PI3K/AKT Signaling in Breast Cancer Molecular Subtyping and Lymph Node Involvement.

Lymph node metastatic involvement persists to be among the most important predictors of recurrence and survival in breast carcinoma (BC). This study is aimed at investigating possible gene expression differences in primary BC between patients with or without lymph node involvement at the time of diagnosis. In a retrospective study, we investigated the potential prognostic role of 9 candidate biomarkers at the mRNA level in a cohort of 305 breast cancer patients, 151 lymph node-negative (LN-) and 154 lymph node-positive (LN+) individuals. The analyzed genes belonged to the RAS pathway (RAF1, ERBB2, PIK3CB, AKT1, AKT2, and AKT3), RB pathway (RB1 and CDK2), and cellular differentiation (KRT8). Their expression profiles were investigated by RT-qPCR and were correlated to immunohistochemically based molecular subtypes and BC clinical and pathological features. The differential expression of several genes in the primary tumor tissue was related to the LN involvement. Some of those genes, including PIK3CB, RB1, and AKT3, were more expressed in LN- BC patients, while some others, notably ERBB2 and AKT1, in LN+ ones. Among the candidate biomarkers, the expression levels of AKT isoforms influenced also patients' survival rates. In detail, higher expression levels of AKT1 and AKT2 negatively influenced overall patients' survival, and in particular, AKT2 expression levels defined a group of luminal B BC patients with shorter cancer-specific survival. On the contrary, longer cancer-specific survival was recorded in luminal A BC patients with higher expression levels of AKT3. That finding was also confirmed by Cox multivariate analysis. The same AKT3 resulted to be a possible candidate predictive biomarker for Tamoxifen response. In conclusion, our study highlighted the complex regulation of the PI3K/AKT pathway in BC and its differences in BC patients with and without lymph node involvement.

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

Desmosomes in verrucous carcinoma of the head and neck.

Verrucous carcinoma (VC) is a variant of squamous cell carcinoma (SCC), characterised by its inability to metastasize. In contrast, hybrid carcinomas, composed of VC and foci of conventional SCC, harbour a metastatic potential. Correct pathohistological diagnosis is therefore crucial for the choice of treatment. There is mounting evidence that desmosomes are involved in several aspects of carcinogenesis. Previous studies have shown an altered expression of desmosomal components in conventional SCC, which was associated with tumour behaviour, but no data have been found on desmosomes in VC. We therefore analysed the expression of desmosomal components in biopsy samples of 21 cases of VC and 5 cases of hybrid carcinoma of the head and neck in comparison to 23 cases of conventional SCC and 47 samples of normal squamous epithelium of similar localisation, using immunohistochemistry and real-time reverse-transcription polymerase chain reaction. We found that the expression patterns of desmosomal components in VC were fairly similar to those in normal epithelium but differed significantly from those in conventional SCC. Immunohistochemical reactions against desmosomal components disclosed the foci of SCC in hybrid carcinomas. In conclusion, we believe that expression patterns of desmosomal components in VC are consistent with its less aggressive behaviour. Differential expression of desmosomal components between VC and SCC makes some desmosomal components potentially useful in the diagnostics of VC, especially for the detection of hybrid carcinoma.

Autopsy reports in extreme longevity.

This study aimed at establishing the prevalence, the type, the severity of various diseases, as well as the main causes of death in the elderly and ultralongevous subjects. The autopsy findings of 140 centenarians (21 males and 119 females) of the age range of 100-109 years were compared to those of 96 elderly subjects (14 males and 82 females) of the age range of 75-95 years. In all cases the clinical diagnosis, the clinical record, the macro- and microscopic findings and the autopsy protocols were evaluated. A lower prevalence (16.3% vs. 39.0%), as well as a slower and less aggressive evolution of neoplastic pathologies (frequency of metastases: 26.0% vs. 55.0%) in the centenarians, as compared to the general aging population, have been found. The chronic-degenerative pathologies, especially the cerebro-degenerative ones were observed more frequently and were of major gravity in the centenarians, compared to the elderly population. The cerebrovascular damage and the consequent cognitive deficit do not influence the survival of the longevous subjects. Intercurrent events or external accidents may interrupt the weak equilibrium of these "frail" subjects.

Nanoelectrode ensembles as recognition platform for electrochemical immunosensors.

In this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.

Mycosis fungoides: is it a Borrelia burgdorferi-associated disease?

Mycosis fungoides (MF) is the most frequently found cutaneous T-cell lymphoma with an unknown aetiology. Several aetiopathogenetic mechanisms have been postulated, including persistent viral or bacterial infections. We looked for evidence of Borrelia burgdorferi (Bb), the aetiologic agent of Lyme disease (LD), in a case study of MF patients from Northeastern Italy, an area with endemic LD. Polymerase chain reaction for the flagellin gene of Bb was used to study formalin-fixed paraffin-embedded lesional skin biopsies from 83 patients with MF and 83 sex- and age-matched healthy controls with homolocalised cutaneous nevi. Borrelia burgdorferi-specific sequence was detected in 15 out of 83 skin samples of patients with MF (18.1%), but in none out of 83 matched healthy controls (P<0.0001). The Bb positivity rates detected in this study support a possible role for Bb in the aetiopathogenesis of MF in a population endemic for LD.

DNA and RNA obtained from Bouin's fixed tissues.

BACKGROUND: The use in many countries of acid fixatives, such as Bouin's solution, has limited the use of archival tissue for molecular analysis. An acidic environment is one of the main causes of DNA degradation. Moreover, RNA extraction is difficult in these types of fixed tissues. AIMS: To amplify DNA and RNA from Bouin's fixed tissues. METHODS: DNA and RNA were extracted from 20 breast cancer samples that had been routinely fixed in Bouin's fixative. Amplification of several genes using primers that produced amplicons of different lengths was carried out using the polymerase chain reaction (PCR) for DNA (with and without restoration) and reverse transcription PCR for RNA. RESULTS: The acid environment of Bouin's fixative damaged both DNA and RNA. However, amplification was successful when the amplicon length was reduced to about 80 bp for RNA and 100-200 bp for DNA, especially if submitted to DNA reconstruction procedures. CONCLUSIONS: It is possible to recover and analyse DNA and RNA from Bouin's fixed and paraffin wax embedded tissues.

Insulin-like growth factor system gene expression in women with type 2 diabetes and breast cancer.

BACKGROUND/AIMS: A twofold increased risk for breast cancer has been reported recently for women with late onset diabetes. Most studies showed that there were differences in serum concentrations of insulin-like growth factors and related proteins between women with and without diabetes who have breast cancer. This study investigated the expression of these markers at the cellular level in a cohort of women with and without type 2 diabetes who underwent biopsy because of a breast lump. METHODS: Relative quantitative analysis of specific mRNA sequences was performed after extraction and reverse transcription polymerase chain reaction amplification from formalin fixed and paraffin wax embedded tissues. Sixty seven breast surgical specimens from women with and without diabetes who did not have cancer and from women with and without diabetes who did have cancer were studied for insulin-like growth factor I (IGF-I), the IGF-I receptor (IGF-IR), insulin-like growth factor binding protein 3 (IGFBP-3), and oestrogen receptor 1 gene expression. RESULTS: The expression of IGF-I and IGF-IR was significantly lower in the cancer groups, whereas there was no significant difference for IGFBP-3 between women with and without cancer. Moreover, there was a good correlation between the expression of IGF-I and IGF-IR in women without cancer: this link was still present in breast tissue from patients with diabetes and cancer, whereas it was lost in patients without diabetes but with cancer. CONCLUSIONS: These differences in IGF-I/IGF-IR expression could contribute to the increased risk for breast cancer in women with type 2 diabetes.

Morphologic and biologic studies on ten cases of verrucous carcinoma of the vulva supporting the theory of a discrete clinico-pathologic entity.

Ten cases of verrucous carcinoma (VC) of the vulva diagnosed from January 1989 to December 1996 were studied. Patient age ranged from 50 to 83 years. The following examinations were performed on buffered formalin-fixed material: 1). in situ DNA hybridization, probes HPV 6/11, 16/18, 31/35/51; and 2). a series of immunohistochemical stainings to demonstrate wild and mutant types of the p53 protein, cytokeratin expression and pattern distribution (AE1 and AE3), and proliferating pattern (MIB 1). In situ DNA hybridization analysis for human papillomavirus 6/11, 16/18, 31/35/51 was negative in all cases. Wild and mutant types of p53 protein transcribed from related oncosuppressor gene were not detected. Keratins AE1 and AE3 showed a peculiar distribution pattern, that is, AE1 was uniformly positive in the surface and intermediate layers, while it was almost negative in the basal layer which-on the contrary-was mainly positive to AE3 keratins. MIB-1 highlighted 10-40% of proliferating cells; however, in all cases, 70-80% of MIB-1 positivity was found in the basal layer of the neoplastic epithelium. These results seem to show the morphofunctional and growth characteristics of neoplastic epithelium, thus stressing that VC should be considered as a discrete entity in vulvar tumors.

PCR analysis in archival postmortem tissues.

BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.

Molecular analyses of sentinel lymph nodes: an open question.

AIMS: To detect micrometastases in the sentinel lymph nodes (SLN) of melanoma patients the authors analysed 52 lymph nodes (47 SLNs and five non-sentinel) and 17 corresponding primary skin melanomas using reverse transcriptase-polymerase chain reaction assays in paraffin-embedded tissues to detect the mRNAs of tyrosinase, MAGE1, MAGE3, MIA, MART-1 and mRNA coding for telomerase catalytic component. RESULTS: Our data show that the use of molecular markers for melanoma micrometastases detection in SLN is still in a very preliminary stage. In comparing the molecular analysis results with the pathological staging we did not find any evident correlation with the expression of the analysed genes in SLN. There are no data for judging the prognostic significance of the detection of circulating tumour cells in patients without clinically recognizable metastases. Despite progress in the field with simultaneous detection of several markers it was assumed that tyrosinase mRNA remains the best target for the detection of metastatic melanoma cells.

In situ polymerase chain reaction detection of transfusion-transmitted virus in liver biopsy.

The potential role of transfusion-transmitted virus (TTV) infection in determining liver damage is poorly understood and no information exists about TTV replication within hepatocytes. In this study, we assess TTV in situ PCR in liver tissue. Twenty-one patients with different degrees of liver damage were studied by both serum TTV-DNA detection and in situ TTV PCR analysis and extractive PCR in liver biopsy paraffin sections (FFPE). Extractive PCR and in situ PCR detected TTV-DNA both in serum and liver tissue of five patients. The presence of TTV in serum matched with that found in the liver and TTV sequences were never found independently in liver or serum. Four out of five TTV-DNA-positive patients have not other known cause of liver damage while in one a coinfection from HCV was observed. Our data indicate that in situ PCR appears to be a reliable tool for the detection of TTV-DNA in FFPE, and may help detecting unknown origin of liver damage.

Non-Hodgkin lymphoma among young adults with and without AIDS in Italy.

To compare the presentation and prognosis of non-Hodgkin lymphoma (NHL) in people with AIDS (PWA) and in the general Italian population, a record linkage study was carried out. The fraction of NHLs attributable to HIV/AIDS was also estimated. Information from the National AIDS Registry (RAIDS) was linked with records from 13 cancer registries (CR), covering about 15% of the Italian population. During the period 1985--94, among PWA ages 15--49, 136 NHLs were identified (8% of all NHLs) and were compared with 1,481 concurrent incident NHL cases of the same age group among non-PWA. Percentages above 13% of all NHLs were registered in the northern areas of Genoa and Varese, i.e., the most heavily affected by the AIDS epidemic. Between 1 year prior to and 3.5 years after AIDS diagnosis, PWA showed an overall standardised incidence ratio (SIR) for NHL of 302. SIR was particularly high (394) within 3 months after AIDS diagnosis and subsequently declined to 170. SIR was somewhat higher in females (428) than in males (280) but similar among intravenous-drug users (299) and other HIV-transmission groups (309). High-grade NHL, particularly immunoblastic and Burkitt's lymphoma, were twice as frequent among PWA than non-PWA. Conversely, low-grade NHL were less frequent. Except for the high proportion of brain localisation, no clear difference emerged in the pattern of NHL presentation site in PWA compared with non-PWA. At variance with NHL in the general population, among PWA histological grade had little impact on survival, which overall appeared to be very poor (2-year survival: 10%; 95% confidence interval: 3%--17%). Our present linkage of RAIDS and CRs represents an efficient tool for the surveillance of trends in incidence and survival of NHL among PWA in Italy.