Thomas Brenn

Cysteine Substitutions in Epidermal Growth Factor–Like Domains of Fibrillin-1: Distinct Effects on Biochemical and Clinical Phenotypes

Fibrillin-1 (FBN1) contains 47 epidermal growth factor (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant fibroblast strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

Differential expression of microRNAs during melanoma progression: miR-200c, miR-205 and miR-211 are downregulated in melanoma and act as tumour suppressors

Background:The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.Methods:To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT–PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.Results:In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.Conclusion:We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

Premature Termination Mutations in FBN1: Distinct Effects on Differential Allelic Expression and on Protein and Clinical Phenotypes

Marfan syndrome (MFS) and other type 1 fibrillinopathies result from mutations in the FBN1 gene, which encodes the connective-tissue microfibrillar protein fibrillin 1. Attempts at correlating genotype with phenotype have suggested considerable heterogeneity. To define the subtype of fibrillinopathy caused by premature termination codon (PTC) mutations, we integrate genotype information and mRNA expression levels with clinical and biochemical phenotypes. By screening the entire FBN1 gene for mutations, we identified 34 probands with PTC mutations. With the exception of two recurrent mutations, these nonsense and frameshift mutations are unique and span the entire FBN1 gene, from IVS2 to IVS63. Allele-specific reverse-transcriptase polymerase chain reaction analyses revealed differential allelic expression in all studied samples, with variable reduction of the mutant transcript. Fibrillin protein synthesis and deposition into the extracellular matrix were studied by pulse-chase analysis of cultured fibroblasts. In the majority of PTC samples, synthesis of normal-sized fibrillin protein was approximately 50% of control levels, but matrix deposition was disproportionately decreased. Probands and mutation-positive relatives were clinically evaluated by means of a standardized protocol. Only 71% (22/31) of probands and 58% (14/24) of the mutation-positive family members met current clinical diagnostic criteria for MFS. When compared with our previously reported study group of 44 individuals with FBN1 cysteine substitutions, the PTC group showed statistically significant differences in the frequency of individual signs, especially in the ocular manifestations. Whereas large-joint hypermobility was more common, lens dislocation and retinal detachment were distinctly less common in the PTC group. We conclude that PTC mutations have a major impact on the pathogenesis of type 1 fibrillinopathies and convey a distinct biochemical, clinical, and prognostic profile.

Postradiation vascular proliferations: an increasing problem

The occurrence of cutaneous vascular lesions is a rare but well‐documented complication of radiation treatment and may be associated with significant morbidity as well as mortality. The overall incidence is low but appears to be rising due to a change in the prevailing treatment of breast carcinoma with increased use of radiation in the setting of breast‐conserving therapy for stage 1 and 2 disease. The spectrum of postradiation vascular lesions is wide and ranges from atypical vascular lesions with reportedly benign clinical behaviour to frank cutaneous angiosarcoma. There is, however, significant clinical as well as histological overlap. It is frequently difficult to classify these postradiation vascular lesions accurately and they create an emerging diagnostic and therapeutic challenge to both pathologists and clinicians. Experience with these vascular lesions is very limited, and this article aims to provide an overview of our current understanding and concept of radiation‐associated vascular lesions with focus on their clinical and histological presentation as well as behaviour and treatment.

Cutaneous epithelioid angiomatous nodule: a distinct lesion in the morphologic spectrum of epithelioid vascular tumors

Cutaneous epithelioid vascular proliferations comprise a morphologic spectrum ranging from benign, reactive to frankly malignant conditions. We present a group of morphologically distinct epithelioid vascular lesions in the skin that defy classification according to current criteria. Clinically, the lesions presented as single (14/15) or multiple (1/15) small (< 1.5 cm, median = 0.5 cm) erythematous to bluish nodules or papules of short duration (weeks to months) without gender predilection (M:F = 1:1). Age distribution ranged from 15 to 79 years (median: 37). Lesions were predominantly located on the trunk (8/15) but also involved the extremities (5/15), face (1/15), and nasal mucosa (1/15). No patient was immunocompromised. Treatment was simple surgical excision. Histologically, the lesions were located superficially in dermis (or submucosa) and consisted of a circumscribed, unilobular, mainly solid proliferation of large polygonal epithelioid endothelial cells with vesicular nuclei and conspicuous nucleoli. Cytoplasm was abundant and contained frequent intracytoplasmic vacuoles. Nuclear atypia was absent. Mitotic figures (up to 5/10HPF) were observed in 8 lesions. Formation of endothelial-lined channels was a focal but constant feature. Also noted were adjacent dilated dermal vessels (8/15), hemosiderin deposition (6/15), and mild fibrosis (9/15). The lesion was accompanied by a chronic inflammatory infiltrate with a lymphoplasmacytic component (14/15), most pronounced at the periphery of the lesion, and varying numbers of eosinophils scattered throughout the lesion (12/15). Eosinophils were conspicuous in only 4 cases. Epidermal hyperplasia was present in 11 cases. By immunohistochemistry, lesional cells stained positively for at least one endothelial marker. Clinical follow-up (median = 30 months) showed no recurrence or metastases. Epithelioid angiomatous nodule is a benign, likely reactive cutaneous lesion in the clinical and morphologic spectrum of epithelioid vascular proliferations. It differs from conventional epithelioid hemangioma both in its clinical presentation with predominant involvement of trunk and extremities as well as its histologic features, which more closely resemble epithelioid angiosarcoma.

Atypical genital nevi. A clinicopathologic analysis of 56 cases

Atypical genital nevi are rare melanocytic lesions that most commonly arise on the vulva of young women. They are currently regarded as nevi of special sites, in that despite histologically worrisome features, their clinical behavior is reportedly benign. However, only few studies with limited follow-up data are available. To better characterize the clinical presentation and behavior of these lesions and to further delineate their histologic features, we retrieved 56 atypical genital nevi arising in the lower female genital tract from our departmental and consultation files. The 56 lesions arose in 55 female patients with a median age of 26 years (range, 6 to 54 y). The dominant histologic feature was a lentiginous and nested junctional component composed of prominent round or fusiform nests, which often showed retraction artifact and/or cellular dyscohesion. Cytologic atypia was mild in 11 cases (20%), moderate in 34 (60%), and severe in 11 (20%). Ten cases (18%) showed focal pagetoid spread, with extension to the granular layer and stratum corneum in 1 case. The atypical junctional melanocytic proliferation was associated with a large common dermal nevus component that dominated the lesion in 26 cases (46%). Adnexal spread (46%) and nuclear atypia of melanocytes situated in the superficial dermis (39%) were relatively common, but dermal mitoses (7%) were uncommon and maturation was present in all cases. A broad zone of dense eosinophilic fibrosis within the superficial dermis was a frequent finding (41%). Clinical follow-up was available in 45 cases (80%) with a median follow-up period of 3.5 years (range, 1 to 16 y). Only 1 lesion recurred, 1.5 years after the initial excision. The original nevus in this patient had only mild cytologic atypia and was present at the margins of excision. The recurrent/persistent nevus was reexcised, and there was no further clinical recurrence in 11.5 additional years of follow-up. Our data support the hypothesis that atypical genital nevi have a benign clinical course despite their occasionally striking cytologic and architectural atypia. Awareness and recognition of this group of melanocytic lesions is important to avoid over diagnosis as melanoma with subsequent wide excision and possibly sentinel lymph node biopsy.

Pleomorphic dermal sarcoma: adverse histologic features predict aggressive behavior and allow distinction from atypical fibroxanthoma

The behavior of atypical fibroxanthoma is benign, if strict diagnostic criteria are applied. Tumors with similar pathologic features but deep subcutaneous invasion, necrosis, and/or lymphovascular or perineural invasion are thought to be associated with adverse outcome and are better regarded as pleomorphic dermal sarcoma or undifferentiated pleomorphic sarcoma of skin. This tumor group is not well documented in the literature, and its characteristics are only poorly defined. To study the clinical and pathologic spectrum more comprehensively, we retrieved 32 pleomorphic dermal sarcomas from our departmental files. The tumors were large (median: 25 mm) and exclusively presented on sun-damaged skin with a strong predilection for the head. Typically, elderly men were affected (median age: 81 y). Histologically, these often ulcerated tumors were poorly marginated, asymmetrical, and deeply invasive into deep subcutaneous, muscular, and/or fascial tissues. The tumors were cellular and composed of pleomorphic epithelioid cells, atypical spindle cells, and multinucleated tumor giant cells in varying proportions. Mitotic count was brisk and often atypical. Tumor necrosis was observed in 53%, lymphovascular invasion in 26%, and perineural infiltration in 29%. The majority of tumors showed a predominance of atypical spindle cells in a fascicular arrangement. A sheet-like growth of pleomorphic epithelioid cells or mixed spindle and epithelioid cell features were less frequently observed. Myxoid and keloidal change, a desmoplastic stromal response, pseudoangiomatous and storiform growth patterns, and admixed osteoclast-like giant cells were additional morphologic features in some cases. No immunoreactivity was noted for multiple cytokeratins, S100, HMB-45, desmin, and CD34. Smooth muscle actin was expressed in 70%, CD31 in 48%, epithelial membrane antigen in 16%, Melan A in 6%, and p63 in 1 case. CD10 was expressed in all cases stained. Follow-up (available for 29 patients; median: 24 mo) showed local recurrence in 28% and a metastatic rate of 10%, mainly in the skin. Progressive metastatic disease was observed in 2 patients. Remission was achieved in 1 patient using systemic chemotherapy. The second patient died in the setting of advanced-stage non-Hodgkin lymphoma. No disease-related mortality was noted. Our data underscore the importance of recognizing adverse histologic features in tumors otherwise resembling atypical fibroxanthoma. Deep subcutaneous invasion, tumor necrosis, and perineural and/or lymphovascular invasion confers at least low-grade malignant potential.

Phase II study of a TLR-9 agonist (1018 ISS) with rituximab in patients with relapsed or refractory follicular lymphoma.

Toll‐like receptor‐9 (TLR‐9) agonists have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody‐dependent cell‐mediated cytotoxicity (ADCC) and T helper cell type 1 shift in the immune response. We combined a TLR‐9 agonist (1018 ISS, 0·2 mg/kg sc weekly × 4 beginning day 8) with standard rituximab (375 mg/m2 weekly × 4) in patients (n = 23) with relapsed/refractory, histologically confirmed follicular lymphoma, and evaluated immunological changes following the combination. Treatment was well‐tolerated with no significant adverse events attributable to therapy. Clinical responses were observed in 48% of patients; the overall median progression‐free survival was 9 months. Biologically relevant increases in ADCC and circulating CD‐3 positive T cells were observed in 35% and 39% of patients, respectively. Forty‐five percent of patients had increased T cells and dendritic cells in skin biopsies of 1018 ISS injection sites 24 h post‐therapy. Pre‐ and post‐biopsies of tumour tissue demonstrated an infiltration of CD8+ T cells and macrophages following treatment. This group of patients had favourable clinical outcome despite adverse prognostic factors. This study is the first to histologically confirm perturbation of the local immune microenvironment following systemic biological therapy of follicular lymphoma.

CpG Oligodeoxynucleotides Alter Lymphocyte and Dendritic Cell Trafficking in Humans

Purpose: CpG oligodeoxynucleotides (CpG-ODN) are being investigated as cancer vaccine adjuvants because they mature plasmacytoid dendritic cells (PDC) into potent antigen-presenting cells. CpG-ODN also induce PDC to secrete chemokines that alter lymphocyte migration. Whether CpG-ODN TLR signals enhance antigen-specific immunity and/or trafficking in humans is unknown. Experimental Design: We conducted a phase I study of CpG-ODN (1018 ISS) given as a vaccine adjuvant with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce T-cell immunity to a peptide vaccine from the tumor-associated antigen hTERT. Results: The adjuvant effect was limited; only 1 of 16 patients showed a high-frequency hTERT-specific tetramer CD8+ T-cell response. However, CpG-ODN induced marked, transient peripheral blood lymphopenia. Biopsies showed dense lymphocytic infiltration at the vaccine site clustered around activated PDC. In vitro, CpG-ODN-treated PDC induced T-cell migration, showing that CpG-ODN stimulation of human PDC was sufficient to chemoattract T cells. Conclusions: Our results show that (a) CpG-ODN with GM-CSF may not be an effective adjuvant strategy for hTERT peptide vaccines but (b) GM-CSF/CpG-ODN causes a PDC-mediated chemokine response that recruits T-cell migration to the peripheral tissues. These findings suggest a novel therapeutic role for targeted injections of CpG-ODN to direct lymphocyte migration to specific sites such as the tumor bed.

Multi-exon deletions of the FBN1 gene in Marfan syndrome.

BackgroundMutations in the fibrillin -1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion.MethodsWe used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exonsResultsTwo novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine) domain and the adjacent 25th calcium-binding EGF-like (6-cysteine) domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44–46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening.ConclusionsTwo novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.