Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers.

Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear whether it confers worse survival in this setting. The prognostic value of KRAS mutations in both MSS and MSI CRC remains unclear. We examined the effect of BRAF and KRAS mutations on survival in stage II and III MSI colon cancer patients. BRAF exon 15 and KRAS exon 2-3 mutation status was assessed in 143 stage II (n = 85) and III (n = 58) MSI colon cancers by high resolution melting analysis and sequencing. The relation between mutation status and cancer-specific (CSS) and overall survival (OS) was analyzed using Kaplan-Meier and Cox regression analysis. BRAF V600E mutations were observed in 51% (n = 73) and KRAS mutations in 16% of cases (n = 23). Patients with double wild-type cancers (dWT; i.e., BRAF and KRAS wild-type) had a highly favourable survival with 5-year CSS of 93% (95% CI 84-100%), while patients with cancers harbouring mutations in either BRAF or KRAS, had 5-year CSS of 76% (95% CI 67-85%). In the subgroup of stage II patients with dWT cancers no cancer-specific deaths were observed. On multivariate analysis, mutation in either BRAF or KRAS vs. dWT remained significantly prognostic. Mutations in BRAF as well as KRAS should be analyzed when considering these genes as prognostic markers in MSI colon cancers.

The curious case of the orange coloured tonsils.

Tangier disease is an extremely rare and severe form of high density lipoprotein deficiency. Even though there is no specific therapy for patients with Tangier disease, it is important to recognize the clinical presentation as patients are at an increased risk of developing atherosclerosis and subsequent CVD. The case discussed in this report, illustrates the importance of recognizing that orange discoloured tonsils are an indication that the patient could be suffering from Tangier's disease.

Maspin is a marker for early recurrence in primary stage III and IV colorectal cancer.

BACKGROUND: Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC. METHODS: Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses. RESULTS: Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location. CONCLUSION: High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

Contributions of natural arsenic sources to surface waters on a high grade arsenic-geochemical anomaly (French Massif Central).

The subwatershed studied drains a non-exploited area of the St-Yrieix-la-Perche gold mining district (French Massif Central) and it is located on an arsenic (As) geochemical anomaly. In this context, it is important to know the geochemical processes involved in the transfer of As from solid environmental compartments to the aquatic system. The stream showed a temporal variation of dissolved As (As(d)) content from 69.4 µg.L(-1) in the low flow period to 7.5 µg.L(-1) in the high flow period. Upstream, ground- and wetland waters had As(d) concentrations up to 215 and 169 µg.L(-1), respectively. The main representative As sources were determined at the subwatershed scale with in-situ monitoring of major and trace element contents in different waters and single extraction experiments. The As sources to stream water could be regrouped into two components: (i) one As-rich group (mainly in the low flow period) with groundwater, gallery exploration outlet waters and wetland waters, and (ii) one As-poor group (mainly in the high flow period) with rainwaters and soil solutions. In the soil profile, As(d) showed a significant decrease from 52.4 µg.L(-1) in the 0-5 cm superficial soil horizon to 14.4 µg.L(-1) in the 135-165 cm deep soil horizon. This decrease may be related to pedogenic processes and suggests an evolution of As-bearing phase stability through the soil profile. Quantification of As(d) fluxes at the subwatershed scale showed that groundwater was the major input (>80%) of As(d) to surface water. Moreover, natural weathering of the As-rich solid phases showed an impact on the As release, mainly from superficial soil horizons with runoff contributing about 5% to As input in surface water.

High lymph node yield is related to microsatellite instability in colon cancer.

BACKGROUND: Lymph node (LN) yield in colon cancer resection specimens is an important indicator of treatment quality and has especially in early-stage patients therapeutic implications. However, underlying disease mechanisms, such as microsatellite instability (MSI), may also influence LN yield, as MSI tumors are known to exhibit more prominent lymphocytic antitumor reactions. The aim of the present study was to investigate the association of LN yield, MSI status, and recurrence rate in colon cancer. METHODS: Clinicopathological data and tumor samples were collected from 332 stage II and III colon cancer patients. DNA was isolated and PCR-based MSI analysis performed. LN yield was defined as "high" when 10 or more LNs were retrieved and "low" in case of fewer than 10 LNs. RESULTS: Tumors with high LN yield were significantly associated with the MSI phenotype (high LN yield: 26.3% MSI tumors vs low LN yield: 15.1% MSI tumors; P=.01), mainly in stage III disease. Stage II patients with high LN yield had a lower recurrence rate compared with those with low LN yield. Patients with MSI tumors tended to develop fewer recurrences compared with those with MSS tumors, mainly in stage II disease. CONCLUSIONS: In the present study, high LN yield was associated with MSI tumors, mainly in stage III patients. Besides adequate surgery and pathology, high LN yield is possibly a feature caused by biologic behavior of MSI tumors.

Fate of arsenic-bearing phases during the suspended transport in a gold mining district (Isle river Basin, France).

Arsenic-rich (~140-1520 mg x kg(-1)) suspended particulate matter (SPM) was collected daily with an automatic sampler in the Upper Isle River (France) draining a former gold mining district in order to better understand the fate of arsenic during the suspended transport (particles smaller than 50 µm). Various techniques at a micrometric scale (EPMA, quantitative SEM-EDS with an automated particle counting including classification system and µXRD) were used to directly characterize As-bearing phases. The most frequent ones were aggregates of fine clay particles. Their mineralogy varied with particle sources involved. These aggregates were formed by chlorite-phlogopite-kaolinite assemblages during the high flow and chlorite-illite-montmorillonite during the low flow. Among all the observed As-carriers in SPM, these clay assemblages were the least As-rich (0.10 up to 1.58 wt.% As) and their median As concentrations suggested that they were less concentrated during the high flow than during the low flow. Iron oxyhydroxides were evidenced by µXRD in these clay aggregates, either as micro- to nano-sized particles and/or as coating. (Mn, Fe)oxyhydroxides were also present as discrete particles. Manganese oxides (0.14-1.26 wt.% As) transport significantly more arsenic during the low flow than during the high flow (0.16-0.79 wt.% As). The occurrence of Fe oxyhydroxide particles appeared more complex. During the low flow, observations on banks and in wetlands of freshly precipitated Fe hydroxides (ferrihydrite-type) presented the highest As concentrations (up to 6.5 wt.% As) but they were barely detected in SPM at a microscale. During the high flow, As-rich Fe-oxyhydroxides (0.10-2.80 wt.% As) were more frequent, reflecting mechanical erosion and transport when the surface water level increased. Arsenic transfers from SPM to corresponding aqueous fraction mostly depend on As-carrier stability. This study shows the temporal occurrence of each type of As-bearing phases in SPM, their As concentrations at a particle scale and abundance according to hydrological periods.

Loss of lamin A/C expression in stage II and III colon cancer is associated with disease recurrence.

AIM OF THE STUDY: Loss of the nuclear lamina protein lamin A/C (LMNA) has been observed in several human malignancies. The present study aimed to investigate associations between LMNA expression and clinical outcome in colon cancer patients. PATIENTS AND METHODS: Clinicopathological data and formalin-fixed paraffin embedded tissues were collected from 370 stage II and III colon cancer patients. Tissue microarrays were constructed, stained for lamin A/C and evaluated microscopically. Microsatellite instability status was determined for 318 tumours. RESULTS: Low levels of LMNA expression were observed in 17.8% of colon tumours, with disease recurrence occurring in 45.5% of stage II and III colon cancer patients with LMNA-low expressing tumours compared to 29.6% of patients with LMNA-high expressing tumours (p=0.01). For stage II patients, disease recurrence was observed for 35.7% of LMNA-low compared to 20.3% of LMNA-high expressing tumours (p=0.03). Microsatellite stable (MSS) tumours exhibited more frequently low LMNA expression than microsatellite instable (MSI) tumours (21% versus 9.8%; p=0.05). Interestingly, disease recurrence among LMNA-low and LMNA-high expressing MSS tumours varied significantly for stage III patients who had not received adjuvant chemotherapy (100% versus 37.8%; p<0.01) while no such difference was observed for patients who received adjuvant chemotherapy (46.7% versus 46.0%; p=0.96). CONCLUSION: These data indicate that low expression of LMNA is associated with an increased disease recurrence in stage II and III colon cancer patients, and suggest that these patients in particular may benefit from adjuvant chemotherapy.

Lymph node negative colorectal cancers with isolated tumor deposits should be classified and treated as stage III.

BACKGROUND: The prognostic role of pericolic or perirectal isolated tumor deposits (ITDs) in node-negative colorectal cancer (CRC) patients is unclear. Rules to define ITDs as regional lymph node metastases changed in subsequent editions of the TNM staging without substantial evidence. Aim of this study was to investigate the correlation between ITDs and disease recurrence in stage II and III CRC patients. MATERIALS AND METHODS: The medical files of 870 CRC patients were reviewed. Number, size, shape, and location pattern of all ITDs in node-negative patients were examined in relation to involvement of vascular structures and nerves. The correlation between ITDs and the development of recurrent disease was investigated. RESULTS: Disease recurrence was observed in 50.0% of stage II patients with ITDs (13 of 26), compared with 24.4% of stage II patients without ITDs (66 of 270) (P < .01). Disease-free survival of ITD-positive stage II patients was comparable with that of stage III patients. Also within stage III, more recurrences were observed in ITD-positive patients compared with ITD-negative patients (65.1 vs. 39.1%, respectively). No correlation was found between size of ITDs and disease recurrence. More recurrences were seen in patients with irregularly shaped ITDs compared with patients with 1 or more smooth ITDs present. CONCLUSIONS: Because of the high risk of disease recurrence, all node-negative stage II patients with ITDs, regardless of size and shape, should be classified as stage III, for whom adjuvant chemotherapy should be considered.

Deconvolution of trace element (As, Cr, Mo, Th, U) sources and pathways to surface waters of a gold mining-influenced watershed.

The Upper Isle River (SW France) drains the second most productive gold-mining district of France. A high resolution survey during one hydrological year of As, Cl(-), Cr, Fe, Mn, Mo, SO(4)(2-), Th and U dissolved concentrations in surface water aimed to better understand pathways of trace element export to the river system downstream from the mining district. Dissolved concentrations of As (up to 35000 ng/L) and Mo (up to 292 ng/L) were about 3-fold higher than the regional dissolved background and showed a negative logarithmic relation with discharge. Dissolved concentrations of Cr (up to 483 ng/L), Th (up to 48 ng/L) and U (up to 184 ng/L) increased with discharge. Geochemical relationships between molar ratios in surface water, geochemical background as well as rain- and groundwater data were combined. The contrasting behavior of distinct element groups was explained by a scenario involving three seasonal components: (i) The high flow component is poorly concentrated in As and Mo but highly concentrated in Cr, Th, U. This has been attributed to diffuse sources such as water-soil interactions, atmospheric inputs, bedrock and bed sediment weathering. Although this component probably also includes a contribution by weathering of sulfide veins, this signal is masked by dilution. (ii) One low flow component presents high SO(4)(2-), Fe, As and Mo and moderate Cr, Th and U concentrations. This component has been attributed to point sources such as mine gallery effluents, mining waste weathering and groundwater inputs from natural and/or mining-induced sulfide oxidation in the ore deposit. (iii) A second low flow component showing high As plus Mo concentrations associated with very low SO(4)(2-), Fe, Cr, Th and U concentrations, probably reflects trace element scavenging by ferric oxyhydroxide formation in the adjacent aquifer. This is supported by the decrease of Fe, Cr, Th and U in surface waters. Flux estimates suggest contrasting element-specific impacts on annual dissolved fluxes. Runoff may account for the major part of annual dissolved As, Mo, Th and U fluxes in the Upper Isle River. Inputs related to sulfide oxidation respectively contributed approximately 30% and approximately 24% to annual As and Mo fluxes. The formation of ferric oxyhydroxides strongly retained Cr, Th and U during the low flow, limiting their dissolved concentrations in surface waters. If this process may eventually decrease As mobility, its impact on dissolved As concentrations in surface water may be limited or/and counterbalanced by As release during sulfide oxidation.

Solid speciation and mobility of potentially toxic elements from natural and contaminated soils: a combined approach.

The study area (Szklary Massif, SW Poland) comprises three sites of different soil provenance: (1) natural serpentine Cambisol, (2) anthroposol situated on waste dump and (3) cultivated Inceptisol developed on glacial tills next to the dump. Potentially toxic elements (PTE) have either lithogenic or anthropogenic origins in these sites. The chemical partitioning of Co, Cr, Cu, Ni, Pb and Zn among solid forms was determined by sequential extractions coupled with direct mineral identifications (SEM, electron microprobe analysis - EMPA, and XRD). Examination of solid residues after several extraction steps was conducted in order to discuss the indirect speciation obtained by the extraction method. Total concentrations of PTE having anthropogenic origin greatly exceed those of lithogenic origin. Mobility of studied PTE is variable in the different environments except for Cr which is always mostly found in residual fractions of extractions. Cu and Pb are more mobile than Cr and Co in all soils. Zn is more stable (Cu>Pb>Ni>Co>Zn>>Cr) in the serpentine soil and cultivated epipedon (Pb>Cu>Zn>Ni>Co>>Cr) than in the anthroposol (Zn>CuPb>Ni>Co>>Cr). PTE of lithogenic origin are generally less mobile than those from anthropogenic origin except Ni which is more mobile in the serpentine soil. Nonetheless, mineral forms of metals better determine their mobility than metal origin. Identification by direct methods of the PTE mineral form was not possible for metals present at low concentrations (Cu, Pb). However, direct mineralogical examinations of the solid residues of several extractions steps improved the assessment of the PTE solid speciation and mobility, particularly for Cr, Ni and Zn.

[Important prognostic significance of a sentinel-node biopsy in patients with malignant melanoma]. / Belangrijke prognostische betekenis van schildwachtklierbiopsie bij patiënten met maligne melanoom.

OBJECTIVE: To determine the prognostic significance of sentinel-node biopsy in patients with malignant melanoma (unlike the United States, a sentinel-node biopsy is still not routinely performed on melanoma patients in the Netherlands, as the outcomes of prospectively randomised clinical trials are being awaited). DESIGN: Retrospective. METHODS: Between 1996 and 2001 a sentinel-node biopsy and a re-excision of the scar of the diagnostic biopsy were performed on all melanoma patients who had a Breslow thickness > or = 1 mm or a Clark level > or = IV. At operation the sentinel node was identified with a gamma probe and patent blue. It was removed and sent for pathological investigation for the presence of melanoma cells. If the sentinel node was tumour positive, a dissection of the regional lymph-node basin was performed. Subsequently, these patients were put forward for the European Organisation for Research and Treatment of Cancer (EORTC) peginterferon alfa(2b) adjuvant treatment study. RESULTS: A sentinel-node biopsy was performed in 61 lymphnode basins in 57 patients (18 male and 39 female; median age: 45 years (range: 9-80)). The median Breslow thickness of the melanomas was 2.2 mm (range: 0.7-13 mm). In 10 of the 61 cases histological examination of the sentinel node demonstrated tumour cells. In 2 additional cases tumour cells were demonstrated only by immunohistochemical studies or complete dissection of the node. Eight regional lymph-node basins were dissected, two of which contained additional metastases. The median follow-up was 36 months (range: 1-68). During follow-up 12 of the 57 patients were found to have metastases, in 8 of these patients the sentinel-node biopsy contained tumour cells. The negative predictive value of a tumourless sentinel node with respect to the later occurrence of distant metastases was 92%. CONCLUSION: The patients with a tumour-positive sentinel node had a poorer prognosis with respect to distant metastases than patients with a tumour-negative node. This is the main reason for performing sentinel-node biopsy: to predict the prognosis of the disease. Therefore sentinel-node biopsy should be incorporated into the treatment of patients with malignant melanoma.

[Erythema multiforme vs. Stevens-Johnson syndrome and toxic epidermal necrolysis: an important diagnostic distinction]. / Erythema multiforme versus Stevens-Johnson-syndroom en toxische epidermale necrolyse; belangrijk diagnostisch onderscheid.

Three patients, a girl aged 10 and two women aged 59 and 64 years, had erythema multiforme, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), respectively. SJS and TEN are rare illnesses with a high morbidity and mortality. The aetiology is mainly iatrogenic: a hypersensitivity reaction to certain pharmaceutical prescriptions. SJS and TEN should be differentiated from the more frequent erythema multiforme, a self-limiting disease without important residual symptoms, which is usually initiated by infection with herpes simplex virus. SJS and TEN are variants in a spectrum of exfoliative dermatoses with epidermal necrosis. SJS and TEN on the one hand and erythema multiforme on the other can be distinguished on the basis of aetiology, clinical symptoms and histopathology. The distinction can, however, be difficult, notably in the early stages. The girl recovered completely. The first woman was treated with corticosteroids and also recovered; she was thought to have developed the syndrome as a reaction to malarial prophylactics. The third patient died, despite extensive treatment, of multiorgan failure and sloughing of 70% of the skin, probably as a reaction to amoxicillin given for pneumococcal pneumonia.

[Less operations required due to perioperative frozen section examination of sentinel nodes in 275 breast cancer patients]. / Minder operaties vereist door peroperatief vriescoupeonderzoek van schildwachtklieren bij 275 patiënten met mammacarcinoom.

OBJECTIVE: To determine the reliability of a peroperative frozen section examinations of sentinel lymph nodes in mammary carcinoma. DESIGN: Retrospective. METHOD: In the Reinier de Graaf Hospital and Diagnostic Centre SSDZ Delft, the Netherlands, the results of frozen section from sentinel lymph node investigations of mammary carcinomas from 1997-2000 were compared with the final pathological results. If axillary dissection had been performed on these patients, the histopathological findings of the dissected lymph nodes were also studied. RESULTS: Frozen sections were made of 287 sentinel lymph nodes from 275 patients. A tumour was found in the sentinel lymph nodes of 64 patients and these patients immediately underwent a complete axillary lymph node dissection. For 31 of these patients a tumour was also found in the other lymph nodes. In 29 of these 31 patients, histological examination had shown extranodal extension. The frozen sections from the sentinel nodes of the remaining 211 patients were considered negative. However, in 13 of these patients, the paraffin sections of the sentinel node nevertheless showed a tumour and the remaining axillary lymph nodes were removed in a second operation. In the last 89 patients studied, the sentinel lymph nodes were cut at four levels and stained immunohistochemically at one level for cytokeratins. Accordingly micrometastases were found in the sentinel lymph nodes of 4 of the 13 patients with (false-)negative frozen sections. False-positive results did not occur. CONCLUSION: The major advantage of the sentinel node method in breast cancer is that for women without metastasis present in the sentinel node, axillary dissection is avoided. By means of a peroperative examination of frozen sections, 83% of the patients with a metastasis in the sentinel lymph node (or about one quarter of all patients) were spared from having a second operation for axillary dissection at a later stage.

[Diagnostic image (54). Sinus thrombosis and hemorrhagic infarction in puerperium ]. / Diagnose in beeld (54). Sinustrombose en hemorragische infarcering in het kraambed ('kraamhoofd').

A 28-year-old woman developed puerperal sinus sagittalis superior thrombosis that caused hemorrhagic cerebral infarction and death by cerebral herniation.

Minerals controlling arsenic and lead solubility in an abandoned gold mine tailings.

Numerous areas have been contaminated by heavy metals and metalloids due to industrial and mining activities. Studies investigating the behavior of such contaminants in the environment have identified speciation as a key factor controlling their mobility, availability and toxicity. Here we characterize As- and Pb-bearing phases resulting from the oxidation of sulfide-rich tailings of a former gold mine (La Petite Faye, France) in order to assess the risk for water quality. Elements were first pre-concentrated by granulometric fractionation (sedimentation in deionized water) and then investigated using X-ray diffraction and electron microprobe analyses. Two main As-Pb-bearing minerals were clearly identified: scorodite (FeAsO4 x 2H2O) and beudantite PbFe3(AsO4)(SO4)(OH)6. Minor amounts of As and Pb were dissolved in deionized water during granulometric fractionation, indicating the possible presence of other soluble Pb-sulfates which could be some of the primary metastable products of sulfide oxidation. This dissolution also provides information about the fate of these phases in the case of intensive leaching of the tailings. Scorodite may not be considered as a relevant candidate for As on-site immobilization, because its solubility largely exceeds drinking water standards whatever the pH. Since beudantite solubility has not yet been determined, an estimation of its solubility product was obtained using the Gibbs free energy of formation of plumbojarosite [Pb0.5Fe3(SO4)2(OH)6]. This estimation suggests that beudantite should efficiently maintain low Pb concentration in waters. However, Pb dissolution in deionized water during the granulometric fractionation led to Pb concentrations much higher than the French and US drinking water standards (2.4 x 10(-7) mol l(-1)), which may be due to dissolution of the suspected metastable Pb-sulfates. Accurate determination of beudantite solubility is now required to improve the Pb risk assessment on such polluted sites.

Value of a panel of antibodies to identify the primary origin of adenocarcinomas presenting as bladder carcinoma.

AIMS: Adenocarcinomas may arise primarily from the urinary bladder, but secondary involvement from adenocarcinomas arising in adjacent organs is more common. In the present study we tried to differentiate primary urinary bladder adenocarcinomas from adenocarcinomas arising from the surrounding organs, based on their antigen profiles in routinely processed, paraffin-embedded tissue specimens. We analysed the staining results using stepwise linear discriminant analysis. METHODS AND RESULTS: We investigated the usefulness of a panel of antibodies against cytokeratin 7, E48, cytokeratin 20, PSA, PSAP, CEA, vimentin, OC125 and HER-2/neu, to discriminate primary bladder adenocarcinoma from adenocarcinomas arising from the prostate, urachus, colon, cervix, ovary and endometrium. In the differential diagnosis with urinary bladder adenocarcinoma, an overall correct classification was reached for 77% and 81% of urachal and colonic carcinomas, respectively, using CEA, for 93% of prostatic adenocarcinomas using PSA, for 82% and 70% of cervical and ovarian adenocarcinomas, respectively, using OC125, and for 91% of endometrial adenocarcinomas using vimentin. Adding other antibodies did not improve the classification results for any of these differential diagnoses. CONCLUSIONS: For the surgical pathologist, a panel of antibodies consisting of CEA, PSA, OC125 and vimentin is helpful to differentiate primary urinary bladder adenocarcinomas from adenocarcinomas originating from prostate and endometrium, less helpful in differentiation with urachal carcinoma, and not helpful in differentiation with colonic, cervical and ovarian carcinoma.

Haemoptysis and an abnormal X-ray after prolonged treatment in the ICU.

We describe a case of Nocardia bronchopneumonia occurring in a patient after 5 weeks of treatment in an ICU. The clinical features were haemoptysis and fever, and an aspecific coarse nodular pattern on the chest roentgenogram. The diagnosis was made, after death, by positive blood cultures for Nocardia asteroides and autopsy showing Nocardia bronchopneumonia.

Efficient expansion of tumor-infiltrating lymphocytes from solid tumors by stimulation with combined CD3 and CD28 monoclonal antibodies.

Combined CD3 and CD28 monoclonal antibodies (mAb) may initiate efficient activation and expansion of tumor-infiltrating lymphocytes (TIL). In this study we compared phenotypical and functional characteristics of TIL from a group of 17 solid human tumors, stimulated either by high-dose recombinant interleukin 2 (rIL-2, 1000 IU/ml) or by a combination of anti-CD3 and anti-CD28 monoclonal antibodies in the presence of low-dose rIL-2 (10 IU/ml). Compared to activation with high-dose rIL-2, stimulation of TIL with CD3/CD28 mAb induced significantly stronger proliferation and yielded higher levels of cell recovery on day 14. Following the CD3/CD28 protocol, expansion of an almost pure population of CD3+ cells was obtained. Whereas CD4+ cells dominated in the first week of culturing, within 4 weeks the CD8+ population increased to over 90%. The specific capacity to kill autologous tumor cells was not increased as compared to the high-dose rIL-2 protocol, but all cultures showed high cytotoxic T cell activity as measured in a CD3-mAb-mediated redirected kill assay. These studies show that combined CD3 and CD28 mAb are superior to rIL-2 with respect to the initiation of expansion of CD8+ cytolytic TIL from solid tumors. Stimulation with specific tumor antigens at a later stage of culturing may further augment the expansion of tumor-specific cytolytic T cells.

Serum antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease are mainly associated with ulcerative colitis. A correlation study between perinuclear antineutrophil cytoplasmic autoantibodies and clinical parameters, medical, and surgical treatment.

Perinuclear antineutrophil cytoplasmic antibodies have recently been demonstrated in the sera of patients with inflammatory bowel disease. Three hundred and sixty six sera obtained from 120 patients with ulcerative colitis, 105 patients suffering from Crohn's disease and 49 non-inflammatory bowel disease controls were tested in two laboratories, using an indirect immunofluorescence assay. In addition, a fixed-neutrophil enzyme linked immunoadsorbent assay (ELISA) was evaluated in one of the two laboratories. The results in the immunofluorescence test showed a high degree of correlation between the two laboratories (Kappa coefficient = 0.8). Ninety five of the 120 (79%) ulcerative colitis patients had a positive test whereas only 14 of the 105 (13%) patients with Crohn's disease were positive. Sera from four patients suffering from primary sclerosing cholangitis were positive as well as four of the 45 control sera (9%). The sensitivity of the perinuclear antineutrophil cytoplasmic antibody immunofluorescence test for the diagnosis of ulcerative colitis was 0.75 with a specificity of 0.88 and a positive predictive value of 0.88 (all sera). In the ELISA technique 37 of 94 ulcerative colitis sera and one of the 68 Crohn's disease sera were positive. In the control group only one of the patients suffering from primary sclerosing cholangitis reacted positively (32 non-inflammatory bowel disease sera tested). The ELISA technique had a high specificity (0.97), but a low sensitivity (0.39). There was no relation of perinuclear antineutrophil cytoplasmic antibodies in ulcerative colitis patients or in Crohn's disease patients with disease activity, duration of illness, localisation, extent of disease, previous bowel operations or medical treatment. The clinical significance of perinuclear antineutrophil cytoplasmic antibody positive and negative subsets in both groups of patients thus remains unexplained. Our study confirms that determination of serum antineutrophil cytoplasmatic antibodies in patients with inflammatory bowel disease may differentiate ulcerative colitis from Crohn's disease. Further immunological studies are needed to explain the absence of these antibodies in a subset of ulcerative colitis patients and their role in the pathogenesis of the disease.