Changes in the use of end points in clinical trials for elderly cancer patients over time.

BACKGROUND: Physicians need well-addressed clinical trials assessing benefits and harm of treatments to avoid under-treatment or over-treatment of elderly patients. The main objectives of this report were to present an overview of end points used in clinical trials dedicated to elderly patients; and to assess the evolution in chosen end points before and after the creation of the International Society of Geriatric Oncology in the early 2000s. PATIENTS AND METHODS: All phases I, II and III trials dedicated to the treatment of cancer among elderly patients published between 2001 and 2004 and between 2011 and 2014 were reviewed. All phase III clinical trials assessing cancer treatments among adults in the same periods were also reviewed to identify subgroup analyses of elderly patients among these trials. RESULTS: Among phase III trials dedicated to elderly patients, overall survival was a common primary end point. Interestingly, tumor centered end points were very common in the first time period and very uncommon in the second time period, whereas composite end points were very uncommon in the first time period but very common in the second time period. Concerningly, disease-specific survival was very infrequently reported in dedicated clinical trials of elderly patients despite their importance in evaluating competing risk of death from non-oncology causes. The use of patient-reported outcomes (PROs) as a primary end point remained very uncommon but the reporting of PROs as a secondary end point tended to increase in the second time period, from 19% to 33% (P = 0.10). Functional status was infrequently reported. CONCLUSION: During the past decade, the use of clinically meaningful end points such as PROs and functional status in elderly patients remained moderate. Yet, the use of PROs as a secondary end point tended to increase between the two time periods.

Inclusion of elderly patients in oncology clinical trials.

BACKGROUND: Physicians need clinical trials assessing benefits and harms of treatments to avoid under-treatment or over-treatment of elderly patients. The main objectives of this report were to examine how data regarding elderly oncology patients were presented in medical literature; and to assess the evolution of this presentation between two time periods. PATIENTS AND METHODS: All phases I, II and III trials dedicated to the treatment of cancer among elderly patients published between 2001 and 2004 and between 2011 and 2014 were reviewed. All phase III clinical trials assessing cancer treatments among adults in the same periods were also reviewed to evaluate potential subgroup analyses in elderly patients in these studies. Key characteristics of interest were extracted by two investigators before descriptive and comparative analyses were undertaken. RESULTS: A total of 1084 trials were included: 366 and 718 from the first and second time period, respectively. Twenty-seven and 193 of these trials were phase I and II trials dedicated to elderly or frail patients, respectively. A large proportion of phase III trials published between 2011 and 2014 reported at least one analysis dedicated to elderly patients (46.7%) versus 19.3% during the first time period. The use of subgroup analyses of elderly patients in phase III trials was the most frequent source of information. Subgroup analyses were more frequent among trials with industrial funding, trials published in high impact factor journal, intercontinental trials and trials with large sample size. The age threshold defining the elderly subgroup increased over time. CONCLUSION: Elderly patients have become a topic of interest during the past decade. However, data available are mostly extracted from subgroup analyses, which can only be regarded as preliminary evidence.

Disseminated arterial calcification and enhanced myogenic response are associated with abcc6 deficiency in a mouse model of pseudoxanthoma elasticum.

OBJECTIVE: Pseudoxanthoma elasticum is an inherited metabolic disorder resulting from ABCC6 gene mutations. It is characterized by progressive calcification and fragmentation of elastic fibers in the skin, retina, and the arterial wall. Despite calcium accumulation in the arteries of patients with pseudoxanthoma elasticum, functional consequences remain unknown. In the present study, we investigated arterial structure and function in Abcc6(-/-) mice, a model of the human disease. APPROACH AND RESULTS: Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry. Expression of genes involved in osteochondrogenic differentiation was measured by polymerase chain reaction. Elastic arterial properties were evaluated by carotid echotracking. Vascular reactivity was evaluated using wire and pressure myography and remodeling using histomorphometry. Arterial calcium accumulation was 1.5- to 2-fold higher in Abcc6(-/-) than in wild-type mice. Calcium accumulated locally leading to punctuate pattern. Old Abcc6(-/-) arteries expressed markers of both osteogenic (Runx2, osteopontin) and chondrogenic lineage (Sox9, type II collagen). Abcc6(-/-) arteries displayed slight increase in arterial stiffness and vasoconstrictor tone in vitro tended to be higher in response to phenylephrine and thromboxane A2. Pressure-induced (myogenic) tone was significantly higher in Abcc6(-/-) arteries than in wild type. Arterial blood pressure was not significantly changed in Abcc6(-/-), despite higher variability. CONCLUSIONS: Scattered arterial calcium depositions are probably a result of osteochondrogenic transdifferentiation of vascular cells. Lower elasticity and increased myogenic tone without major changes in agonist-dependent contraction evidenced in aged Abcc6(-/-) mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis in the long term.

High-frequency (20-50 MHz) ultrasonography of pseudoxanthoma elasticum skin lesions.

BACKGROUND: In most patients pseudoxanthoma elasticum (PXE) manifests with yellowish cutaneous papules and dermal elastorrhexis on skin biopsy. In a small number of cases there are no skin manifestations on clinical examination, and establishing a diagnosis of PXE in such patients is challenging. High-frequency ultrasonography (HFUS) may be of use in predicting skin areas that would yield a biopsy specimen positive for elastorrhexis. OBJECTIVES: To describe characteristics of clinically visible PXE skin using HFUS, and to evaluate its relevance for diagnosis. METHODS: HFUS was performed in a cohort of patients with PXE and in controls at a referral centre. HFUS images of PXE skin were compared with those of other conditions. Five operators blind-scored multiple HFUS images of photoprotected or photoexposed skin from patients with PXE and controls. The diagnostic indices (sensitivity, specificity, likelihood ratios, interobserver agreement) were calculated. RESULTS: The HFUS changes considered as diagnostic for PXE were primarily oval homogeneous hypoechogenic areas in the mid-dermis. The size of these areas closely matched the extent of the histological changes. The sensitivity and specificity of the diagnostic items and interobserver agreement were high, particularly in photoprotected skin. Dermal hypoechogenicity in PXE could be related to high hydration of connective tissue due to the presence of glycosaminoglycans despite elastic fibre mineralization. CONCLUSIONS: HFUS provides suggestive images of PXE skin lesions. HFUS should now be studied to determine whether it is a potentially valuable technique for the noninvasive identification of elastorrhexis in patients with PXE in whom skin involvement is clinically minimal or absent.

The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties.

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.

[Increased haemoglobin A2 levels in pseudoxanthoma elasticum]. / Augmentation de l'hémoglobine A2 au cours du pseudoxanthome élastique.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is normally associated with mutations in the ABCC6 gene. A PXE phenotype without mutations in ABCC6 has been described in Greek and Italian patients presenting with beta thalassemia. We attempted to determine the incidence of beta thalassemia in a cohort of French patients with PXE. PATIENTS AND METHODS: Fifty patients with PXE were included in the study. Laboratory examinations comprised hemoglobin electrophoresis, ABCC6 gene study and in some studies: mutation analysis, beta-globin gene. RESULTS: No cases of beta thalassemia were diagnosed in this cohort of French patients with PXE. However, 20% of the latter exhibited a significant but isolated (i.e. without microcytic anemia) increase of hemoglobin A2 (HbA2). Statistical comparisons showed no difference in terms of geographical origin or severity of PXE between patients with high levels of HbA2 and those with normal levels of HbA2 other than the extent of cutaneous involvement. Study of the beta-globin gene displayed mutations only in the two patients with the highest recorded levels of HbA2. ABCC6 + beta-globin digenism was ruled out of the pathogenesis of PXE. DISCUSSION: The PXE phenotype seen in some patients with beta thalassemia appears to be associated with epigenetic modification of ABCC6 transcription and depends specifically on the beta globin locus. Isolated increase in HbA2 is probably a laboratory marker for PXE. Here again, a functional epigenetic reaction between ABCC6 and the beta-globin locus was suspected. However, these reciprocal interactions are clearly unequal since the change in ABCC6 transcription occurring during the course of beta thalassaemia is responsible for a PXE phenotype while increased HbA2 during the course of PXE has no clinical consequences.

The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family.

ATP-binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes. Of the fifty-six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail. Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug-resistance associated proteins MRP1, MRP2, MRP3 and MRP6. To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP. In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults. Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP-dependent, vanadate-sensitive organic anion transporter.

A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum.

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

The mouse lysyl oxidase-like 2 gene (mLOXL2) maps to chromosome 14 and is highly expressed in skin, lung and thymus.

The predicted amino acid sequence derived from a mouse expressed sequence tag (EST) contig contained two domains that are highly conserved among members of the lysyl oxidase gene family: a copper binding-site with four histidines and a catalytic domain that includes a tryptophan residue. This new cDNA sequence showed the highest level of sequence homology with the human loxl2 cDNA and suggested that it encoded the mouse equivalent of hLOXL2. The mLOXL2 gene was mapped to chromosome 14 by radiation hybrid analysis. The mLOXL2 locus was tightly linked with a LOD score over 9 to the marker D14Mit32. The mLOXL2 gene is expressed as a 4-kb mRNA in almost all tissues analyzed, with highest levels of mRNA in skin, lung and thymus.

Pseudoxanthoma elasticum maps to an 820-kb region of the p13.1 region of chromosome 16.

We have performed linkage analysis on 21 families with pseudoxanthoma elasticum (PXE) using 10 polymorphic markers located on chromosome 16p13.1. The gene responsible for the PXE phenotype was localized to an 8-cM region of 16p13.1 between markers D16S500 and D16S3041 with a maximum lod score of 8.1 at a recombination fraction of 0.04 for marker D16S3017. The lack of any locus heterogeneity suggests that the major predisposing allele for the PXE phenotype is located in this region. Haplotype studies of a total of 36 PXE families identified several recombinations that further confined the PXE gene to a region (< 1 cM) between markers D16S3060 and D16S79. This PXE locus was identified within a single YAC clone and several overlapping BAC recombinants. From sequence analysis of these BAC recombinants, it is clear that the distance between markers D16S3060 and D16S79 is about 820 kb and contains a total of nine genes including three pseudogenes. We predict that mutations in one of the expressed genes in the locus will be responsible for the PXE phenotype in these families.

Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE).

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

Pyroglutamic acid and iron regulate the expression of the pcp gene in Pseudomonas fluorescens MFO.

Pyrrolidone carboxyl peptidase (Pcp) is an aminopeptidase (EC 3.4.11.8) able to specifically remove the L-pyroglutamyl residue from the amino-terminus of polypeptides. Since nothing was known concerning the regulation and function of Pcps, a mutant of a milk-isolated strain lacking Pcp activity (Pseudomonas fluorescens MB1), was constructed by homologous recombination using a transcriptional fusion between pcp and a reporter gene (uidA). The wild-type and mutant strains were grown in synthetic media and in milk to investigate the environmental effects on pcp transcription. The expression of pcp and of the transcriptional fusion pcp::uidA was not sensitive to environmental conditions like temperature, osmolarity or nitrogen and phosphate starvation but was induced by the product of the enzymatic activity, pyroglutamic acid (pGlu). The expression of the native gene and the fusion in inducing conditions was also controlled by the iron concentration. The identification in the pcp promoter sequence of putative ferric uptake regulator (Fur) binding sites suggests a transcriptional regulation in a Fur-dependent fashion. Two other putative regulatory stretches, corresponding to inverted repeated sequences with perfect and imperfect symmetry, were also identified. pGlu and iron are therefore at least two of the transcriptional effectors of pcp expression.

Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase.

On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments. Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the synthetic chromogenic substrate pyroglutamyl-beta-naphthylamide and the dipeptide pyroglutamyl-alanine. Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp. These residues are not essential for the catalytic activity. Replacement of Asp-89 by Asn and Ala resulted in enzymes which retained nearly 25% of activity and which had no activity, respectively. Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes. Proteins with changes of Glu-81 to Gln and Asp-94 to Asn were not detectable in crude extract and were probably unstable in bacteria. Our results are consistent with the proposal that Cys-144 and His-166 constitute the nucleophilic and imidazole residues of the Pcp active site, while residue Glu-81, Asp-89, or Asp-94 might constitute the third part of the active site. These results lead us to propose Pcps as a new class of thiol aminopeptidases.