Spontaneous and pronase‐induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines

A subgroup of HER2‐overexpressing breast tumours co‐expresses p95 $^{{\rm{HER2}}}$ , a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95 $^{{\rm{HER2}}}$ expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2‐positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95 $^{{\rm{HER2}}}$ expression. Immunohistochemistry was performed on parallel formalin‐fixed, paraffin‐embedded sections of the same case series using antibodies directed against either the intra‐ or extra‐cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185 $^{{\rm{HER2}}}$ ‐positive/p95 $^{{\rm{HER2}}}$ ‐positive samples than in the p185 $^{{\rm{HER2}}}$ ‐positive/p95 $^{{\rm{HER2}}}$ ‐negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95 $^{{\rm{HER2}}}$ ‐positive cases. Conversely, the percentage of 2+ scored cells was higher in p95 $^{{\rm{HER2}}}$ ‐negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2‐HER3 dimers was studied using a proximity‐ligation assay. In vitro experiments showed that short‐term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2–HER3 dimers, and did not interfere with pertuzumab‐binding capacity. In conclusion, the presence of p95 $^{{\rm{HER2}}}$ as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing

Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G₂₂₅₄ constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G₂₂₅₄ nucleotide but with an A₂₂₅₄ nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A₂₂₅₄ and or G₂₂₅₄ strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G₂₂₅₄ mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases.     

Osteosarcoma of the hands and feet: a distinct clinico-pathological subgroup

Osteosarcomas of hands or feet are rare, and seemingly these cases differ in presentation and behavior compared to those in usual locations. The clinico-pathological presentation of patients with osteosarcomas of the hand or foot was studied and compared with published cases. Forty osteosarcomas were identified among 4,221 cases, representing 0.95 % of all osteosarcomas. Thirty of these were well documented. Mean age at diagnosis was 43 years (hands) and 36 years (feet) and male–female ratio was 1.2:1 and 2.0:1, respectively. In the hand, 62 % of the osteosarcomas presented in the metacarpals and 23 % in the phalanges, and only two cases occurred in the carpal bones. Distribution in the foot was tarsal bones 56 %, metatarsal bones 33 %, and phalanges 11 %.Of the cases in the hand 54 % were of high grade and of those in the foot 71 %. Survival of osteosarcomas of the hand or foot was 81 %. Only patients with high-grade osteosarcoma died of the disease. Histological grade was the only significant variable related to survival. High-grade osteosarcoma of the hand or feet should be treated similar to those in conventional sites. Osteosarcomas of hands or feet are rare and in a relative high proportion are of low grade. Survival in high-grade cases is comparable to that in conventional sites.     

Daytime course of sleepiness in de novo Parkinson's disease patients

Normal subjects show an increase of sleepiness in the morning, early afternoon and before sleep. In the advanced stages of Parkinson's disease (PD) the mean level of sleepiness is quite high, while with respect to healthy subjects it seems to be unchanged in the early stages. The aim of this study was to evaluate the time–course of the sleepiness level during the wakefulness period in untreated patients with early‐stage Parkinson's disease. Eighteen Parkinson's disease patients who had never been treated before with dopaminergic drugs (male = 9, female = 9, age: 68.39 ± 1.89, mean ± standard error) and 18 healthy subjects (male = 9, female = 9, age: 67.22 ± 1.98) were recruited for this study. All subjects underwent continuous actigraphic recording for three consecutive days, during which they also completed the Karolinska Sleepiness Scale (KSS) once an hour throughout wakefulness. Our results showed a higher level of sleepiness in the patients than the controls in the hours following awakening and in the early afternoon, specifically at 08:00 and 14:00 hours (08:00 hours, PD patients, KSS: 3 ± 0.3 versus healthy subjects, KSS: 2 ± 0.2, P < 0.05; 14:00 hours, PD patients, KSS: 4.4 ± 0.5 versus healthy subjects, KSS: 3 ± 0.3, P < 0.05). We suggest that some daytime hours are sensitive windows showing the first increase of sleepiness which will spread later to the whole daytime.     

Beyond the joints,the extra-articular manifestations in rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory disease typically affecting the joints, but the systemic inflammatory process may involve other tissues and organs. Many extra-articular manifestations are recognized, which are related to worse long outcomes. Rheumatoid nodules are the most common extra-articular feature, found in about 30% of patients. Secondary Sjögren's syndrome and pulmonary manifestations are observed in almost 10% of patients, also in the early disease. Active RA with high disease activity has been associated with an increased risk of such features. Male gender, smoking habit, severe joint disease, worse function, high pro-inflammatory markers levels, high titer of rheumatoid factor, and HLA-related shared epitope have been reported as clinical predictors of occurrence of these rheumatoid complications. In addition, there is a little evidence deriving from randomized controlled trials in this field, thus the therapeutic strategy is mainly empiric and based on small case series and retrospective studies. However, considering that these extra-articular manifestations are usually related to the more active and severe RA, an aggressive therapeutic strategy is usually employed in view of the poor outcomes of these patients.The extra-articular features of RA remain, despite the improvement of joint damage, a major diagnostic and therapeutic challenge, since these are associated with a poor prognosis and need to be early recognized and promptly managed.     

Chronic treatment with nitric oxide-releasing aspirin reduces plasma low-density lipoprotein oxidation and oxidative stress,arterial oxidation-specific epitopes,and atherogenesis in hypercholesterolemic mice

The effects of chronic treatment with nitric oxide-containing aspirin (NO-aspirin, NCX-4016) in comparison with regular aspirin or placebo on the development of a chronic disease such as atherosclerosis were investigated in hypercholesterolemic low-density lipoprotein (LDL)-receptor-deficient mice. Male mice were assigned randomly to receive in a volume of 10 ml/kg either placebo (n = 10), 30 mg/kg/day NO-aspirin (n = 10), or 18 mg/kg/day of regular aspirin (n = 10). After 12 weeks of treatment, the computer-assisted imaging analysis revealed that NO-aspirin reduced the aortic cumulative lesion area by 39.8 +/- 12.3% compared with that of the placebo (P < 0.001). Regular aspirin did not reduce significantly aortic lesions (-5.1 +/- 2.3%) compared with the placebo [P = 0.867, not significant (NS)]. Furthermore, NO-aspirin reduced significantly plasma LDL oxidation compared with aspirin and placebo, as shown by the significant reduction of malondialdehyde content (P < 0.001) as well as by the prolongation of lag-time (P < 0.01). Similarly, systemic oxidative stress, measured by plasma isoprostanes, was significantly reduced by treatment with NCX-4016 (P < 0.05). More importantly, mice treated with NO-aspirin revealed by immunohistochemical analysis of aortic serial sections a significant decrease in the intimal presence of oxidation-specific epitopes of oxLDL (E06 monoclonal antibody, P < 0.01), and macrophages-derived foam cells (F4/80 monoclonal antibody, P < 0.05), compared with placebo or aspirin. These data indicate that enhanced NO release by chronic treatment with the NO-containing aspirin has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic mice.     

Viral load 1 week after liver transplantation, donor age and rejections correlate with the outcome of recurrent hepatitis C.

BACKGROUND: Early identification of patients at a higher risk of rapidly progressive recurrent hepatitis post liver transplantation (LT) could help to tailor antiviral therapy. METHODS: We studied the correlation between early post-LT viral load and the histological and clinical outcomes of 49 consecutive patients (34 males, median age 55 years) in whom viraemia was monitored at days 0, 1, 7, 30, 180 and 365 after LT. RESULTS: Hepatitis C recurred at histology in 38 of 42 (90.5%) patients. Early viral load after LT was higher in patients with rapidly progressive hepatitis C recurrence (day 7 median HCV-RNA levels: 5.84 vs 4.93 Log(10) IU/ml, P=0.003). Day 7 HCV-RNA levels >/=2.5 x 10(5) IU/ml, donor age >60 years and rejection episodes were independently associated with progression to cirrhosis within one year post-LT [P=0.018, odds ratio (OR) 27.59; P=0.043, OR 13.85 and P=0.048, OR 9.95, respectively]. Day 7 viraemia and rejection episodes were independently associated with 5-years survival. Day 7 viraemia, in combination with acute hepatitis and/or donor age, showed 80% sensitivity, 94% specificity and 90.5% diagnostic accuracy to identify severe recurrence. CONCLUSIONS: Early post-LT HCV-RNA correlates with the severity of hepatitis C recurrence and in combination with donor age (>60 years) and rejections, identifies patients with a high risk of severe recurrence and candidates of cost-effective pre-emptive antiviral therapy.     

Lack of correlation between intracavitary thrombosis detected by cross sectional echocardiography and systemic emboli in patients with dilated cardiomyopathy.

The correlation between intracavitary thrombosis detected by cross sectional echocardiography and systemic embolism was studied in 126 consecutive patients with idiopathic dilated cardiomyopathy who were examined from January 1980 to September 1987. A total of 1041 serial echocardiograms were obtained with 3.5 and 5 MHz transducers. The mean follow up period was 41.2 months. The survival rate was 88% at two years and 56% at five years. Echocardiography showed intracavitary thrombi in 14 (11.1%) patients; 13 were mural and 11 were localised at the apex of the left ventricle. Twelve patients (8.4%) had systemic emboli; this corresponded to an incidence of new embolic events of 1.4 for 100 patient-years. Patients with intracavitary thrombi or systemic emboli were treated with oral anticoagulants, as were nine in functional class IV of the New York Heart Association, for 61 patient-years. The cumulative observation period for the whole population study was 418 patient-years. None of the patients with intracavitary thrombosis had embolic complications and none of those with embolism had intracavitary thrombi. Rates of intracavitary thrombosis and systemic embolism in this series were low and there was no overlap between the two events. This may have been because the patients did not have severe dilated cardiomyopathy, because echocardiography did not detect all the thrombi, or because patients were treated with oral anticoagulants. The presence of intracardiac thrombosis detected by cross sectional echocardiography is not predictive of systemic embolism in patients with idiopathic dilated cardiomyopathy. Criteria for the use of the anticoagulant treatment remain largely empirical in these cases.