Axillary dissection in the context of the biology of lymph node metastases.

BACKGROUND: Modern breast surgery, as the primary treatment of invasive breast carcinoma, has been evolving over the last century. Aggressive radical surgery, which included chest wall resection, complete axillary clearance and internal mammary node dissection, has slowly changed to a less aggressive approach. This has been based on an improved understanding of the biology of the disease. Over the years, randomized prospective trials, performed at centers all over the world, have demonstrated that axillary dissection does not impact on the overall survival while it helps with loco-regional control of breast cancer. Its major role, at the present time, is limited to staging and prognostication; functions that are equally well served by the limited approach of a sentinel node biopsy. SOURCES: This review is based on the available medical literature involving the biology and organ specificity of the metastatic process, not only in breast cancer but also in other malignancies. In addition, studies pertaining to clinical breast cancer, and the role of surgery in its treatment, were reviewed. The ongoing trials on the role of sentinel node biopsy in the management of the clinically node negative patients are discussed. CONCLUSIONS: This review covers the history, pathophysiology, and clinical basis of the current role of axillary dissection for invasive breast cancer. From the data presented we hope that the medical community will agree that there is no therapeutic role for extended axillary dissection at the current time.

Biologic and clinical significance of lymphadenectomy.

Interest in the lymphatic system and its relationship to metastases has developed owing to renewed interest in sentinel node biopsy. This article summarizes the anatomy, physiology, and biology of the lymphatic system and lymph node metastases, and reviews studies of lymph node metastases and surgical resection of cancers in different anatomic sites. On the basis of these studies, the authors conclude that lymph node metastasis functions as an indicator of prognosis, not the controlling or determining factor of prognosis. Thus, varying degrees of treatment of regional lymph nodes and metastases do not seem to be controlling factors in the outcome of cancer.

Complement activation and subclassification of tissue immunoglobulin G in the abdominal aortic aneurysm.

Features of autoimmunity in abdominal aortic aneurysm (AAA) have been described, including increases in IgG content. The present experiments were carried out to determine (1) whether the increases in IgG are subclass specific and (2) whether the IgG complex is associated with an increase in the isoforms of complement C3. Seven AAA, four athero-occlusive (AOD), and two normal (NL) aortic tissue extracts were evaluated for immunoreactive complement (C3) components, both by ELISA and by Western immunoblots (probed with a polyclonal goat anti-human C3). The extracts were also assayed for each of the four subclasses of IgG by ELISA (monoclonal mouse anti-human IgGs). Compared to the amounts of IgG by subclass in normal aorta, AAAs had increases of 193-fold in IgG1, 160-fold in IgG2, 389-fold in IgG3, and 627-fold in IgG4. Increases relative to AOD by subclass were smaller, but each subclass was statistically significantly elevated (P < 0.01) over NL or over AOD. There was a 125-fold increase in immunoreactive C3 by ELISA in AAA vs NL, and Western immunoblotting techniques revealed the presence of multiple C3 degradation products. Increases in IgG1, 2, and 3 may be responsible for activation of complement in AAA by the classical pathway. Since the complement system is one of the major effector pathways of inflammation, the presence of complement-fixing IgG subclasses along with increased C3 in the aneurysm wall may be an important mechanism promoting matrix proteolysis in AAA.

Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification.

Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp). The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986. This change did not affect drug sensitivity. The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype. The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere. Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome. Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp. Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy.

Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells. Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity. Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.

Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells.

The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins.

Intracellular distribution and pharmacokinetics of daunorubicin in anthracycline-sensitive and -resistant HL-60 cells.

Anthracycline-sensitive (HL-60) and -resistant (HL-60/AR) cells, which do not overexpress the P-glycoprotein, each transport and distribute daunorubicin (DNR) into distinct intracellular locations, as visualized by digitized video fluorescence microscopy. At pH 7.4, the fluorescence of DNR in HL-60 cells appears distributed diffusely in both the nucleus and cytoplasm. In contrast, HL-60/AR cells show much less fluorescence in the nucleus and cytoplasm; most of the fluorescence localizes first to the Golgi apparatus and is then gradually shifted to the lysosomes and/or mitochondria. In pharmacokinetic studies, HL-60/AR cells exposed to different extracellular concentrations of [14C]DNR consistently accumulated less radioactive drug than the parent HL-60 cells. Incubation of HL-60/AR cells with sodium azide and deoxyglucose blocked the efflux of [14C]DNR and also prevented the shift of DNR fluorescence from the Golgi apparatus to the lysosomes/mitochondria. The efflux and the intracellular shift of DNR could also be inhibited by lowering the temperature to 18 degrees C, which stops endosomal membrane fusion. When DNR was allowed to accumulate in HL-60 or HL-60/AR cells at pH 5 there was an increase in the proportion of drug fluorescence in the membranes of both HL-60 and HL-60/AR cells; a decrease in the amount of drug retained by HL-60, but not by HL-60/AR cells; and a decrease in the cytostatic effects of DNR on both HL-60 and HL-60/AR cells. These data suggest that DNR resistance is associated with a failure of DNR to pass through membranes and to bind to cytoplasmic and nuclear structures. Instead, most of the drug is taken up by the Golgi apparatus from which it is then shifted to the lysosomes or to mitochondria, or out of the cell.

Role of glutathione and dependent enzymes in anthracycline-resistant HL60/AR cells.

We studied the cellular enzymatic defenses against anthracycline-induced free radical damage in the HL60 human myelogenous leukemia cell line and in its anthracycline-resistant subline, HL60/AR. Intracellular glutathione (GSH) levels and gamma-glutamyl transpeptidase activity were lower in HL60/AR than in HL60 cells. Glutathione-S-transferase (GST) and glutathione peroxidase activities were similar in both cell lines. The intracellular distribution of GSH/GST was visualized by digitized video fluorescence microscopy, utilizing the fluorescent probe monochlorobimane fluorescence microscopy, utilizing the fluorescent probe monochlorobimane (MBCl), which is specifically conjugated to GSH by GST. In HL60 cells stained with the MBCl probe, a bright diffuse cytoplasmic and nuclear fluorescence pattern was observed, whereas in HL60/AR cells, the fluorescence was mostly localized to the Golgi apparatus with a lesser component of diffuse cytoplasmic and nuclear fluorescence. Pretreatment of HL60/AR cells with buthionine sulfoximine (BSO) partially reversed resistance to daunorubicin. This effect of BSO on resistance was associated not only with the abolition of localized MBCl fluorescence to the Golgi apparatus but also with increased intracellular accumulation and retention of daunorubicin. The results of our studies demonstrate that inhibition of GSH synthesis in HL60/AR cells results in significant sensitization to daunorubicin and suggest that changes in the intracellular distribution of GSH/GST and/or increased drug retention may be involved in mediating this effect.

Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan.

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.