A micromorphometry-based concept for routine classification of sentinel lymph node metastases and its clinical relevance for patients with melanoma.

BACKGROUND: The sentinel lymph nodes (SLNs) as the primary targets for lymphatic metastases can be removed selectively by gamma probe-guided sentinel lymph nodectomy (SLNE) in nearly all patients with cutaneous melanoma. Correspondingly high standards in terms of specificity, sensitivity, and microstaging are required for the evaluation of SLNs. METHODS: Since 1995, the authors have performed SLNE in 389 lymph node regions (LNRs) on 342 patients with melanoma. The harvested 636 SLNs and a further 1394 nonsentinel lymph nodes (non-SLNs) were evaluated by standardized, semiserial section histology, including immunohistochemistry. For each LNR, this technique permitted routine S classification using two simple morphometric parameters: the number of tumor-involved, 1-mm slices of the SLNs (n) and the centripetal depth of metastatic cell invasion (d). S1 was defined by 1 < or = n < or = 2 and d < or = 1 mm, equivalent to localized peripheral tumor cell deposits; S2 was defined by n > 2 and d < or = 1 mm, indicating more extended peripheral metastases; S3 was defined by d > 1 mm in SNLs with deeper metastatic infiltration; and S0 meant no detectable tumor cells (n = 0). RESULTS: The authors diagnosed 325 SLNs as S0, 24 SLNs as S1, 22 SLNs as S2, and 18 SLNs as S3. The occurrence of at least one melanoma-positive non-SLN subsequent regional completion lymph node dissection (RCLND) significantly increased from 0 of 12 in S1 SLNs to 2 of 13 in S2 SLNs and 9 of 15 in S3 SLNs (P = 0.001; chi-square test). Like the T classification of the primary melanoma, the S classification proved to be a highly significant predictor for distant metastasis (P < 0.001). It turned out to be an independent factor of influence on distant metastasis and survival in multivariate Cox analyses, which included tumor thickness, primary tumor site, patient gender, and patient age as covariates. CONCLUSIONS: The data presented recommend the S-staging concept as a promising option to fill a gap between the T and conventional N component of the pTNM classification. If its predictive capacity can be confirmed in multicenter studies, then the S classification may become the decisive criterion for or against RCLND, and a combined T plus S staging system will help to improve prognostically relevant stratification of melanoma patients in adjuvant therapy trials.

Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo.

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.

Multivariate prognostic analysis of stage I(E) primary non-Hodgkin's lymphomas of the nasal cavity.

From January 1968 to December 1997, a total of 71 patients with stage I(E) (Ann Arbor staging system, 1971) primary non-Hodgkin's lymphomas of the nasal cavity received treatment in the Cancer Center of Sun Yat-Sen University of Medical Sciences. Thirty-seven lesions were limited to the nasal cavity (limited I(E)), whereas the other 34 were extended to the structure out of the nasal cavity (extended I(E)). Forty-four patients were treated with radiochemotherapy and 27 with radiotherapy alone. Kaplan-Meier methods were used in the survival analysis. Multivariate analysis was carried out using the Cox proportional hazard model. The 5- and 10-year survival rates were 71.85% and 59.67% for the patients with a complete response to irradiation, and both were 13.89% for the patients with residue lesions (p = 0.0004). The 5- and 10-year survival rates were 69.81% and 56.72% for limited I(E), and 40.65% and 35.57% for extended I(E) (p = 0.0047). The prognosis was better for those younger than 44 years (p = 0.0003). The 10-year survival rates for radiotherapy alone and combined radiochemotherapy are 52% and 75% for limited I(E) versus 37.58% and 45% for extended I(E) (p = 0.0644). B symptoms did not significantly affect clinical outcome (p = 0.729). Multivariate analysis showed that complete response of local lesion after radiotherapy, invasion of the primary tumor to adjacent structures, and patients' age were independent prognostic factors. Our study showed that radiotherapy is the main treatment method for the primary non-Hodgkin's lymphomas of the nasal cavity; the addition of chemotherapy may improve long-term survival. The local tumor response to radiotherapy, whether the extranasal structures were invaded, and patients' age were independent prognostic factors.

The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.

The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.

c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization.

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.