Decision making in the management of recurrent head and neck cancer

Despite substantial improvements in head and neck squamous cell carcinoma (HNSCC) treatment, the major obstacle to long‐term survival remains disease recurrence. Salvage options are often limited due to prior therapy and the escalated morbidity of retreatment. The costs of treatment must be measured against the anticipated quality and quantity of life recovered, even with resectable disease. This review surveys the recurrent HNSCC literature to better guide decision making. Across multiple studies, negative prognostic factors include impaired performance status, advanced recurrent stage, brief disease‐free interval, previous chemotherapy, and nonlaryngeal sites of recurrence. When possible, surgical salvage remains the principal option for durable disease control, quality of life preservation, and cure. Nonsurgical therapies have also demonstrated measurable improvements in locoregional control. Interpretation of salvage literature must be tempered by recognition of significant selection bias. The decision for salvage therapy must be individualized, with management that involves well‐informed patients resulting in the best outcomes. © 2013 Wiley Periodicals, Inc. Head Neck 36 : 144–151, 2014     

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H₂S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H₂S biogenesis.Cystathionine β-synthase (CBS; EC 4.2.1.22) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of the hydroxyl group of l-serine (Ser) by l-homocysteine (Hcy), yielding cystathionine (Cth) (1). A deficient activity of human CBS (hCBS) is the cause of classical homocystinuria [CBS-deficient homocystinuria (CBSDH); Online Mendelian Inheritance in Man (OMIM) no. 236200], an autosomal, recessive inborn error of sulfur amino acid metabolism, characterized by increased levels of Hcy in plasma and urine. CBSDH manifests as a combination of connective tissue defects, skeletal deformities, vascular thrombosis, and mental retardation (2).The hCBS is a homotetrameric enzyme whose subunits are organized into three structural domains. The N-terminal region binds heme and is thought to function in redox sensing and/or enzyme folding (3, 4). The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5, 6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7–9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10, 11). The CBS motif pair, commonly known as a “Bateman module” (12, 13), is responsible for CBS subunit tetramerization (14, 15). The presence of pathogenic missense mutations in this region often does not impair enzyme activity but typically interferes with binding of AdoMet and/or the enzyme’s activation by AdoMet (15–17). Removal of the regulatory region leads to a dimer with much increased activity (14, 15). Recently, we showed that removal of residues 516–525, forming a flexible loop of the CBS2 motif of hCBS, yields dimeric species (hCBSΔ516–525) with intact AdoMet binding capacity and activity responsiveness to AdoMet similar to a native hCBS WT (18).hCBS is regulated by a complex molecular mechanism that remains poorly understood. More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a “basal” state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound “activated” state, where the AdoMet-induced conformational change would allow for enzyme activation (16, 19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20, 21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18). In the basal state, the Bateman modules from each hCBS unit are far apart and do not interact with each other, being placed just above the entrance of the catalytic site of the complementary subunit, thus hampering the access of substrates into this cavity. Our hCBSΔ516–525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23, 24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16, 19, 25–27).To solve the molecular mechanism of hCBS regulation by AdoMet, we have analyzed the crystals of an engineered hCBSΔ516–525 protein that bears the mutation E201S, which potentially weakens and/or disrupts the interaction between the Bateman module and the catalytic core (Fig. 1A), thus favoring the activation of the enzyme. The data presented here fill a long-sought structural gap by unraveling the crystal structure of AdoMet-bound hCBS, thus providing the overall fold of the enzyme in its activated conformation and the identity of the AdoMet binding sites. Comparison with the structures of hCBS in basal conformation and constitutively activated dCBS was instrumental in the understanding of the regulatory role played by the C-terminal domain as well as the effect of some of the pathogenic mutations in the activation and/or inhibition of this key molecule of transsulfuration.Open in a separate windowFig. 1.Interactions between protein domains in basal hCBS. (A) In hCBSΔ516–525, residues Y484, N463, and S466 anchor the Bateman module (blue) to the protein core (gray) through H-bonds with the residues E201 and D198 from the loop L191–202, thus occluding the entrance to the catalytic pocket. (B) The CBS-specific activity of selected hCBS variants in the absence (blue bars) and the presence (red bars) of 300 µM AdoMet. hCBS enzyme species marked with “Δ” lack residues 516–525 and form dimers.     

Patient-important activity and participation outcomes in clinical trials involving children with chronic conditions

Purpose

Children with chronic conditions experience medical issues over long-term periods of time which can have lasting emotional and social consequences impacting daily life and functioning. Activities and participation outcomes are needed in order to comprehensively assess child-important health in clinical trials. Our objective was to review the extent to which activity and participation outcomes are included in clinical trials of childhood chronic disease and to determine what trial characteristics are associated with their use.

Methods

A review of a large clinical trial registration database (clinicaltrials.gov) was conducted over the 2010 calendar year. The measures used to assess primary and secondary endpoints were coded according to the ICF classification system. Trial characteristics that might be associated with activity and participation outcome use such as sponsorship type, intervention type, health condition, whether the trial was focused on pediatric patients, phase of trial and sample size were also extracted and explored with univariable and multivariable regressions.

Results

Four hundred and ninety-nine trials met inclusion criteria, 495 of which had complete information about hypothesized predictors. Only 36 out of 495 trials included an activity and participation outcome as part of the trial evaluation process. Both univariable and multivariable regression models showed that non-drug trials and late phase of trial (phase IV) showed the strongest likelihood with whether a trial would include an activity and participation outcome.

Discussion

Most registered clinical trials for children with chronic or ongoing medical conditions do not include a comprehensive approach to health outcomes assessment, especially drug trials and early phase trials. Outcome measures in pediatric clinical trials are lagging relative to World Health Organization standards for comprehensive health evaluation.     

Disturbed B and T cell homeostasis and neogenesis in patients with ataxia telangiectasia

Objective

Ataxia telangiectasia (AT) is a rare genetic, multi-system disorder characterized by neurodegeneration, chromosome instability, B and T cell immunodeficiency and a predisposition to cancer. We examined immunologic parameters reflecting cell development and proliferation and their relevancy to the clinical phenotype in affected individuals.

Patients and Methods

AT patients from the AT National Clinic in Israel underwent immunological investigation. Their T and B cell workup included lymphocyte subset counts, immunoglobulin levels, responses to mitogenic stimulations, TCR-Vβ families and BCR immunoglobulin heavy chain spectratyping, TCR rearrangement excision circles (TRECs) and Kappa-deleting recombination excision circles (KRECs).

Results

Thirty-seven AT patients (median age 12.7 years, range 4.2–25.1) were evaluated. CD20 B and CD3 T lymphocytes were decreased in 67 % and 64 % of the patients, respectively, while only 33 % of the patients had reduced lymphoproliferative responses. Almost all AT patients displayed extremely low TRECs and KRECs levels, irrespective of their age. Those levels were correlated to one another and to the amounts of CD3+ and CD20+ cells, respectively. Abnormal TCR-Vβ repertoires were found with different degrees of clonality or reduced expression in these AT patients. There was no clear clustering of expansions to specific TCR-Vβ genes. PCR spectratyping analysis of the FR2 IgH BCR gene rearrangements in peripheral blood was abnormal in 50 % of the patients.

Conclusion

The immunodeficiency associated with AT is combined, remains low over time and not progressive. It is characterized by low TREC and KREC copies suggestive of abnormal T and B cell neogenesis.     

Impact of COMT genotype on serotonin-1A receptor binding investigated with PET

Alterations of the inhibitory serotonin-1A receptor (5-HT_1A) constitute a solid finding in neuropsychiatric research, particularly in the field of mood and anxiety disorders. Manifold factors influencing the density of this receptor have been identified, e.g., steroid hormones, sunlight exposure and genetic variants of serotonin-related genes. Given the close interactions between serotonergic and dopaminergic neurotransmission, we investigated whether a common single-nucleotide-polymorphism of the catechol-O-methyltransferase (COMT) gene (VAL158MET or rs4680) coding for a key enzyme of the dopamine network that is associated with the pathogenesis of mood disorders and antidepressant treatment response, directly affects 5-HT_1A receptor binding potential. Fifty-two healthy individuals (38 female, mean age ± standard deviation = 40.48 ± 14.87) were measured via positron emission tomography using the radioligand [carbonyl-¹¹C]WAY-100635. Genotyping for rs4680 was performed using DNA isolated from whole blood with the MassARRAY platform of the software SEQUENOM^®. Whole brain voxel-wise ANOVA resulted in a main effect of genotype on 5-HT_1A binding. Compared to A carriers (AA + AG) of rs4680, homozygote G subjects showed higher 5-HT_1A binding potential in the posterior cingulate cortex (F _(2,49) = 17.7, p = 0.05, FWE corrected), the orbitofrontal cortex, the anterior cingulate cortex, the insula, the amygdala and the hippocampus (voxel-level: p < 0.01 uncorrected, t > 2.4; cluster-level: p < 0.05 FWE corrected). In light of the frequently reported alterations of 5-HT_1A binding in anxiety and mood disorders, this study proposes a potential implication of the COMT genotype, more specifically the VAL158MET polymorphism, via modulation of the serotonergic neurotransmission.     

Loss of heterozygosity at locus F13B on chromosome 1q in human medulloblastoma

Medulloblastoma is a primitive neuroectodermal tumor of the cerebellum with poorly understood pathogenesis. Previous studies have reported loss of heterozygosity (LOH) on chromosome arms 17p, 11p and 9q and cytogenetic abnormalities of chromosome 1 in medulloblastoma. We have used the polymerase chain reaction to amplify 10 microsatellites on the short arm and B microsatellites on the long arm of chromosome 1 to assess allelic loss in 22 medulloblastomas. Loss of heterozygosity (LOH) on chromosome 1 was found in 9 cases. Eight medulloblastomas (36%) showed an interstitial LOH on chromosome 1q. The common region of overlap was mapped between DISI604 and DIS237 and included the locus F13B in the chromosomal region 1q31–q32.1. An additional tumor had LOH in a proximal region of 1p, but did not exhibit LOH on 1q. None of the medulloblastomas exhibited LOH of the telomeric portion of chromosome 1p, which has been associated with several other human malignancies. Our data suggest the presence of a putative tumor suppressor gene located near the locus F13B on chromosome arm 1q that appears to be involved in the pathogenesis of medulloblastoma. © 1996 Wiley-Liss, Inc.     

Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene

Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively. The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts. APC exon 15-positive “classic” p300^apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IEI, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope. In contrast with this immunoreactivity, 2 novel high m.w. products of approx. 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL. A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160^apc molecules. The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively. This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15. We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains. © 1996 Wiley-Liss, Inc.