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Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

Background Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes may overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective To investigate the ability of whole-exome screening methods to detect disease-causing variants in individuals with PIDDs. Methods Individuals with PIDDs from 278 families from 22 countries were investigated using whole-exome sequencing (WES). Computational CNV prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic copy number variants (CNVs). Analytic approaches initially focused on 475 known or candidate PIDD genes, but were non-exclusive and were further tailored based upon clinical data, family history and immunophenotyping. Results A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on the molecular findings. Twelve PIDD-causing CNVs were detected, including seven smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes, permitted detection of low-grade constitutional, somatic and revertant mosaicism, and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

Survivors of the heritable form of retinoblastoma subsequently develop second primary osteosarcomas at substantially greater frequency than either the general population or survivors of nonheritable retinoblastoma. Here we present molecular genetic evidence that the development of these two disparate tumor types involves specific somatic loss of constitutional heterozygosity for the region of human chromosome 13 that includes the RBI locus. Similar events occur during the genesis of nonheritable osteosarcoma but not in several other embryonal tumors or sarcomas. These findings suggest that a conceptual approach toward defining the number of genes whose recessive mutant forms predispose to cancer is the molecular genetic analysis of clinically associated tumor types. They also suggest that the molecular basis of mixed cancer families may be the differential expression of a single pleiotropic recessive mutation by tissue specific mitotic segregation abnormalities.Retinoblastoma is one of a group of childhood tumors to which predisposition can be inherited as an autosomal dominant trait (1). This form of the disease generally affects both eyes, whereas the spontaneous form usually affects a single eye. The occurrence of retinoblastoma as both heritable and sporadic forms has allowed detailed statistical analyses, which indicate a relationship between these two forms of the disease (2-5). A locus, RBJ, which plays a role in the development of this tumor, has been localized to the q14 band of human chromosome 13 through segregation analysis (6) and by the identification of specific deletions of this chromosomal region in some retinoblastoma patients or their tumors (7)(8)(9).A model to unify these observations has been proposed (10), based on the previous hypothesis that as few as two mutations are required for malignant transformation (2-4). In this model, bilateral retinoblastoma cases have inherited a germinal mutation of the RBJ locus, which predisposes each cell to transformation by a further event. Evidence has recently been presented that suggests that these predisposing mutations are recessive at the cellular level, are expressed upon loss of the homologous wild-type allele (10-14), and occur as germ-line events in heritable cases and somatic events in sporadic cases. Several somatic chromosomal mechanisms effect this loss, including nondisjunctional loss of the homolog carrying the wild-type allele, followed by reduplication of the mutant chromosome, and mitotic recombination between the two homologs (10); the net result is a cell that is homozygous for the mutant allele at the RBI locus (14).Advances in surgical and radiotherapeutic techniques have led to survival rates exceeding 90% for retinoblastoma patients. However, survivors of the heritable, but not the spontaneous, form are at a substantially increased risk for the development of independent, second tumors (15)(16)(17)(18)(19)(20). In a recent longitudinal survey of 693 bilateral retinoblastoma cases (20), 15% developed independent second pr...

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins.Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two KS alleles, leading to a Pro:Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals.Linkage analyses mapped the defect to this type H keratin gene (peak logarithm of odds score at 4) = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type H keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.Epidermolysis bullosa simplex (EBS) has been subdivided into three major types (1-3). (i) EBS Dowling-Meara (EBS-DM) is the most severe type, typified by blistering over whole body regions and keratin filament clumping and cytolysis in basal cells. (ii) EBS Kobner (EBS-K) is also characterized by generalized blistering and basal cell cytolysis, but with fewer abnormalities in basal cell keratin networks. (iii) EBS WeberCockayne (EBS-WC) is the mildest form, with blistering concentrated primarily in palmar and plantar regions, and very minor keratin filament perturbations. An unusual and rare form of EBS with generalized blistering and hyper-and hypopigmented spots over the body surface was first reported by Fischer and Gedde-Dahl (4). The unifying feature of all of these EBS subtypes is basal cell cytolysis, with a largely normal differentiation process in the suprabasal layers.

Biologic and clinical observations suggest that combining imatinib with IFN-␣ may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN␣2b (Peg-IFN-␣2b) 50 g weekly and imatinib 400 mg daily (n ‫؍‬ 56) or to receive imatinib 400 mg daily monotherapy (n ‫؍‬ 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg-IFN-␣2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg-IFN-␣2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P ‫؍‬ .002). The MMR rate increased with the duration of Peg-IFN␣2b treatment (< 12-week MMR rate 67%, > 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg-IFN-␣2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg-IFN-␣2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent. (Blood. 2011;118(12):3228-3235)

Abstract. Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than the collagen deposited in the tissue. In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cutaneous cells in vitro and identified the propeptide using domain-specific antibodies. For this purpose, two bacterial fusion proteins containing unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised against them. Immunoblotting showed that the anti-NC-2 antibodies reacted with procollagen VII isolated from cultured keratinocytes, but not with collagen VII extracted from the skin. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes, but the basement membrane zone of normal skin remained negative. The staining could not be rendered positive by chemical or enzymatic unmasking of potential hidden epitopes in the skin, indicating that most of the NC-2 domain is absent from normal skin. In contrast, a positive staining with NC-2 antibodies was observed in the skin of a patient with dystrophic epidermolysis bullosa, who carried a 14-bp deletion at one of the intron-exon junctions of the collagen VII gene. This aberration led to an in-frame skipping of exon 115 from the mRNA and eliminated 29 amino acids from the NC-2 domain which include the putative cleavage site for the physiological processing enzyme, procollagen C-proteinase. The resuits indicate that in normal human skin, the removal of the NC-2 domain from procollagen VII precedes its deposition at the dermal-epidermal junction. Furthermore, they suggest that an aberration in the procollagen VII cleavage interferes with the normal fibrillogenesis of the anchoring fibrils.CHORIN6 fibrils extend from the lamina densa of the epidermal basement membrane to the papillary connective tissue, thus ensuring tight cohesion between the basement membrane and the dermis in the skin. The main structural protein of the fibrils is collagen VII, a homotrimeric collagen containing three identical al(VII) chains (4,5,31). This collagen is synthesized mainly by epidermal keratinocytes (20,30). The primary synthetic product is larger than the collagen deposited in the tissue, and has been called procollagen VII in analogy to other members of the collagen protein family (5,18,20). repeat sequence, a large globular NH2-terminal NC-1 domain, and a small globular COOH-terminal NC-2 domain (24,25). Based on the cDNA-derived amino acid sequence, the following molecular masses have been calculated for the three procollagen VII domains: NC-1, 133 kD; collagenous domain, 145 kD; and NC-2, ~18 kD (7,12). Rotary shadowing electron micrographs of anchoring fibrils from tissue extracts suggested that NC-2 cleavage is necessary for correct assembly of collagen VII (24,25). During fibrillogenesis, collagen VII molecules form antiparallel tail-to-tail dimers with a small carboxy-term...

Background: Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of skin disorders. Several mutant genes have been identified in ARCI, but the association between genotype and phenotype is poorly understood. Methods: To investigate genotype-phenotype correlations in ARCI, we selected 27 patients from 18 families with specific ultrastructural features of the epidermis. The characteristic findings using electron microscopy (EM) were abnormal lamellar bodies and elongated membranes in the stratum granulosum, classified as ARCI EM type III. DNA samples from a subset of affected individuals were screened for homozygous genomic regions, and a candidate gene region was identified on chromosome 5q33. The region coincides with the ichthyin gene, previously reported as mutated in ARCI. Results: Mutation screening of ichthyin revealed missense or splice-site mutations in affected members from 16 of 18 (89%) families with characteristics of ARCI EM type III. In a control group of 18 patients with ARCI without EM findings consistent with type III, we identified one patient homozygous for a missense mutation in ichthyin. Discussion: Our findings indicate a strong association between ultrastructural abnormalities in the granular layer of epidermis and ichthyin mutations. The results also suggest that EM provides a tool for specific diagnosis in a genetically homogenous subgroup of patients with ARCI.