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.

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.

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.

A possible role for polyamines in cartilage in the mechanism of calcification.

The role of polyamines in cartilage is not known: they may be somehow related to the mechanism of calcification. In epiphyseal cartilage from calf scapulas, they are more concentrated in the ossifying area, where calcification takes place, than in the resting region. Spermidine is present in greater amounts than spermine and putrescine. Since ornithine decarboxylase (EC 4.1.1.17) is measurable only in the resting region of the tissue, it is in this area that polyamine biosynthesis occurs, while they accumulate in the ossifying area. Immunohistochemical evidence is obtained that only in the ossifying zone is spermidine extracellular. It is at this level that the matrix is rearranged to become calcified, and proteoglycans are dissociated and partially removed. The effect of polyamines on solutions of proteoglycan subunits has been studied in vitro by following variations of turbidity and viscosity. While in the presence of putrescine the specific viscosity decreases to asymptotic values, in the presence of either 30 mM spermidine or 2.5-10 mM spermine, the decrement is more marked. At the same concentrations, increase of the turbidity of proteoglycan subunit solutions was observed. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen: at 15 mM concentration, about 90% of proteoglycans were removed from the column. Alkaline phosphatase activity, which plays an important role in calcification, is enhanced by spermidine and spermine. These results obtained in vitro support the hypothesis that polyamines may be related to calcification of preosseous cartilage.

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.

Catalytic subunit of telomerase expression is related to RNA component expression.

Telomere length is maintained by the ribonucleoprotein enzyme telomerase. The RNA component of telomerase (hTR) is widespread, and only the expression of the mRNA encoding the catalytic protein subunit (hTRT) is correlated with telomerase activity. We have studied the level of expression of hTR and hTRT in four different models of neoplastic and preneoplastic lesions using the RT-PCR method on RNA extracted from paraffin-embedded human tissues after microdissection. The expression at the mRNA level was compared with the enzymatic activity. Our results suggest that there may be a reciprocal control at the transcriptional level of the expression of hTRT and hTR which in turn is associated with tumor progression.

RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification.

Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. In this report, we show that RNA isolated from surgical or autopsy tissues, routinely processed by fixation and paraffin embedding, is not completely degraded. RNA fragments around 100-200 bases in length are still present even in organs late fixed and very rich in RNase, such as the pancreas. Here we describe a general protocol to obtain RNA from single 6-8-microns tissue sections. The RNA extracted can be analyzed for the presence of specific sequences by reverse transcription and amplification with the PCR. We studied the retinoblastoma gene expression in 38 human pancreas specimens from surgical or autopsy origin.

RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis.

Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general method for quantitation of specific RNA sequence extracted from single 6-8-micron human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific product after reverse transcription (RT)-PCR must be established. We usually perform RT with avian myeloblastosis virus (AMV)-RT, using specific antisense primers and a variable number of cycles of PCR amplification. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplification product and the number of cycles. The quantity of specific product is standardized relative to beta-actin mRNA to normalize for the degree of RNA degradation, which can be quite different among samples. The amplification products were quantified by dot blot and 32P-labeled hybridization probe or by capillary electrophoresis with a laser-induced fluorescence detector. The intratest variation range was for the dot blot mean +/- 10% standard deviation (SD) and for the capillary electrophoresis mean +/- 3% SD.

Air pollution and lung cancer in Trieste, Italy: spatial analysis of risk as a function of distance from sources.

To investigate the relationship between four sources of environmental pollution (shipyard, iron foundry, incinerator, and city center) and lung cancer risk, we conducted a case-control study of decreased men in Trieste, Italy. We identified 755 cases of lung cancer and 755 controls through the local autopsy registry. Information on smoking habits, occupational history, and place of residence were obtained from the subject's next of kin. The case-control design was used to properly account for subject-specific confounders, which represent a major problem in geographical analysis. Spatial models were used to evaluate the effect of sources of pollution on lung cancer after adjustment for age, smoking habits, likelihood of exposure to occupational carcinogens, and levels of air particulate. The models are based on distance from the sources and enable estimation of the risk gradient and directional effects separately for each source. The risk of lung cancer was highly related to the city center (p = 0.0243), with an excess relative risk at zero distance of 2.2 and a smooth decrease moving away from the source (-0.015), and related to the incinerator (p = 0.0098), with an excess relative risk of 6.7 in the source and a very steep decrease (-0.176). These results are consistent with findings of previous analyses and provide further evidence that air pollution is a moderate risk factor of lung cancer.

Cigarette smoking and histologic type of lung cancer in men.

STUDY OBJECTIVES: To determine whether intensity, duration, age at initiation, and cessation of cigarette smoking act differently in the development of various histologic types of lung cancer. DESIGN: A case-control study among deceased men who underwent autopsy, a procedure that involves approximately 73% of all local deaths. SETTING: The Province of Trieste in northeastern Italy PARTICIPANTS: Seven hundred fifty-five patients with lung cancer, including 267 with squamous cell carcinoma, 218 with small cell carcinoma, 90 with large cell carcinoma, 158 with adenocarcinoma, and 22 with other histologic types, and 755 control subjects who had died of causes other than chronic lung diseases and certain tumors. Information on smoking habits, residential history, and occupational exposure was obtained from each subject's next of kin. RESULTS: Compared with nonsmokers, the odds ratio (OR) for current smokers was 13.4 for all types combined, 18.8 for squamous cell carcinoma, 14.3 for small cell carcinoma, 34.3 for large cell carcinoma, and 7.9 for adenocarcinoma. Intensity of smoking, duration, age at starting, and dose were all directly associated with all histologic types of lung cancer, although the OR was lower for adenocarcinoma than for other cell types. When results were restricted to ever smokers, exposure-response curves were similar across histologic types. The risk of lung cancer attributable to smoking was 88% for all types combined, 91% for squamous cell carcinoma, 89% for small cell carcinoma, 95% for large cell carcinoma, and 82% for adenocarcinoma. CONCLUSIONS: This study confirms that cigarette smoking causes all types of lung cancer, but the proportion of cases attributable to smoking is lower for adenocarcinoma than for other types, due to a higher proportion of nonsmokers.

Cohort effect of HCV infection in liver cirrhosis assessed by a 25 year study.

BACKGROUND: The role of HCV infection in the development of chronic liver disease is still unclear. OBJECTIVES: Assess the presence of HCV infection in patients with liver cirrhosis. STUDY DESIGN: 123 cases of cirrhotic liver randomly selected over a 25 years period (1969-1994) from the autopsy archives of the Pathology Department of the University of Trieste, Italy, were analyzed for the presence of HCV viral genome. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissues of the cirrhotic liver. Genotype analysis for HCV was performed after RT-PCR by dot-blot hybridization with the three major genotype-specific probes (G1, G2 and G3). RESULTS: The overall HCV genome frequency was 50.4% (62/123). The positivity was quite constant in the 1969-1979 period (35-38%), rose to 65% in 1984, peaked to 77% in 1989 (P<0.005 vs. the previous decade), and decreased to 50% in 1994. HCV genotype G1 was found in 89% of the 62 positive samples. The mean age of death of HCV-positive and HCV-negative patients was comparable (69+/-12 vs. 67+/-16 years, NS). CONCLUSIONS: These data show an increasing frequency of HCV infection in cirrhotic liver tissues from 1969 to 1994, which peaked in 1989. The genotype G1 was the almost uniquely associated with cirrhosis. These findings indicate that the HCV infection occurred around the late 1950s-early 1960s, thus supporting the hypothesis of a cohort effect. HCV infection seems not to alter the natural history of liver cirrhosis as indicated by the comparable age at death of HCV positive and HCV negative patients.

Molecular characterization of intraductal breast carcinomas.

In situ duct carcinoma (DCIS) is a heterogeneous group of lesions which has recently been subdivided into three types: well-differentiated (type I), intermediately differentiated (type II) and poorly differentiated (type III) DCIS. Fourteen cases of DCIS and 11 of DCIS with minimal invasion were analysed for mRNA levels of beta-actin, EGFR, c-cerbB2, MTS1, k-ras, RB, BRCA1, cyclin E, and c-myc genes. A microdissection technique was used on paraffin-embedded tissue. A statistically significantly higher expression of cyclin E oncogene and MTS1 tumor suppressor gene was seen in type III DCIS than in the other types, while no significant differences in the mRNA expression patterns of the other genes were observed. These data are consistent with the fact that poorly differentiated DCIS is a readily recognizable class of tumours that have a particularly aggressive behaviour and probably unique histogenesis.

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.

Causes of death and clinical diagnostic errors in extreme aged hospitalized people: a retrospective clinical-necropsy survey.

BACKGROUND: There are little data on causes of death in extreme aged. We compared, using autopsy findings, main cause of death, overall disease status, and accuracy rate of clinical diagnoses in extreme aged and persons dying at younger ages. METHODS: We reviewed the complete clinical and autopsy records of 114 consecutive inpatients (97 women, 17 men, age range 97-106, mean 99, median 98) who died in Trieste, Italy, and represented 99% of all extreme-aged person deaths in the hospital and 70% in the area. The control group included 151 patients (66 women, 85 men, age range 65-74, mean 70, median 70) who died during the same period in that hospital. RESULTS: Vascular and respiratory diseases together caused 84% of deaths in extreme aged. The main causes of death were pneumonia (n = 40, 35%), pulmonary embolism (n = 16, 14%), stroke (n = 12, 11%), and myocardial infarction (n = 8, 7%). Cancer was responsible for 6% (7/114) of deaths in extreme aged and 42% (64/151) in the control group. In 5% of extreme aged, autopsy findings did not explain death. The premortem diagnostic accuracy rate for clinical diagnoses was good in 44% of extreme aged, sufficient in 18%, poor in 28%, and not evaluable in 10%, and was significantly different from controls. Pneumonia, pulmonary embolism, and myocardial infarction were markedly underestimated by clinicians in both groups. CONCLUSIONS: Extreme aged die mainly of cardiovascular and respiratory diseases and, in most cases, of acute events. Senescence is a rare cause of death. Death from cancer is substantially lower than in persons dying at younger ages. In contrast to no autopsy studies, most extreme aged in our study were found to have specific diseases that explained their deaths.

Cancer in the frail patient. A coming epidemic.

With the aging of the population, frailty has emerged as a new clinical entity. The frail person has exhausted any functional reserve. Current criteria for the recognition of frailty include age of over 85 years, dependence in one or more activities of daily living, three or more comorbid conditions, and the presence of one or more geriatric syndromes. It is calculated that there are approximately 6 million frail patients in the United States and approximately 400,000 of them have cancer. Management of cancer in the frail person is mainly comprised of palliation, which may include some forms of chemotherapy, such as navelbine, gemcitabine, or low-dose taxanes.

Cancer Incidence and Mortality in the Elderly: Profile of the Italian Problem.

In Italy, 28% of all deaths are due to cancer. Of the 15% of the population that is 65 years or older, 65% of cancer deaths occur in men and 69.5% in women. The proportional mortality and incidence of cancer in Italy varies with increasing age. All patients over 65 years are found to be at increased risk for intestinal cancers. In older men, the proportion of lung and urinary tract cancers decreases, while prostate, intestinal and gastric cancers increases. Prostate cancer predominates in men over 85 years and intestinal and gastric cancers are relatively more common than lung cancer. In women over age 74, intestinal cancer is most common. Breast, genital and lung cancers decrease rapidly with age, while stomach, liver, gallbladder and pancreatic cancers increase with age. The ratio of incidence to mortality for all cancers studied is shown to equalize with increased age, the probable result of lower hospitalization rates and less adequate cancer therapy for the elderly.

Reduced expression of CDKN2a/P16INK4a in mycosis fungoides.

Alterations in the CDKN2a gene have been demonstrated in a wide range of human tumors including hematopoietic malignancies. To verify whether altered CDKN2a expression is involved in the pathogenesis of mycosis fungoides (MF), we examined mRNA expression in 20 patients with MF by RT-PCR and dot blot hybridization. CDKN2a mRNA expression was undetectable in 5 of the 20 patients (25%), intermediate in 13 (65%) and high in 2 (10%). Immunohistochemical studies, which were performed in ten patients, revealed that in the four patients showing no mRNA, p16INK4a was expressed in <1% of neoplastic lymphocytes whereas in the four patients with an intermediate mRNA level, specific nuclear staining was present in 1-25% of tumor cells. In the two patients with high levels of CDKN2a mRNA, >25% of neoplastic lymphocytes stained positively. No direct correlation between clinicopathological and molecular findings was evident in our patients. DNA mutational analysis revealed no alterations in a total of six patients examined. Our results indicate that the lack of CDKN2a expression, as found in 25% of the patients, may have a pathogenetic role in MF even though the absence of CDKN2a mRNA was not associated with point mutations or minor gene deletions.

Cancer of the oldest old. What we have learned from autopsy studies.

Cancer in the oldest old is a novel problem, due to the recent burgeoning of the population aged 85 and older. This article addresses three critical questions related to cancer in the oldest old: Does the incidence of cancer increase after age 95? Is cancer a common cause of death for the oldest old? Is cancer accurately diagnosed in the oldest old? The authors analyzed a group of 507 autopsies of elderly, divided in three age groups, 75 90 years, 95 99, and over 99 (centenarians). The prevalence of cancer was 35% among the younger persons and 20% and 16% respectively for those aged 95 99 and for the centenarians. A fourth of the patients in the younger group died from cancer but only 9.5% of the people between 95 and 98 years and 7.1% of the centenarians died from cancer. The cancer was the direct cause of death for 67% of the younger persons and 41% of patients belonging to the two oldest groups. The prevalence of metastases was 63% for tumors occurring in persons aged 75 90, 32% in persons aged 95 98, and 29% in the centenarians. Cancer had been accurately diagnosed prior to death in 67.4% of persons aged 75 90, in 38.5% of those aged 95 99, and 29.4% of the centenarian. Cancer as cause of death had been underestimated in 16% of the cases in the younger persons and in almost 50% of cases of the oldest old. This study suggests that the incidence of cancer and the importance of cancer as a cause of death may decline after age 95 and that the clinical diagnoses underestimate significantly both the incidence of cancer and the prevalence of cancer deaths in the oldest old.

Invasive cancer of the uterine cervix in the Province of Trieste: epidemiological considerations.

The epidemiological behaviour of the invasive cancer of the uterine cervix in the Province of Trieste is analysed. During this study period, 488 cases of this neoplasia were diagnosed with an incidence rate standardized for the European population of 20.25 degrees/0000. This value is within the average ones recorded in other geographic regions. The age group which is most affected is that from 60 to 64 years. By analyzing the overall incidence rate of the first two years if compared with the last two years of our study period, a 29% decrease is recorded, with an annual average decrease of 2.6%. This value is one of the highest reported in the literature. This decrease is recorded in the age groups over 40, while no decrease is recorded in women under age 40.