). Immunohistochemistry was performed on synovium of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 16 SpA patients and ten healthy controls, and on paired synovial biopsies of eight SpA patients, before and after anti‐TNFα therapy. Phenotype and cytokine production were analysed by flow cytometry. CD163+ macrophages were increased in the synovial lining and sublining in SpA versus RA, as well as in colonic lamina propria in SpA versus controls. The number of CD163+ macrophages in the synovial sublining correlated with C‐reactive protein levels and erythrocyte sedimentation rate. Paralleling the increase of CD163, HLA‐DR was increased in the synovial lining and sublining of SpA. In contrast, the co‐stimulatory molecules CD80 and CD86 and the dendritic cell markers CD1a and CD83 were scarce in SpA synovium. Flow cytometry indicated that CD163+ macrophages expressed high levels of HLA‐DR and could produce in vitro tumour necrosis factor α (TNFα) but not interleukin‐10 (IL‐10). Finally, anti‐TNFα therapy in vivo induced a decrease of CD163+ macrophages in the synovial lining and sublining. In conclusion, macrophages expressing the scavenger receptor CD163 are increased in synovium and in colonic mucosa in SpA, highlighting the relationship between joint and gut in this disease. The correlation with inflammatory parameters, the expression of HLA‐DR, the production of TNFα but not IL‐10, and the reduction by anti‐TNFα therapy support a role for CD163+ macrophages in the synovial inflammation in SpA. Copyright

Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

BackgroundWith the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas.MethodsFormalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers.ResultsEGFR immunoreactivity was present in 36/43 (83.7%) of anal canal and in 20/24 (83.3%) of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23), but could be identified in 4 of 24 tonsil tumours.From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified.ConclusionEGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified. This indicates that EGFR and K-RAS mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in anal canal and tonsil squamous cell carcinoma.

Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers

Chemical agents (DTT, EDTA) and enzymes (collagenase, DNAse) are often used for the generation of a single cell suspension from tissue samples. In this study, flow cytometry was used to examine the effect of chemical agents and enzymes on the expression of cell membrane markers of T lymphocytes from tonsils and peripheral blood. Expression of CD4, CD8, CD25, CD38, L-selectin, CD44, alphaEbeta7 and alpha4beta7 were studied. Incubation of lymphocytes with DTT and EDTA resulted in a decrease of CD38, alphaEbeta7 and alpha4beta7 expression. Incubation with collagenase A and DNAse resulted in a decrease of CD25, L-selectin, alphaEbeta7 and alpha4beta7. The results of this study indicate that a careful interpretation is necessary for phenotypic descriptions of lymphocyte populations obtained by enzymatic isolation techniques.

Detection of Microsatellite Instability in Colorectal Cancer Using an Alternative Multiplex Assay of Quasi-Monomorphic Mononucleotide Markers

Colorectal malignancies demonstrating microsatellite instability (MSI) have a very heterogeneous histological appearance, better prognosis, and altered response to therapy. Consequently, identification of the MSI phenotype is both relevant and interesting as a screening and prognostic tool and as a potential predictive factor of chemotherapeutic response. Several groups have argued for the exclusive use of mononucleotide markers for MSI analysis. In this study, an alternative MSI typing multiplex system of mononucleotide microsatellite repeats was developed. This system obviates the need to compare allelic profiles between tumor and matching normal DNA, rendering MSI analysis amenable to high throughput. The quasi-monomorphic allelic distribution of five alternative mononucleotide markers was evaluated in genomic DNA. Only SEC63 and CAT25 were found to be quasi-monomorphic and were thus combined with BAT25 and BAT26 from the Bethesda panel. Consequently, 177 colorectal cancer samples previously analyzed by the Bethesda panel were tested for MSI using this alternative mononucleotide panel. In an attempt to resolve discordant cases, immunohistochemistry of MLH1, MSH2, and MSH6 was performed. The concordance between both panels reached 99.4% when microsatellite stability and MSI-L were grouped together. These new markers were subsequently multiplexed in a single polymerase chain reaction assay. The resulting mononucleotide fluorescent multiplex MSI assay has high accuracy, reliability, and throughput, thus reducing the time and cost involved in MSI testing.

99mTc-(CO)3 His-Annexin A5 Micro-SPECT Demonstrates Increased Cell Death by Irinotecan During the Vascular Normalization Window Caused by Bevacizumab

Colorectal tumors are dependent on angiogenesis for growth, and vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Antiangiogenic drugs can induce a transient normalization of the tumor vasculature with improved delivery of coadministered chemotherapy. The efficacy of antihuman VEGF antibody (bevacizumab) with or without irinotecan was evaluated in a colorectal cancer xenograft using 99mTc-(CO)3 His-annexin A5. Methods: Colo205-bearing mice were treated with a single dose of bevacizumab (5 mg/kg) during 2, 4, or 6 d. Microvessel density, pericyte coverage (α-smooth-muscle actin immunostaining), collagen-covered tumor vessels (Masson trichrome staining), and tumor hypoxic fraction (pimonidazole staining) were determined at the 3 different time points after treatment with bevacizumab. To investigate the possible synergistic effects of combination therapy with bevacizumab and irinotecan, Colo205-bearing mice were treated with a single dose of bevacizumab 2, 4, or 6 d before administration of a single dose of irinotecan (100 mg/kg) or 0.9% NaCl. The apoptosis-detecting radiotracer 99mTc-(CO)3 His-annexin A5 was injected (18.5 MBq) in mice 12, 24, and 48 h after the start of the irinotecan or NaCl treatment, and micro-SPECT was subsequently performed 3.5 h after injection of the radiotracer. Results were correlated to histologic analysis for apoptosis (caspase-3 activation). Results: Four days after bevacizumab administration, microvessel density decreased significantly, and α-smooth-muscle actin and collagen-covered vessels, compared with control tumors, were increased, suggesting normalization of the tumor vasculature. Hypoxic fraction was slightly reduced 4 d after treatment with bevacizumab. SPECT analyses demonstrated a significant increase in tumoral 99mTc-(CO)3 His-annexin A5 uptake 4 d after bevacizumab treatment and 24 h after irinotecan administration (232.78 ± 24.82 percentage injected dose/tumor weight [g]/body weight [kg], P < 0.05), compared with each monotherapy, indicating a synergistic effect of both therapies. Conclusion: 99mTc-(CO)3 His-annexin A5 micro-SPECT demonstrates increased antitumor activity of irinotecan during the transient vascular normalization period caused by bevacizumab. Our data outline the importance of timing of combined anti-VEGF treatment with chemotherapy.

In vivo imaging of apoptosis in oncology: an update.

In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that 99mTc-hydrazinonicoti-namide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither 31 P MRS nor 1 H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.

Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non-heme iron proteins

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides. It contains 125 amino acid residues of which five are cysteines. The first four, Cys‐9, Cys‐12, Cys‐28 and Cys‐29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas. The remaining Cys‐115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mössbauer and Raman spectroscopy.

In vitro and in vivo evaluation of [99mTc]-labeled tricarbonyl His-annexin A5 as an imaging agent for the detection of phosphatidylserine-expressing cells

INTRODUCTION Apoptosis is one of the mechanisms behind successful chemotherapy and radiation treatment. Radiolabeled annexin A5 has been demonstrated to be a successful tool in the detection of apoptosis following chemotherapy in vivo. METHODS His-tagged annexin A5 was labeled with [(99m)Tc]-tricarbonyl and evaluated as apoptosis imaging radiotracer in vitro and in vivo. The binding of the radiotracer was evaluated in Colo205 cells stimulated with 5-FU (1 mM) for 4 and 24 h, and confirmed by flow cytometry. Biodistribution and dosimetric studies were performed in healthy nude mice (n=5) via planar scintigraphy. [(99m)Tc]-(CO)(3) His-annexin A5 was also evaluated for in vivo imaging of spontaneous apoptosis in Colo205-bearing mice (n=12). RESULTS The labeling procedure yielded a compound with 95-99% radiochemical purity and good in vitro stability. In vitro binding experiments indicated that the radiotracer retained its PS-binding activity. [(99m)Tc]-(CO)(3) His-annexin A5 rapidly cleared from the blood and predominantly accumulated in the kidneys. Absorbed dose (per organ) was found to be 116 ± 64 μGy/MBq for the kidneys and 10.38 ± 0.50 μGy/MBq for the liver. The effective dose was 7.00 ± 0.28 μSv/MBq. Spontaneous apoptosis in Colo205-bearing mice was visualised by [(99m)Tc]-(CO)(3) His-annexin A5 SPECT and correlated well with caspase-3 immunostaining (R=0.867, P<.01). CONCLUSION [(99m)Tc]-(CO)(3) His-annexin A5 may be a useful novel radioligand for the in vivo detection of cell death associated with PS expression. A simple, noninvasive way of detecting apoptosis in vivo could have many applications including a better understanding of the extent and timing of apoptosis in response to cancer therapies and assessment of early tumor response.

Single-Photon Emission Computed Tomographic Imaging of the Early Time Course of Therapy-Induced Cell Death Using Technetium 99m Tricarbonyl His-Annexin A5 in a Colorectal Cancer Xenograft Model

As apoptosis occurs over an interval of time after administration of apoptosis-inducing therapy in tumors, the changes in technetium 99m (99mTc)-tricarbonyl (CO)3 His-annexin A5 (His-ann A5) accumulation over time were examined. Colo205-bearing mice were divided into six treatment groups: (1) control, (2) 5-fluorouracil (5-FU; 250 mg/kg), (3) irinotecan (100 mg/kg), (4) oxaliplatin (30 mg/kg), (5) bevacizumab (5 mg/kg), and (6) panitumumab (6 mg/kg). 99mTc-(CO)3 His-ann A5 was injected 4, 8, 12, 24, and 48 hours posttreatment, and micro–single-photon emission computed tomography was performed. Immunostaining of caspase-3 (apoptosis), survivin (antiapoptosis), and LC3-II (autophagy marker) was also performed. Different dynamics of 99mTc-(CO)3 His-ann A5 uptake were observed in this colorectal cancer xenograft model, in response to a single dose of three different chemotherapeutics (5-FU, irinotecan, and oxaliplatin). Bevacizumab-treated mice showed no increased uptake of the radiotracer, and a peak of 99mTc-(CO)3 His-ann A5 uptake in panitumumab-treated mice was observed 24 hours posttreatment, as confirmed by caspase-3 immunostaining. For irinotecan-, oxaliplatin-, and bevacizumab-treated tumors, a significant correlation was established between the radiotracer uptake and caspase-3 immunostaining (r = .8, p < .05; r = .9, p < .001; r = .9, p < .001, respectively). For 5-FU- and panitumumabtreated mice, the correlation coefficients were r = .7 (p = .18) and r = .7 (p = .19), respectively. Optimal timing of annexin A5 imaging after the start of different treatments in the Colo205 model was determined.