A subset of high-grade pulmonary neuroendocrine carcinomas shows up-regulation of matrix metalloproteinase-7 associated with nuclear β-catenin immunoreactivity,independent of EGFR and HER-2 gene amplification or expression

Nuclear translocation of β-catenin has been correlated with epidermal growth factor receptor (EGFR) overexpression/activation in nonsmall cell lung cancer. Less is known on β-catenin transactivation in high-grade pulmonary neuroendocrine tumors and on the status of β-catenin activating EGFR and human epidermal growth factor receptor 2 (HER-2) or β-catenin target genes cyclin D1 and matrix metalloproteinase-7 (MMP-7). β-catenin immunoreactivity was evaluated in 51 large-cell neuroendocrine carcinomas (LCNEC) and 45 small-cell lung carcinomas (SCLC). Nineteen cases were assessed for β-catenin gene exon 3 mutations, expression of MMP-7, and expression/gene amplification of EGFR, HER-2, and cyclin D1. β-catenin was expressed in all 96 high-grade neuroendocrine tumors, the vast majority (94%) showing >50% immunopositive cells. A disarrayed immunoreactivity, however, was commonly encountered consisting in variably altered membrane-associated patterns of staining along with progressive accumulation of cytoplasmic immunoreactivity. In LCNEC, but not in SCLC, the disarrayed patterns correlated with EGFR and HER-2 protein expression. β-catenin nuclear accumulation was found in nine tumors, including seven LCNEC and two SCLC, and was always associated with disarrayed immunoreactivity and increased MMP-7, but not cyclin D1 expression. These cases, however, did not show β-catenin gene mutations or EGFR and HER-2 gene amplification or expression. No association was found between nuclear β-catenin and any clinicopathological variable including patients' survival. The subcellular compartmentalization of β-catenin is profoundly altered in high-grade pulmonary neuroendocrine tumors. A minor subset of these tumors shows β-catenin nuclear accumulation in association with increased expression of MMP-7, but not of cyclin D1, independent of EGFR and HER-2 gene amplification or expression. The authors have no significant financial or other relationship with the manufacturers of any commercial products or commercial services presented in this paper     

A comprehensive in vitro characterization of pancreatic ductal carcinoma cell line biological behavior and its correlation with the structural and genetic profile

There are a large number of stable pancreatic ductal carcinoma cell lines (PDCL) that are used by researchers worldwide. Detailed data about their differentiation status and genetic alterations are present in the literature, but a systematic correlation with cell biological behavior is often lacking. PDCL (n=12) were clustered by source of tumor cell (ascites, primary tumor, metastasis), and the data of functional cell biology were correlated with the reported structural and genetic profiles. Major histocompatibility complex expression, chemosensitivity and aneuploidia appeared to be related to the source of PDCL, and proliferative capacity appeared to be related to the grade of differentiation. No correlation between genetic/structural features of PDCL and biological behavior was found. All the cell lines appeared generally insensitive to in vitro treatment with 5-fluorouracil and showed variable degrees of susceptibility to gemcitabine, raltitrexed and oxaliplatin. All the PDCL showed resistance to Fas-mediated apoptosis but were significantly sensitive to the pro-apoptotic effect of inflammatory cytokines [interleukin (IL)-1, tumor necrosis factor (TNF) and interferon ]. PDCL were characterized for the secretion of several factors relevant to the tumor-immune cross talk. Vascular endothelial growth factor, CCL2, CCL5 and transforming growth factor were the factors most frequently released; less frequent was the secretion of CXCL8, CCL22, IL-6 and sporadically CXCL12, IL-10 and hepatocyte growth factor. The cytokines IL-1 and TNF were always undetectable. In conclusion, a clear correlation between structural/genetic features and function could not be detected, suggesting the weakness of a morphological classification for the in vitro studies of pancreatic cancer.     

Vasculogenic mimicry has no prognostic significance in pT3 and pT4 cutaneous melanoma

The concept of vasculogenic mimicry has been introduced to define periodic acid-Schiff (PAS)-positive channels and loops lined by tumor cells, instead of endothelium, able to contribute to microcirculation in uveal melanomas. Previous studies have shown that the PAS-positive patterns are associated with a poor prognosis in uveal melanoma. The aim of the current study was to investigate whether vasculogenic mimicry has a prognostic impact in pT3 and pT4 cutaneous melanoma. Fifteen patients with pT3 and pT4 cutaneous melanoma who did not experience progression after 10 years of follow-up and 30 matched controls who underwent progression were selected. Tumor sections were stained with PAS reaction, omitting the nuclear counterstaining. For immunohistochemistry, sections were stained with CD31, CD105 (endoglin), and laminin. Differences in the distribution of the PAS-positive patterns and a series of clinicopathological variables were evaluated by the Pearson chi(2) and Mann-Whitney U tests. We observed PAS-positive linear sheets, arcs, elliptical loops, and networks encircling roundish to oval aggregates of melanoma cells. The overall distribution of the PAS-positive patterns did not match with the blood microvessels' architecture as detected by immunohistochemical analysis. No statistically significant differences in the distribution of PAS-positive patterns were found between cases and controls. The presence of a parallel pattern correlated significantly with thickness (P = 0.04), whereas an inverse correlation was found with vessel area (P = 0.05). In conclusion, our results suggest that there is a mismatch between vasculogenic mimicry and tumor angiogenesis and do not support any prognostic role of vasculogenic mimicry in thick cutaneous melanoma.     

Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.     

Serum IgA preferentially binds to cationic polypeptides in IgA nephropathy.

The observation of negatively charged IgA in the mesangium of patients with primary IgA nephropathy (IgA-GN) prompted us to study the charge of serum IgA in IgA-GN, Henoch Schönlein purpura (HSP), alcoholic liver cirrhosis (ALC), membranous nephropathy (MGN) and systemic lupus erythematosus (SLE). Since no abnormal distribution of IgA isoelectric points was detected by isoelectric focusing studies, we developed a sensitive charge-dependent assay using plates coated with either cationized BSA (cBSA) or poly-L-lysine. In 15 IgA-GN sera, the amount of IgA reacting specifically with cBSA (cBSA-IgA) was almost linearly correlated with the poly-L-lysine-binding IgA (r = 0.97, P = 0.0006), suggesting that both assays detect charge-dependent interactions and thus probably measure anionic IgA. Significantly high serum levels of cBSA-IgA were observed in 56% of IgA-GN patients and in 40% of ALC patients. In contrast, normal serum levels of cBSA-IgA were detected in HSP, MGN and SLE. Both, the mono- or polymeric IgA bound to cBSA in a patient''s serum studied. Contrasting with the presence of anionic IgA, no increase of cBSA-IgG was observed in IgA-GN. IgA rheumatoid factor (IgA-RF) assay showed high levels in IgA-GN (39%) and in ALC (25%). IgA-RF levels did not correlate with the amount of cBSA-IgA. When 18 patients with IgA-GN were tested after kidney transplantation, increased levels of cBSA-IgA and/or IgA-RF were found to be associated with the recurrence of mesangial IgA deposits in the graft. This suggests that both negatively charged IgA and IgA-RF may play a role in the recurrence of mesangial IgA deposits.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

New multiple congenital anomalies: Mental retardation syndrome (MCA/MR) with facio-cutaneous-skeletal involvement

Five unrelated patients (a male and 4 females) were affected with a previously undefined multiple congenital anomalies/mental retardation syndrome which has been designated the facio-cutaneous-skeletal (FCS) syndrome and which includes mental retardation with specific sociable, humorous behavior, characteristic facial appearance, excessive generalized skin, postnatal growth failure, and skeletal involvement. Consanguinity was noted in 2 patients, thus autosomal recessive inheritance is suggested. © 1992 Wiley-Liss, Inc.     

A comprehensive haplotype analysis of CYP19 and breast cancer risk: the Multiethnic Cohort

The CYP19 gene encodes for aromatase (P450arom), a key steroidogenic enzyme that catalyzes the final step of estrogen biosynthesis. Apart from rare mutations in CYP19 which result in severe phenotypes associated with estrogen insufficiency, little is known about whether common variation in CYP19 is associated with risk of hormone-related diseases. In this study, we employed a haplotype-based approach to search for common disease-associated variants in this candidate breast cancer susceptibility gene among African-American, Hawaiian, Japanese, Latina and White women in the Multiethnic Cohort Study (MEC). We utilized 74 densely spaced single-nucleotide polymorphisms (SNPs) (one every approximately 2.6 kb) spanning 189.4 kb of the CYP19 locus to characterize linkage disequilibrium (LD) and haplotype patterns among 69-70 individuals from each ethnic population. We detected four regions of strong LD (blocks 1-4) that were quite closely conserved across populations. Within each block there was a limited diversity of common haplotypes (5 to 10 with a frequency >/=5%) and most haplotypes were observed to be shared across populations. Twenty-five haplotype-tagging SNPs (htSNPs) were selected to predict the common haplotypes with high probability (average Rh2=0.92) and genotyped in a breast cancer case-control study in the MEC (cases, n=1355; controls, n=2580). We first performed global tests for differences in risk according to the common haplotypes and observed significant haplotype-effects in block 2 [P=0.01; haplotypes 2b (OR=1.23; 95% CI, 1.07-1.40), 2d (OR=1.28; 95% CI, 1.01-1.62)]. We also found a common long-range haplotype comprised of block-specific haplotypes 2b and 3c to be associated with increased risk of breast cancer (haplotype 2b-3c: OR=1.31; 95% CI, 1.11-1.54). Our findings suggest the hypothesis that women with the long-range CYP19 haplotype 2b-3c may be carriers of a predisposing breast cancer susceptibility allele.     

Resistance to serum bactericidal activity distinguishes Brazilian purpuric fever (BPF) case strains of Haemophilus influenzae biogroup aegyptius (H. aegyptius) from non-BPF strains. Brazilian Purpuric Fever Study Group.

We studied the ability of normal human serum to lyse H. influenzae biogroup aegyptius (H. aegyptius) isolates recovered from patients with Brazilian purpuric fever (BPF clone) or non-BPF clone strains. BPF clone isolates, although similar to non-BPF clone isolates with regard to the ability to fix C3 to their surfaces, could be distinguished from non-BPF clone strains by their resistance to lysis in vitro following incubation with normal adult human serum.     

Recombinant tumor necrosis factor alpha does not inhibit the growth of African trypanosomes in axenic cultures

Mice whose tumor necrosis factor alpha (TNF-alpha) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-alpha directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-alpha on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-alpha used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-alpha lots tested, one lot of mouse TNF-alpha inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-alpha inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-alpha, as well as human TNF-alpha, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-alpha/ml of medium). These results suggest that exogenously added recombinant TNF-alpha generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-alpha on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.