Quantitative lymph node burden as a 'very-high-risk' factor identifying head and neck cancer patients benefiting from postoperative chemoradiation.

Background: Adjuvant chemoradiation (CRT) is standard for head and neck squamous cell carcinoma (HNSCC) patients with positive margins or extranodal extension (ENE) following surgery. However, emerging evidence suggests the number of positive lymph nodes (LNs) is the dominant determinant of survival in non-oropharyngeal HNSCC and thus may better identify those benefiting from treatment intensification. Patients and methods: Patients from the National Cancer Database diagnosed with non-oropharyngeal HNSCC (oral cavity, larynx, hypopharynx) between 2004 and 2014 and undergoing surgical resection, neck dissection, and postoperative radiotherapy (RT) were included. Multivariable regression with first-order interaction terms was used to model the interaction between postoperative CRT and continuous number of positive LNs with respect to overall survival. Results: In total, 7144 patients met inclusion criteria. In multivariable analysis, increasing number of positive LNs was associated with both increasing mortality (P < 0.001) and increasing benefit from postoperative CRT versus RT alone (interaction P < 0.001). While there was no benefit from postoperative CRT in patients with 0-2 LN+ [hazard ratio (HR) 0.96, 95% confidence interval (CI) 0.86-1.07, P = 0.47], increased benefit was seen in those with 3-5 LN+ (HR 0.84, 95% CI 0.70-1.00, P = 0.05) and those with ≥6 LN+ (HR 0.65, 95% CI 0.51-0.82, P < 0.001) in multivariable models. By contrast, margin status and ENE did not reliably identify patients benefitting from postoperative CRT based on statistical tests of interaction. Even in patients with ENE, positive margins, or both, only those with ≥6 LN+ had improved survival with postoperative CRT. Conclusion: Increasing metastatic nodal burden was associated with increased benefit from CRT compared with RT alone, surpassing conventional high-risk factors in identifying patients benefiting from CRT. Stratification by metastatic LN number may characterize a very-high-risk patient cohort best suited for treatment intensification.

Possible kinetic mechanism of human placental alkaline phosphatase in vivo as implemented in reverse micelles.

Human placental alkaline phosphatase is an integral membrane protein. It catalyzes the hydrolysis of phosphoester linkage of a broad-range substrate. We have embedded the enzyme in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. Linear Lineweaver-Burk and Eadie-Hofstee plots for the substrate and linear Arrhenius plot for the temperature-dependent enzyme reaction were obtained in reverse micelles suggesting that the substrate diffusion limitation was not a rate-limiting step in the system and exchange of materials between reverse micelles was very rapid. The catalytic constant (kcat) of the enzyme was decreased, and the Km for the substrate was increased in reverse micelles, both in an exponential way with the [H2O]/ AOT] ratio (omega O). The enzyme was more stable in reverse micelles than in aqueous solution at 30 degrees C but was unstable at higher temperature (65 degrees C). The activation energy of the enzyme in reverse micelles was 46.5 +/- 2.6 kJ/mol, which was about 20 kJ/mol higher than that in aqueous solution and reflected in the lower Kcat value at low omega O. The binding affinity between the substrate 4-nitrophenyl phosphate and the enzyme in reverse micelles was decreased as implemented by the higher Km and higher Ki for phosphate values. In aqueous solution, the log kcat/pH plot suggested that amino acid residues with pKa values of 9.03 +/- 0.03 and 11.37 +/- 0.10 are involved in catalysis. The former should be deprotonated and the latter should be protonated for the reaction to proceed. In reverse micelles, both the above-mentioned pKa values were detected. However, both groups have to deprotonated to give the optimum catalytic function. In aqueous solution, the enzyme's specificity was highly dependent on pH and buffer. The pKa value of the amino acid residues involved in substrate binding was found to be 8.69 +/- 0.07 in carbonate buffer, but changed to 9.80 +/- 0.06 in Tris buffer. Our results suggested that the rate-limiting step of the enzyme-catalyzed reaction may be changed from phosphate releasing in aqueous solution to another critical step in the reverse micelles.