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.

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.

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.

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.

Locoregional administration of etoposide, but not of interleukin 2, facilitates active specific immunization in guinea pigs with advanced carcinoma.

Using a guinea pig line 10 hepatocellular carcinoma model for advanced metastatic disease, we studied the therapeutic effect of local cytotoxic drug treatment at the tumor site as compared to, and in combination with, active specific immunization. In addition, locoregional treatment with interleukin 2 (IL-2) was studied. Intratumoral administration of the cytotoxic drug etoposide (VP-16), but not of IL-2, when started in a late stage of tumor growth and continued for 3 wk, caused full regression of all intradermally implanted tumors and cured a small number of animals (14%). When the primary tumor was removed at the onset of treatment, administration of VP-16 and, to a lesser degree, IL-2 at the former tumor site led to improvement of cure rates (up to 30%). Complete cure always coincided with the induction of antitumor immunity. Since both VP-16 and IL-2, when locally administered, strongly augment T-cell-mediated immune responses, the observed therapeutic effect was partially attributed to potentiation of a T-cell-mediated antitumor response. Active specific immunization (ASI) using viable irradiated tumor cells admixed with Bacillus Calmette-Guérin also aims at induction of specific antitumor immunity. In late-stage disease, ASI alone induced cure rates of 39%. Combination of ASI with local cytotoxic drug treatment, but not with locoregional administration of IL-2 at the former primary tumor site, led to very high cure rates (up to 78%). Cured animals were always resistant to a second challenge with line 10 tumor cells. Routinely, one systemic injection with cyclophosphamide was given at the start of all treatment protocols. Omission of CY strongly reduced the cure rates obtained with ASI and locoregional VP-16 treatment. The high cure rates likely relate to the fact that locally administered cytotoxic drugs are capable of reversing immune tolerance, besides exerting direct antitumor action within tumor-draining lymphoid tissues. The present results therefore support our view that local cytotoxic drug treatment should be further explored for its incorporation in antitumor therapies such as ASI, aiming at maximal clinical benefit and minimal toxicities.

Tumour localisation with 131I-labelled human IgM monoclonal antibody 16.88 in advanced colorectal cancer patients.

Human IgM monoclonal antibody 16.88 recognised an intracellular antigen strongly expressed in colorectal cancer tissue in 51% of our patients. Tumour localisation was carried out with 185 MBq 131I-16.88 (8 mg) in 20 of these patients with advanced disease. In 16 patients (80%) immunoscintigraphy was positive in at least one organ site with disease. Of all sites, 55% could be visualized. In general, lesions less than 3 cm could not be detected. Sequential immunoscintigrams of liver metastases showed variable patterns. Initial "cold" lesions corresponded to liver metastases with poor blood supply as indicated by 99mTc-sulphur-colloid and 99mTc-HMPAO scintigraphy, respectively. The mean (S.D.) biological half-life (whole body clearance of radioactivity) was 37.6 (5.0) h. A second infusion of 131I-16.88 with the addition of high doses of unlabelled 16.88 could be done safely, but did not result in better visualisation of tumour lesions or affect radioactivity clearance from the body.

Specific and nonspecific T-cell-mediated suppression of antihost immune reactivity in graft-versus-host reaction.

Intravenous immunization of mice with irradiated (2000 rads) allogeneic lymphoid cells induces the generation of suppressor T cells. Such suppressor T cells are capable of suppressing the antihost immune reactivity during acute and delayed graft-versus-host reactions. These suppressor T cells are strictly antigen-specific as far as their activation is concerned, but also suppress the reaction against unrelated antigens presented by the irradiated host.

Suppression of antigraft immunity by preimmunization. II. Characterization of the suppressor cells.

Immunization of mice with irradiated (20 Gy) or non-irradiated allogeneic spleen cells i.v. induces delayed-type hypersensitivity (DTH)-reactive T cells, as well as suppressor T cells, against histocompatibility antigens. The suppressor T cells are unable to suppress the induction and functional activity of the simultaneously activated DTH-reactive T cells. However, the suppressor T cells do suppress the generation of DTH-reactive T cells after subsequent s.c. immunization of the same mice, and after transfer into secondary recipients. Systemic transfer of suppressor T cells is effective the first few days after their induction, and affects the afferent limb of the DTH response. The population of suppressor T cells, which is essential for the systemic transfer of suppression, appeared to be Lyt-1+2+. Splenectomy experiments showed that the spleen is not essential for induction of the suppressor T cells. The precursors of the suppressor T cells belong to the pool of recirculating T lymphocytes; they are insensitive to adult thymectomy and can be depleted by antithymocyte serum treatment.

Suppression of antigraft immunity by preimmunization. I. Kinetic aspects and specificity.

Intravenous injection of 2,000 rad of irradiated allogeneic cells can suppress the development of antigraft delayed-type hypersensitivity (DTH) to major and minor histocompatibility (H) antigens which normally arises after s.c. immunization. Secondary type DTH responses to minor H antigens were also largely suppressed by an i.v. injection of irradiated allogeneic cells 1 week preceding the s.c. priming injection. The extent of suppression of primary DTH to allogeneic H-2-incompatible cells depended on the dose of i.v. injected irradiated cells. After a dose of 1 x 10(7) irradiated spleen cells i.v., the suppression persisted for at least 40 days. Intravenous injection of cells incompatible for minor H antigens could not suppress the DTH to H-2 alloantigens and vice versa. Suppression of DTH to H-2 alloantigens was haplotype specific. Proliferation studies indicated that the immunosuppressed mice do not respond upon s.c. immunization with an increased proliferative activity in the draining lymph nodes, in contrast to nonsuppressed mice. The data suggest that i.v. preimmunization with allogeneic cells induces specific suppression of antigraft immunity acting at the induction stage of the immune response.

Suppression of antigraft immunity by preimmunization. IV. Persistence by long-lived recirculating suppressor T cells.

Delayed-type hypersensitivity (DTH) against histocompatibility (H) antigens in mice, which normally arises after s.c. immunization, can be prevented by i.v. preimmunization with irradiated spleen cells carrying the relevant H antigens. We have previously shown that during the first week after i.v. preimmunization the nonresponsiveness is due to suppressor T lymphocytes. The induced state of nonresponsiveness, however, is long-lasting. In this study we investigated whether this long-lasting state of nonresponsiveness of DTH is associated with suppressor T lymphocytes or is caused by inactivation of the relevant clones of alloreactive T cells. This was done by parabiosis of nonresponsive and naive mice and by transfer of thoracic duct lymphocytes from nonresponsive mice, harvested at various intervals after the i.v. immunization. At a long interval after the i.v. immunization, the state of nonresponsiveness could still be transferred to syngeneic naive mice by parabiosis, as well as by transfer of thoracic duct lymphocytes. Selective elimination of the T cells from the latter by treatment with anti-Thy-1.2 plus complement prevented the transfer of the state of nonresponsiveness, indicating that suppressor T cells were involved.

Lipopolysaccharide-induced suppression of DTH-reactivity to histocompatibility antigens. I. Kinetic aspects and specificity.

The effects of bacterial lipopolysaccharide (LPS) on the development of DTH-reactivity to alloantigens in mice were investigated. DTH to a particular set of alloantigens could be suppressed by treatment of responder mice with a single intravenous (i.v.) injection of 100 micrograms LPS and a simultaneous subcutaneous (s.c.) injection of the appropriate allogeneic spleen cells. The suppression lasted at least 60 days and affected the afferent limb of the DTH response as well as the efferent limb. The suppression could be adoptively transferred to naive syngeneic recipient mice by spleen cells, but not by immune serum, and proved to be antigen-specific. In spite of this specificity, the DTH-response against unrelated "third-party" alloantigens could be suppressed as well, provided the latter were presented during the induction-phase of DTH together with the alloantigens that had been used for the induction of suppression.

Characterization of suppressor T cells in graft-versus-host reactions.

Intravenous (i.v.) immunization of mice with irradiated (2000 rads) allogeneic lymphoid cells induces the generation of suppressor T cells. Such suppressor T cells can suppress the anti-host delayed type hypersensitivity (DTH) by other T cells during acute Graft-versus-Host reactions in irradiated recipient mice. Suppression of anti-host DTH is detectable at least 50 days after i.v. induction of the state of suppression in the donors. The generation of suppressor T cells in the donors is associated with proliferation. Furthermore, the suppressor T cells need to proliferate further in the irradiated allogeneic hosts in order to display a maximal suppressive effect. Two types of suppressor T cells were found to be required for suppression of the anti-host DTH response: a Lyt-1+2- cell population and a Lyt-1-2+ cell population.

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.

The long-lasting state of specific nonresponsiveness induced by intravenous immunization with alloantigens is due to the generation of recirculating suppressor T cells.

We investigated whether the long-lasting state of nonresponsiveness that is induced by intravenous immunization with alloantigens is mediated by suppressor T cells, or is caused by inactivation or deletion of the relevant alloreactive T cell clones. The data from parabiosis and thoracic duct drainage experiments suggest that the state of nonresponsiveness depends on recirculating non-proliferating Ts memory cells.

[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.

[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.

[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.

[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.