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Cyclin D1 regulates G1–S progression. In many carcinomas it is overexpressed and it might even correlate with prognosis. However, the amplification of CCND1 contributes to the loss of cell cycle control only in a small fraction of malignant gliomas. Cyclin D1 can be immunohistochemically demonstrated by DCS-6 mAb. In astrocytic gliomas the fraction of tumor cells with positive nuclei is almost null in well differentiated tumors and increases with the increase of proliferation rate that occurs in anaplasia. The correct evaluation of this fraction is hindered by the positive staining of normal oligodendrocytes and microglia cells. The cyclin D1-positive staining of normal oligodendrocytes and microglia cells has been studied in a series of 20 oligodendrogliomas, five diffuse astrocytomas and five oligoastrocytomas and in 10 samples of normal cortex and white matter, using cyclin D1 DCS-6 mAb, Feulgen reaction and CR3.43 mAb for microglia cells. As well as microglial nuclei, the nuclei of normal oligodendrocytes of the cortex and white matter, including peri-neuronal satellites and pericapillary cells, were immunostained by DCS-6 mAb. In infiltrative areas of oligodendrogliomas, normal, cyclin D1-positive oligodendrocytes and cyclin D1-negative tumor cells coexisted. In anaplastic oligodendrogliomas, cycling tumor oligodendrocytes may regain the capacity to express cyclin D1, which is thus positive in some tumor cells. The occurrence of positive oligodendrocytes in the peripheral parts of tumors can be useful in distinguishing astrocytomas from oligoastrocytomas. [ABSTRACT FROM AUTHOR]

Intrafibral vacuoles are the morphological hallmark in a wide variety of human skeletal muscle disorders with different etiology. In most cases, differential diagnosis is feasible with a routine histochemical work up of muscle biopsy. Ultrastructural analysis is an important confirmatory tool, but it is not widely available. Immunohistochemical stainings for p62, LAMP2 and LC3 are commonly available as tissutal marker for autophagy. We compared the immunohistochemical patterns for autophagic markers p62, LC3 and LAMP2 with routine histochemical markers in 39 biopsies from patients with definite diagnoses of glycogen storage disease type 2 (LOPD or Pompe disease, PD), sporadic inclusion body myositis (sIBM), oculo-pharyngeal muscular dystrophy (OPMD) and necrotizing myopathy (NM). Moreover, we also analyzed muscles of 10 normal controls. In PD group, LC3 and LAMP2 showed an higher percentage of positive fibers, whereas p62 was limited to a minority of fibers, thus paralleling the results of histochemical stainings; in NM group, LAMP2 and LC-3 were diffusely and unspecifically expressed in necrotic fibers, with p62 significantly expressed only in two cases. OPMD biopsies did not reveal any significant positivity. The most interesting results were observed in sIBM group, where p62 was expressed in all cases, even in fibers without other markers positivity. There results, although limited to a small number of cases, suggest that the contemporary use of p62, LAMP2 and LC-3 staining may have an adjunctive role in characterizing muscle fiber vacuoles, revealing autophagic pathway activation and providing further clues for the understanding of pathogenetic mechanisms.s.

Background: Pompe's disease is an inherited metabolic myopathy caused by acid α-glucosidase deficiency. Early diagnosis optimizes the treatment effectiveness., Methods: One-hundred-thirty-seven consecutive patients with unexplained hyperCKemia underwent the assessment of acid α-glucosidase activity on dried blood spot. Second tier confirmatory testing in positive patients included the assessment of α-glucosidase activity on lymphocytes or muscle tissue and molecular analysis., Results: Three patients were diagnosed with later-onset Pompe's disease, revealing 2.2% prevalence in asymptomatic hyperCKemia. Moreover, three patients positive to the screening revealed abnormal biochemical second tier testing, but were heterozygous for the common c.-32-13T>G mutation at molecular level., Conclusions: The selective screening for later-onset Pompe's disease in asymptomatic hyperCKemia allowed the identification of affected patients in a pre-clinical stage. Additionally, the identification of carriers with biochemical alterations related to Pompe's disease extends the spectrum of its manifestations to heterozygous subjects., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16-p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14ARF was inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14ARF pathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14ARF inactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.

Four members of a family were found to carry the A3243G mtDNA mutation. Clinical features varied from typical MELAS to myoclonic epilepsy to simple deafness without neurological signs. Several other members of the family had symptoms consistent with a mitochondrial disease. Muscle biopsy in 3 of the 4 patients showed the most prominent mitochondrial alterations with partial deficiency of cytochrome c oxidase in the case with the mildest phenotype. Mitochondrial DNA analysis detected a variable percentage of A3243G mutation, roughly correlating with the phenotype. The interesting feature of the family lies in the great intrafamilial variability of the severity of clinical expression, encompassing MELAS and MERRF features, associated with the A3243G mtDNA mutation. A search for the most common mtDNA mutations is recommended in all patients featuring incomplete MELAS or MERRF syndromes and in all familial cases presenting minimal clinical signs.

We report a case of late onset of Becker's muscular dystrophy (BMD), diagnosed at the age of 60, which showed a very mild clinical course. Remarkably, the immunohistochemical pattern did not show significant alterations, while Western blotting disclosed low molecular weight dystrophin. DNA analysis showed a deletion of the exons 45-53 of the Xp21 gene, which is fairly typical of Becker's muscular dystrophy but not predictable of clinical course. The possibility of Xp21 muscular dystrophy must be considered in all myopathies of uncertain cause, also in elderly patients.

Sixteen cases of ependymoma were studied for CDKN2A/p16 inactivation by immunohistochemistry using a p16 monoclonal antibody, by homozygous deletion (HD) assay and 5'CpG promoter methylation assay (methylation-specific PCR). Three out of 16 cases were p16 immuno-negative: two corresponded to grade II ependymomas and one to grade III. The latter ependymoma, characterized by a high Ki-67/MIB-1 LI, was the only one of the whole series to show CDKN2A HD. No promoter methylation was found in the two immuno-negative cases without CDKN2A HD. Alternative mechanisms, such as point mutations or alterations in p16 post-translational regulation, may be responsible for p16 inactivation. Since in our series just one out of eight anaplastic cases showed negative immunostaining and CDKN2A HD, p16/CDKN2A inactivation may not play an important role in the malignant transformation of ependymomas. Amplification of CCNDI and CDK4, p27/Kipl degradation and TP53 mutations were previously studied by other authors and were demonstrated not to correlate with anaplasia. Up to date, molecular genetic studies have not been useful in recognizing the anaplastic variant in ependymomas.

The cell-cycle regulator p16 inhibits the complex cdk4-cyclin D1 and controls G1-S transition. In human tumors, p16 inactivation is often accomplished by homozygous deletion (HD) of its encoding gene, CDKN2A. Methylation of the 5' CpG island promoter has been proposed as an alternative mechanism of inactivation. Expression of p16, CDKN2A HD and 5' CDKN2A CpG island methylation was studied in 25 oligodendrogliomas by immunohistochemistry and PCR amplification. Ten oligodendrogliomas were p16-immunonegative, and CDKN2A HD was determined in 8 of these cases. In the 2 immunonegative cases without HD, no CpG island methylation was found. The absence of CpG island methylation in the p16-immunonegative cases without HD suggests either non-genetic regulation of p16 or different genetic changes. CDKN2A HD did not correlate with histological grading (p = n.s.); however, it showed a correlation with survival (p = 0.03), supporting an important role of CDKN2A in the prognosis of oligodendrogliomas., (Copyright 2000 Wiley-Liss, Inc.)

Recent data suggest that death of muscle cells during development and in selected pathological conditions occurs via apoptosis. We investigated the occurrence of apoptosis in normal and pathological human skeletal muscle, using in situ end-labeling (ISEL) to detect DNA fragmentation, and immunohistochemistry for the expression of tissue transglutaminase and interleukin-1beta-converting enzyme (ICE)-like proteases. In normal subjects, apoptotic myonuclei were occasionally observed as evidence of normal tissue turnover. Myonuclear apoptosis due to a deficit of trophic support from nerve cells also occurred in spinal muscular atrophies. No apoptosis of muscle cells was found in dystrophinopathies, myotonic dystrophy and inflammatory myopathies, suggesting that death of myofibers in those conditions is not due to activation of a gene-directed program of death. In dystrophinopathies and inflammatory myopathies, apoptosis was found in interstitial mononuclear cells, as a likely mechanism of clearance of the inflammatory infiltrates.

The metachromatic dye-Ca++ATPase method, that in normal muscle distinguishes fiber types on the basis of their metachromatic or orthochromatic staining, was applied to 382 pathological muscle biopsies. Results were compared in serial sections with those obtained with the conventional ammonium sulphide method. In pathological muscle the metachromatic dye-Ca++ATPase method confirms the advantages already showed in normal muscle: fast and easy performance, neat fiber typing, simultaneous staining of nuclei. Moreover in pathological muscle the metachromatic dye-Ca++ATPase method showed some muscle changes which are missed by the conventional ammonium sulphide one, namely central nuclei, macrophagic invasion and some structural abnormalities. This allows immediate correlation between those alterations and fiber typing.

A 61-year-old woman presented progressive distal weakness and wasting of her upper limbs spreading to the shoulder girdle and to the neck muscles. Creatine kinase, cerebrospinal fluid, spinal X-ray and spinal nuclear magnetic resonance were normal. Electromyography showed myopathic alterations. Muscle biopsy showed abundant rods in many fibres. Prednisone 75 mg/die for two months did not modify the symptoms.

A 9-year-old boy complained of exertional myalgias and described two episodes of myoglobinuria. His family history was negative for neuromuscular diseases. The findings of a neurological examination were normal. Serum creatine kinase was increased, ECG was normal, EMG showed slight "myopathic" signs. Muscle biopsy disclosed a small group of basophilic fibres as the only abnormality. Muscle glycolytic enzymes and carnitine palmitoyl transferase were normal. Immunoblotting using antidystrophin antibody demonstrated a protein with low molecular weight. Genomic DNA analysis showed a deletion of the HindIII fragments spanning from exon 45 to exon 48. Eight years after the first observation the patient has diffuse muscle hypertrophy without muscle weakness.

Two groups of monocular strabismic patients were followed-up by means of both PL and orthoptic examinations. The first group underwent a full-day occlusion except 1 to 4 hours; the second was occluded less than 4 hours per day. The first treatment regimen proved to be faster in getting pseudo-isoacuity and alternating fixation, but was followed by relative occlusion amblyopia and/or shifting of the dominant eye in three patients. PL techniques proved to be particularly useful in strictly monitoring these patients.

A 20-year-old patient was born with epidermolysis bullosa and a severe, slowly progressive muscle disease. Skin biopsy demonstrated junctional epidermolysis bullosa. Muscle biopsy demonstrated degenerative changes with increase in connective tissue, fibre size variability, rods and cytoplasmic bodies, central nuclei. In muscle biopsy dystrophin, chondroitin unsulphate, chondroitin 4-sulphate, chondroitin 6-sulphate, heparan sulphate, collagen III, collagen IV and VI, laminin, and fibronectin were normally distributed. This is the first report of the association of a form of congenital muscular dystrophy with junctional epidermolysis bullosa and, together with the previous reports of muscle involvement in epidermolysis bullosa simplex and dystrophica, it suggests the existence of a syndrome characterized by the contemporaneous presence of skin and muscle involvement.

A computerized version of preferential looking (CPL) was developed by the authors. Formal parameters (spatial frequency, luminance, contrast of the stimuli; randomized procedure; computerized statistical control) and preliminary binocular acuity results in 69 healthy children (6-36 months) are compared to those of OPL, FPL and ACP version. Low cost, standardized procedure, statistical control of visual acuity estimates and the need of one operator only are among CPL advantages.

A 35-year-old man with severe progressive dilating cardiomyopathy and no clinical signs of muscle disease underwent muscular investigations because of markedly increased serum creatine kinase. Muscle biopsy demonstrated Becker type muscular dystrophy with dystrophin of low molecular weight. Genetic analysis showed a deletion spanning from exon 45 to exon 46 in the Xp21 region. Xp21 Becker type muscular dystrophy must be considered in the differential diagnosis of dilating cardiomyopathy.