Gene Status in HER2 Equivocal Breast Carcinomas: Impact of Distinct Recommendations and Contribution of a Polymerase Chain Reaction‐Based Method

Background.

The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status.

Methods.

A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA.

Results.

HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas.

Conclusion.

The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.     

MBL2 gene polymorphisms protect against development of thrombocytopenia associated with severe dengue phenotype

Dengue disease can clinically evolve from an asymptomatic and mild disease, known as dengue fever (DF), to a severe disease known as dengue hemorrhagic fever (DHF). Recent evidence has shown how host genetic factors can be correlated with severe dengue susceptibility or protection. Many of these genes, such as CD209, TNF-a, vitamin D receptor, and FC gamma receptor IIA, are components of the innate immune system, suggesting that innate responses might have a role in dengue pathogenesis. MBL2 gene polymorphisms have been shown to modulate susceptibility or protection in many viral diseases. We investigated the involvement of MBL2 gene in the dengue clinical outcome through the analysis of MBL2 exon 1 polymorphisms (at codons 52, 54, and 57) known to be associated with reduced serum levels of the MBL protein. The genotypes of 110 well-characterized dengue-positive patients were statistically analyzed to establish possible correlations between MBL2 polymorphisms and parameters such as sex, type of infection (primary or secondary response), race/ethnicity, course of infection, and age. We found significant correlations between wild-type AA MBL2 genotype and age as associated risk factors for development of dengue-related thrombocytopenia.     

MBL2 genetic screening in patients with recurrent vaginal infections

PROBLEM: Mannose-binding lectin (MBL) is an important component of the innate immunity, present at the mucosal level in vagina: a common pathogen's entry point. METHOD OF STUDY: We used a rapid genotyping method based on melting temperature assay to search for three single nucleotide polymorphisms (SNPs) located in the first exon of the MBL2 gene and we also measured MBL serum levels in patients with recurrent bacterial vaginosis (rBV) and recurrent vulvovaginal candidiasis (rVVC). RESULTS: Detected frequencies of MBL2 SNPs were comparable to the ones already reported for the Italian population and no significant differences were found between rVVC, rBV and controls. MBL serum levels did not show significant differences between the studied groups. CONCLUSION: No correlation for the screened mutations has been found neither in protecting nor in favoring the infection in rVVC and rBV patients. Our data demonstrate a lack of association between functional polymorphisms in the first exon of MBL2 gene, MBL deficiency, VVC and rBV.     

Electrocardiographic Criteria of True Left Bundle Branch Block: A Simple Sign to Predict a Better Clinical and Instrumental Response to CRT

Background: Cardiac resynchronization therapy (CRT) has proved to be very effective in improving morbidity and mortality in patients affected with severe congestive heart failure. Its efficacy has been shown to be greater in patients with left bundle branch block (LBBB). The aim of our study was to verify if newly proposed criteria for true LBBB identify patients with a better clinical and instrumental response to CRT. Methods: Between May 2007 and April 2011, 111 patients with left ventricular ejection fraction (LVEF) ≤ 35% and LBBB morphology received a CRT device and were divided into two groups according to QRS morphology. Group 1 (61 patients) consisted of patients with "true" LBBB morphology; group 2 (50 patients) consisted of patients with "false" LBBB. The primary endpoint was the utility of criteria for true LBBB to predict a composite endpoint of all-cause mortality and hospital admission with heart failure. The secondary endpoint was the utility of the same criteria to predict an absolute increase in LVEF ≥ 10%. Results: "False" LBBB morphology and a dose of bisoprolol <5 mg at last follow-up were the only parameters related to clinical outcome in multivariate analysis (respectively: hazard ratio [HR] 3.98, confidence interval [CI] 95% 1.51-10.48; HR 0.15, CI 95% 0.05-0.43). "True" LBBB morphology was the only variable significantly related to a greater increase in LVEF (HR 4.57, CI 95% 1.36-8.28). Conclusion: True LBBB morphology is related to a higher event-free survival rate in CRT patients and better echocardiographic response. (PACE 2012; 35:927-934).     

The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms

The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade.     

Laser ablation of the biliary tree: in vivo proof of concept as potential treatment of unresectable cholangiocarcinoma

Objectives: The palliative treatment of cholangiocarcinoma is based on stent placement with well-known procedure-related complications. Consequently, alternative energy-based techniques were put forward with controversial long-term results. This study aims to evaluate the safety and effectiveness of biliary tree laser ablation (LA) in terms of: (i) absence of perforation, (ii) temperature increase, (iii) induced thermal damage in in vivo models.

Materials and methods: The common bile duct and cystic ducts of two pigs were ablated with a diode laser (circumferential irradiation pattern) for 6 and 3?min at 7?W. Laser settings were chosen from previous ex vivo experiments. Local temperature was monitored through a fibre Bragg grating (FBG) sensor embedded into the laser delivery probe. Histopathological analysis of the ablated specimen was performed through in situ endomicroscopy, haematoxylin and eosin (H&;E) and nicotinamide adenine dinucleotide (NADH) stains.

Results: Temperature reached a plateau of 53?°C with consequent thermal damage on the application area, regardless of laser settings and application sites. No perforation was detected macroscopically or microscopically. At the H&;E stain, wall integrity was always preserved. The NADH stain allowed to evaluate damage extension. It turned out that the ablation spreading width depended on application time and duct diameter. In situ endomicroscopy revealed a clear distinction between ablated and non-ablated areas.

Conclusions: The temperature distribution obtained through LA proved to induce a safe and effective intraductal coagulative necrosis of biliary ducts. These results represent the basis for further experiments on tumour-bearing models for the treatment of obstructive cholangiocarcinoma.