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The surgeon's role in primary breast cancer.

During the 1960s, it was suggested that, at least in clinical stage I disease, simple mastectomy or even wide excision, plus radiotherapy to the breast and gland fields resulted in the same distant recurrence rate and survival as the orthodox radical operation. It was thought that local control of the disease was not important in terms of ultimate prognosis and it was felt that minimal surgery incurred no penalty. It was forecast that breast cancer would soon be treated by radiotherapy alone, possibly even without removal of the tumour. Four developments have materially affected the situation. 1. It has been shown that assays of hormone receptors should be carried out on all primary tumours. 2. In clinical stage II disease, it has been demonstrated that restricted surgery results in a penalty in terms of distant recurrence and survival. 3. It is now realised that the number of involved axillary lymph nodes must be known to evaluate future prognosis. 4. Adjuvant chemo or endocrine therapy have proved to be effective in treating patients with heavy axillary node involvement. These developments mean that the effective treatment of early breast cancer must entail removal of the tumour, achieve local control of disease and include axillary node dissection. Currently, only the modified radical mastectomy effectively equates with these three aims. Nevertheless, it is possible that tumour excision, irradiation to the tumour bed and breast, and axillary node dissection would achieve the same object whilst conserving the breast.

Axilla↗

Induction of cytotoxicity of the renal hilar lymph nodes by pedal subcutaneous administration of interleukin-2 in patients with renal cancer.

The authors attempted to increase the cytotoxicity of the renal hilar lymph nodes in ten patients with renal cell carcinoma by administration of recombinant interleukin-2 (IL-2) into the subcutaneous tissue of the instep. A total of 5000 to 21,000 units was given over 3 to 12 preoperative days. Mononuclear cells separated from the lymph nodes excised from the renal hilum at surgery served as the effector cells. Fresh autologous tumor cells and three cultured cell lines, K562, Raji, and ACHN, served as the target cells. Cytotoxicity was measured by a chromium-releasing assay. The results showed that the mononuclear cells from the lymph nodes of the patients given IL-2 became cytotoxic to all target cells. Surface markers of lymphocytes in the lymph nodes were unchanged. Peripheral blood lymphocytes showed increased cytotoxicity only against Raji cells. Adverse effects were mild or moderate and included local redness, high fever, liver dysfunction, and toxic erythema.

Adult↗

Serum TA90 immune complex assay can predict outcome after resection of thick (> or =4 mm) primary melanoma and sentinel lymphadenectomy.

BACKGROUND: We hypothesized that the postoperative serum level of TA90-IC, an immune complex of a 90-kDa tumor-associated antigen and its antibody, might have a significant correlation with recurrence and survival in patients with thick primary melanomas. METHODS: We used our prospective melanoma database to identify all patients who underwent wide local excision and sentinel lymphadenectomy for primary melanomas > or =4 mm and from whom sera had been collected and cryopreserved within 6 months after surgery. These sera were analyzed in a blinded fashion for TA90-IC status by using our double-determinant enzyme-linked immunosorbent assay. Results were correlated with disease-free survival (DFS) and overall survival (OS). Standard prognostic factors for melanoma were then compared with TA90-IC status for the prediction of DFS and OS. RESULTS: The sensitivity and specificity of the TA90-IC assay for predicting recurrence were 70% and 85%, respectively. Five-year DFS and OS rates were higher for the TA90-IC-negative group than the positive group. The differences in DFS and OS between the TA90-IC-negative and -positive groups were significant. At a median follow-up of 25 months, multivariate analysis identified postoperative TA90-IC status and sex as significant predictors of DFS. TA90-IC status was the only independent prognostic factor with multivariate analysis. CONCLUSIONS: TA90-IC status after resection of thick primary melanoma accurately predicts outcome. A positive postoperative TA90-IC level might affect a decision regarding adjuvant therapy, regardless of regional nodal status.

Adult↗

Immunologic responses following respiratory sensitization to house dust mite allergens in mice.

Allergens from the house dust mite Dermatophagoides pteronyssinus are a major cause of human respiratory diseases, including asthma. In order to help in understanding the early events in allergen sensitization, a murine model of allergic respiratory disease was developed. Mice were immunized by intranasal inoculation of Der p 1 or Der p 2 on days 0, 3, 7, 10, 14, 17, 21 and 29. T cell reactivity was determined using in vitro assays of allergen-specific cytokine production by cells from the spleen, the draining superficial cervical lymph nodes (SCLN) and the non-draining brachial and inguinal nodes. The cytokines assayed in supernatants were IL-4, as a measure of Th2 activation, IL-2 as a measure of Th 1 activation, and IL-3/GM-CSF as an overall marker of T cell stimulation. There was evidence of local and systemic T cell activation by day 7, with the release of IL-2 and of IL-3/GM-CSF by SCLN and spleen cells, respectively. IL-4 production by SCLN and spleen cells was not evident until day 21. T cell sensitization in non-draining node groups was not detected. Intradermal skin tests were performed at the above specified times and positive wheal responses indicated that specific IgE was present from day 3. The above data suggest that respiratory immunization to allergen is rapid and is associated with early systemic sensitization. In this model both Th1 and Th2 responses were evident, with the Th1 response occurring early and the Th2 following after repeated immunizations.

Allergens↗

Contribution of the spleen, lymph nodes and bone marrow to the antibody response in collagen-induced arthritis in the rat.

The relative contribution of different lymphoid tissues to the anti-CII antibody response was studied in rats with arthritis produced as a result of immunizing them with collagen type II (CII). Antibody production was measured by maintaining lymphoid cells in short-term culture in collagen-coated microculture wells: the antibody they secreted was determined directly by a modified ELISA. Systemic sensitization to CII was established within a week of immunization, and a stronger response in the local draining lymph nodes relative to the spleen was associated with the development of clinical disease. From experiments involving splenectomy and adoptive cell transfer, the spleen was ascribed a suppressive role in controlling both arthritis and total antibody production. The bone marrow was found to be an important site of antibody production and the greater production of antibody by cells from tibial marrow in limbs with arthritis, compared with healthy limbs, argues for a local immune response to degrading joint antigens that may have systemic suppressive or protective properties. It is concluded that local immunity reflects the state of disease and that the antibodies produced by different lymphoid tissues may be made in response to different stimuli, and that the antibodies in turn may have different pathological effects.

Animals↗

The mouse lymph node homing receptor is identical with the lymphocyte cell surface marker Ly-22: role of the EGF domain in endothelial binding.

The lymph node homing receptor core polypeptide (mLHRc) is composed of a tandem collection of domains: a lectin domain, an epidermal growth factor (EGF) domain, and two repeats common in complement regulatory proteins. Here we demonstrate localization of mLHRc to chromosome 1, the portion syntenic with chromosome 1 in man. This locus is inseparable in mouse strains from the murine lymphocyte cell surface marker Ly-22. The data indicate that Ly-22 is an allelic determinant on the LHR resulting from a single amino acid interchange within the EGF domain. Cross-blocking experiments demonstrate that anti-Ly-22 and MEL-14 recognize independent epitopes and that Ly-22 is distinct from the carbohydrate binding region. Application of anti-Ly-22 in the in vitro binding assay shows inhibition of binding of lymphocytes to high endothelial venules (HEVs). The localization of the Ly-22 epitope in this novel chimeric protein suggests direct participation of the EGF domain in the adhesion of lymphocytes to HEV.

Amino Acid Sequence↗

In vivo immune responses of mice during carcinogenesis by ultraviolet irradiation.

In these experiments, we tested in various in vivo assays the immune responses of inbred C3H/HeN(MTV-) (C3H-) mice during carcinogenesis by chronic exposure to UV irradiation. Although the UV-treated mice were unable to reject syngeneic UV-induced tumor transplants, they rejected H-2-incompatible tumor allografts and H-2-compatible skin allografts. The primary hemagglutinin response to sheep red blood cells was normal in these mice, as were the induction of a local graft-versus-host reaction with lymphoid cells from UV-irradiated donors and the induction of an inflammatory response to dimethyl sulfoxide in the footpads of UV-treated mice. An early transient depression of two reactions in UV-irradiated mice occurred: delayed hypersensitivity to dinitrochlorobenzene measured by footpad swelling and the graft-versus-host reaction in UV-irradiated recipients measured by the use of the popliteal lymph node weight gain assay. Both of these reactions returned to a normal level before the development of primary tumors. We conclude that the inability of UV-irradiated mice to reject syngeneic and autochthonous UV-induced tumors was not due to a generalized immunosuppressive effect of chronic UV irradiation.

Animals↗

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer.

The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg(-1) protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.

Adult↗

Interferon-gamma release in sympathetically denervated rat submaxillary lymph nodes.

OBJECTIVE: To examine the regulation of interferon (IFN)-gamma release by cells derived from submaxillary lymph nodes of rats subjected to an acute or chronic superior cervical ganglionectomy (SCGx). METHODS: A unilateral SCGx and a contralateral sham operation were performed. Twenty hours or 7 days later cells from submaxillary lymph nodes were incubated for 24 h without any additional treatment (experiment 1), after adding lipopolysaccharide or concanavalin A (experiment 2) or after adding norepinephrine (NE, 10(-8) M; experiment 3). IFN-gamma concentration in the culture media was measured by ELISA. RESULTS: Compared to controls, cells obtained from lymph nodes at a time of degeneration of sympathetic nerve terminals released more IFN-gamma, whereas those derived from chronically SCGx lymph nodes released less IFN-gamma. Stimulation of IFN-gamma release by mitogens was detectable in the innervated or acutely denervated lymph nodes, but not in chronically denervated lymph nodes. When the effect of 10(-8) M NE on IFN-gamma release was tested, the neurotransmitter augmented cytokine release in cells prepared from chronically denervated lymph nodes only. CONCLUSION: The microenvironment provided by local sympathetic nerves is essential to enable an appropriate IFN-gamma release by submaxillary lymph node cells to occur.

Animals↗

Development of plasmacytoid cells with Russell bodies in autoimmune "viable motheaten" mice.

Mice homozygous for the autosomal recessive mutation "viable motheaten" (mev) are severely immunodeficient, show polyclonal B-cell activation, and express multiple autoantibodies over a maximum life span of 25 weeks. Lymphoid tissues from these mice contain large numbers of atypical plasma cells in which discrete glycoprotein inclusions are found within the endoplasmic reticulum. Such plasma cells are termed "Mott cells," and the inclusions are called "Russell bodies." Dense accumulations of Mott cells are present in the marginal zones of the spleen and in the lymph nodes of mev/mev mice. Russell bodies in Mott cells from mev/mev mice contain immunoglobulin (Ig) as shown by immunofluorescence microscopy at the light-microscopic level and by indirect protein A-immunogold localization at the electron-microscopic level. Ultrastructural analyses reveal the presence of amorphous, lamellar, and crystalline Russell bodies. These Ig crystals have a periodicity of 150-190 A. Lymph node cell preparations which were enriched in Mott cells by velocity sedimentation failed to secrete Ig in a polyclonal reverse plaque assay. An obligate role of the thymus in Mott cell development is evidenced by the absence of Mott cells in neonatally thymectomized mev/mev mice and in mice doubly homozygous for the nude (nu) and mev mutations. These data suggest that Mott cells in mev/mev mice are thymic-dependent plasmacytoid cells resulting from chronic B-cell activation accompanied by impaired Ig secretion.

Age Factors↗

Why certain dyes are useful for localizing the sentinel lymph node.

UNLABELLED: Location of the sentinel lymph node in malignant melanoma and early breast cancer patients requires separate injections of radiocolloid and blue dye. These agents are administered at alternative times because of their different rates of transit. This study has elucidated why particular dyes are absorbed by the lymphatic system from an investigation of dye molecular structure as a function of protein binding ability. METHODS: A dye-protein binding assay was developed using size-exclusion chromatography and ultraviolet spectrophotometry and applied to a series of 20 sulfonic acid group-containing dyes. Radiochemical analyses were also used with 3 99mTc-labeled dyes to rationalize which functional groups are involved in the protein binding reaction. RESULTS: Methylene blue resulted in no protein affinity at 37 degrees C, whereas disulfonate dyes separated by 1 atom such as Patent blue or Indigo carmine gave <30% binding. Optimum protein binding (84%-100%) was achieved with those dyes containing at least 2 sulfonic acid groups separated by 2-6 atoms in their chemical structure. Seven symmetric tetrasulfonic acid azo dyes were examined, including Evans blue, to result in 59%-71% binding. CONCLUSION: Ionizable groups (sulfonic acids) that are present in the structure of dyes are directly involved in dye-protein binding. At the molecular level, there is a sulfonation reaction between sulfonic acid dyes and amino groups on the protein surface to form sulfonamide complexes. This reaction shows how the soluble dyes Evans blue and Patent blue are trapped in lymph after subdermal injection during the sentinel node biopsy procedure.

Animals↗

S100 protein serum levels in cutaneous malignant melanoma.

We investigated the utility of serum S100 determined by means of immunoradiometric assay in a cohort of 438 patients affected by cutaneous melanoma (126 untreated and 312 previously treated). Using 0.2 microg/l cut-off value, determined in 134 healthy blood donors, the sensitivity was 4.2% in stage I patients (4/94), 5.3% in stage II patients (1/19), and 38.5% in stage III patients (5/13). Even though the sensitivity increased progressively from stage I to stage II and III, these differences were not statistically significant. The prognostic significance of S100 evaluation at diagnosis was investigated in terms of survival but no statistical correlation between S100 basal levels and survival was found. In the 312 previously treated patients serum S100 levels were correlated to disease extent, high levels of the marker were observed in 42.8% (9/21) of patients with local recurrence, in 32% (16/50) of patients with lymph node and/or in-transit metastases, in 77.3% (17/22) of patients with distant metastases, and in patients with NED, the specificity of the marker was 96.8% (212/219). The difference between these groups were statistically significant. In conclusion, S100 protein was abnormally high in patients with metastatic malignant melanoma. Serial S100 measurements in a follow-up study are necessary to test the importance of the protein in the management of patients with metastatic malignant melanoma.

Adolescent↗

A positive role for PEA3 in HER2-mediated breast tumour progression.

Overexpression of HER2 is associated with an adverse prognosis in breast cancer. Despite this, the mechanism of its transcriptional regulation remains poorly understood. PEA3, a MAP kinase (MAPK)-activated member of the Ets transcription factor family has been implicated in the transcriptional regulation of HER2. The direction of its modulation remains controversial. We assessed relative levels of PEA3 expression and DNA binding in primary breast cultures derived from patient tumours (n=18) in the presence of an activated MAPK pathway using Western blotting and shift analysis. Expression of PEA3 in breast tumours from patients of known HER2 status (n=107) was examined by immunohistochemistry. In primary breast cancer cell cultures, growth factors induced interaction between PEA3 and its DNA response element. Upregulation of PEA3 expression in the presence of growth factors associated with HER2 positivity and axillary lymph node metastasis (P=0.034 and 0.049, respectively). PEA3 expression in breast cancer tissue associated with reduced disease-free survival (P<0.001), Grade III tumours (P<0.0001) and axillary lymph node metastasis (P=0.026). Co-expression of PEA3 and HER2 significantly associated with rate of recurrence compared to patients who expressed HER2 alone (P=0.0039). These data support a positive role for PEA3 in HER2-mediated oncogenesis in breast cancer.

Axilla↗

99Tc(m) labelled HL91 versus computed tomography and biopsy for the visualization of tumour recurrence of squamous head and neck carcinoma.

This phase I pilot study reports on (1) the safety and feasibility of 99Tc(m)-HL91, an amine oxime core radioligand that has shown oxygen dependent binding, and imaging; and (2) its usefulness for the visualization of local tumour recurrence of a biopsy proven squamous cell carcinoma of the head and neck (SCCHN) as compared to spiral computed tomogaphy (CT) and biopsy. Nine men (mean age 33 years, range 34-74 years) were prospectively included. For safety measurements, vital signs were recorded and serum chemical analysis carried out, with a complete blood cell count and urine analysis, and an ECG was performed prior to injection of 99Tc(m)-HL91 and repeated during the investigation. Single photon emission computed tomography (SPECT) scans of the head and neck, and of a standard, were performed at 2 h and 4 h post-injection of 740 MBq 99Tc(m)-HL91. Tumour-to-normal tissue background (T/N) ratios and percentage uptake were measured for all 99Tc(m)-HL91 scans. Spiral CT scans were obtained using a Somaton 4+ Siemens scanner within 1 week from the 99Tc(m)-HL91 scans. Based on CT and the 99Tc(m)-HL91 scan findings guided biopsies were performed. No adverse or subjective side effects were noticed. Vital signs, ECG findings, clinical laboratory, blood and urine assays remained stable in all patients. Spiral CT suggested local recurrence in 5/9 patients accompanied by nodal involvement in three, all of which proved positive on biopsy. 99Tc(m)-HL91 scintigraphy was false positive in one patient and true positive (TP) in 3/5 local recurrences and two out of three sites of lymph node involvement depicted by spiral CT. The mean T/N ratios at 2 h and 4 h in TPs were 1.28 (range 1.1-1.66) and 1.40 (range 1.0-1.6), respectively. The corresponding absolute percentages of 99Tc(m)-HL91 lesional uptake at 2 h and 4 h were mu = 0.05% (SD = 0.03%) and mu = 0.048% (SD = 0.035%). The findings suggest 99Tc(m)-HL91 is a safe radioligand and that metabolic binding in a large fraction but not all of local SCCHN recurrences may be expected. The inference that tumour 99Tc(m)-HL91 avidity could be a non-invasive measure of tumour hypoxia deserves however independent confirmation with needle oximetry.

Adult↗

Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone.

Using quantitative techniques we have shown elsewhere that dendritic cells (DC) migrate from blood into the spleen, under the control of T cells. Here we traced the localization of DC within the spleen and sought to explain the means by which they entered. DC were labeled with a fluorochrome, Hoescht 33342, and injected intravenously. Spleens were removed 3 or 24 h later and DC were visualized within particular areas that were defined by mAbs and FITC anti-Igs. At 3 h most DC were in the red pulp, whereas by 24 h the majority had homed to T-dependent areas of the white pulp and may have become interdigitating cells. Lymphoid DC, isolated from spleen and perhaps normally present in blood, may thus be a migratory stage distinct from the relatively fixed interdigitating cells. We also developed a frozen section assay to investigate the interaction of DC with various lymphoid elements. When DC were incubated on sections of spleen, at 37 degrees C but not at 4 degrees C they attached specifically within the marginal zone and did not bind to T areas; in contrast, macrophages attached only to red pulp and T cells did not bind specifically. However, DC did not bind to sections of mesenteric lymph node, whereas T cells localized in particular regions at 4 degrees C but not at 37 degrees C, probably the high endothelial venules. DC may thus express "homing receptors," similar to those of T cells, for certain endothelia. We propose that T cells can modify the vascular endothelium in certain areas to allow egress of DC from the bloodstream.

Animals↗

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer.

The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.

Adenoviruses, Human↗

CpG-oligodinucleotides as an effective adjuvant in pigs for intramuscular immunizations.

In this study, the effect of two oligodeoxynucleotide (ODN) sequences 5'GCT-AGA-CGT-TAG-CGT-3' (CpG-ODN) and 5'-GCT-AGA-GCT-TAG-GCT-3' (GpC-ODN) on the antigen-specific antibody and cellular immune response after intramuscular immunizations with OVA was analyzed in pigs. Pigs immunized with OVA supplemented with these ODNs showed a significantly enhanced primary antibody response in comparison with the control group which received OVA without ODN. This enhanced primary antibody response appeared ODN-sequence-independent as similar effects were seen in both ODN-groups. The OVA-specific antibody titers obtained after a single injection of antigen combined with either of both ODNs were as high as the titers in the control group after two injections. Furthermore, the ODN-supplemented animals showed significantly higher OVA-specific IgA antibodies in their saliva and nasal secretions at some time points after the first immunization. Proliferation assays showed that CpG- as well as GpC-ODN significantly enhanced the antigen-specific as well as the mitogen-induced proliferation in different lymphoid tissues. Furthermore, 48h after the third immunization the CpG-group showed a significantly decreased IL-6 mRNA expression in cells of the local draining lymph node but no significant difference in TGF-beta (Th3-like) and IL-10 (Th2-like). The ODN injected animals showed the tendency to have higher IFN-gamma (Th1-like) mRNA-expression in comparison with the control group. To our knowledge, these are the first in vivo studies in pigs, which demonstrate the appropriateness of CpG-ODN as immunostimulating adjuvants in vaccines for farm animals.

Adjuvants, Immunologic↗

[Gnathostomiasis after a stay in Thailand].

During a 4-week holiday in Thailand a 13-year-old boy from Germany briefly fell ill with diarrhoea without blood-tinged stools and fever. 5 months later oedematous, reddish swellings occurred, at first on the right upper arm, later over the right side of the chest. Especially at night these swellings were associated with severe local and generalized itching. Bilaterally the axillary lymph-nodes were hard and slightly enlarged. There was a marked eosinophilia of 56%, the white count was 22,700/microliters and the serum level of IgE was raised to 1777 IU/ml. No worm eggs or larvae were found in the stool. Enzyme-linked immunosorbent assay, using larvae of Gnathostoma spinigerum as antigen, demonstrated antibodies against this nematode. Gnathostomiasis having been diagnosed treatment was started with albendazole (200 mg twice daily for 21 days), after which the boy was symptom free. After 2 months the eosinophilia had regressed to 10%, the white cell count was normal and the antibody titre had fallen.

Adolescent↗