Phosphorylated mTOR is associated to androgen receptor expression in early triple-negative breast cancer.

The significance of phosphorylated mTOR (p-mTOR) expression is unknown in triple-negative breast carcinoma (TNBC). The aims of the present study were to assess the expression of p-mTOR in early TNBC and to evaluate possible correlations between androgen receptor (AR) expression, clinicopathological parameters and disease outcome. Between January 2009 and December 2013, all consecutive patients who were diagnosed and completed the treatment of invasive TNBC at our institution were eligible for this analysis. Patients with stage IV disease were excluded. The evaluation of p-mTOR immunohistochemical staining was semi-quantitatively considering both the percentage of positive tumor cells (range, 0-100%) and staining intensity (range, 0-3+). Ninety-eight TNBC patients were included. Approximately 33% of cases were p-mTOR positive and there was no association between positive immunostaining for p-mTOR and DFS (p=0.74) and OS (p=0.81). p-mTOR positivity was associated with small tumor size (p=0.03) and AR expression (p=0.04). High expression of p-mTOR may drive tumor proliferation in almost one third of TNBC. The biological association between mTOR activation and AR pathway suggests that there may exist a subgroup of TNBC in which the combination of both AR antagonism and mTOR inhibition should have a synergistic effect on cell growth and tumor progression.

Performance of BOADICEA and BRCAPRO genetic models and of empirical criteria based on cancer family history for predicting BRCA mutation carrier probabilities: a retrospective study in a sample of Italian cancer genetics clinics.

PURPOSE: To evaluate in current practice the performance of BOADICEA and BRCAPRO risk models and empirical criteria based on cancer family history for the selection of individuals for BRCA genetic testing. PATIENTS AND METHODS: The probability of BRCA mutation according to the three tools was retrospectively estimated in 918 index cases consecutively undergone BRCA testing at 15 Italian cancer genetics clinics between 2006 and 2008. RESULTS: 179 of 918 cases (19.5%) carried BRCA mutations. With the strict use of the criteria based on cancer family history 173 BRCA (21.9%) mutations would have been detected in 789 individuals. At the commonly used 10% threshold of BRCA mutation carrier probability, the genetic models showed a similar performance [PPV (38% and 37%), sensitivity (76% and 77%) and specificity (70% and 69%)]. Their strict use would have avoided around 60% of the tests but would have missed approximately 1 every 4 carriers. CONCLUSION: Our data highlight the complexity of BRCA testing referral in routine practice and question the strict use of genetic models for BRCA risk assessment.

An intronic mutation in MLH1 associated with familial colon and breast cancer.

Single base substitutions can lead to missense mutations, silent mutations or intronic mutations, whose significance is uncertain. Aberrant splicing can occur due to mutations that disrupt or create canonical splice sites or splicing regulatory sequences. The assessment of their pathogenic role may be difficult, and is further complicated by the phenomenon of alternative splicing. We describe an HNPCC patient, with early-onset colorectal cancer and a strong family history of colorectal and breast tumors, who harbours a germ line MLH1 intronic variant (IVS9 c.790 +4A>T). The proband, together with 2 relatives affected by colorectal-cancer and 1 by breast cancer, have been investigated for microsatellite instability, immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification. The effect of the intronic variant was analyzed both by splicing prediction software and by hybrid minigene splicing assay. In this family, we found a novel MLH1 germline intronic variant (IVS9 c.790 +4A>T) in intron 9, consisting of an A to T transversion, in position +4 of the splice donor site of MLH1. The mutation is associated with the lack of expression of the MLH1 protein and MSI in tumour tissues. Furthermore, our results suggest that this substitution leads to a complete skip of both exon 9 and 10 of the mutant allele. Our findings suggest that this intronic variant plays a pathogenic role.

Molecular targets in Gastrointestinal Stromal Tumors (GIST) therapy.

Gastrointestinal Stromal Tumors (GISTs) are the most common mesenchimal tumors of the gastrointestinal tract. Such tumors usually have activating mutations in either KIT (75-80%) or Platelet Derived Growth Factor Receptor alpha (PDGFRa) (5-10%) which lead to ligand-independent signal transduction. Targeting these activated proteins with Imatinib mesylate, a small-molecule kinase inhibitor, has proven useful in the treatment of recurrent or metastatic GISTs. However, more than half of patients develop resistance to Imatinib after about 2 years. Therefore, other targets have been studying in order to implement the therapeutical armamentarium for this disease. Sunitinib malate is an oral multikinase inhibitor that targets several receptor tyrosine kinases and has proved to prolong survival in Imatinib-resistant patients. Other molecules, such as Nilotinib, Sorafenib and Dasatinib were shown to be useful in Imatinib resistant mutant cell lines and the results of their activity in humans are being awaited. Recent evidence suggests that GIST cells acquire the capability to escape from the control of KIT and PDGFRa through the activation of alternative pathways. Therefore, further effort should be invested in the discovery of new signaling pathways, such as AXL, MET, IGF-R, which might be involved in the evolution of the disease. After a description of KIT and PDGFRa as known targets of anti-GIST treatments, we review other mechanisms and mediators that might be potential targets of new therapies, providing a comprehensive revision of the new molecular strategies under investigation.

Insulin-like growth factor (IGF) 1 and 2 help to predict disease outcome in GIST patients.

BACKGROUND: The expression of the insulin-like growth factor (IGF) system has never been studied in gastrointestinal stromal tumors (GISTs). PATIENTS AND METHODS: We studied the immunohistochemical expression of IGF1 receptor (IGFR-I), IGF1 and IGF2 in 94 samples of GISTs. IGF1 and IGF2 expression was scored in three classes: negative (N), moderate (M) and strong (S), according to staining intensity and extent. RESULTS: IGFR-I was overexpressed in all cases. IGF1 and IGF2 expression was absent in 25 and 48 cases, moderate in 29 and 16 cases and strong in 40 and 30 cases, respectively. Strong IGF1 expression significantly correlated with higher mitotic index (P = 0.0001), larger (P = 0.01), higher risk (P = 0.0002), metastatic (P = 0.0001) and relapsed (P = 0.04) GISTs. Strong IGF2 expression correlated with higher mitotic index (P = 0.05) and higher risk GISTs (P = 0.001). The Kaplan-Meier analysis (N versus M versus S) showed a significant worsening of the disease-free survival (DFS) with the increase of IGF1 (P = 0.02) and IGF2 (P = 0.02) expression. In the subgroup of patients with operated high-risk GISTs, there was a better trend in DFS for patients affected by GISTs with negative IGF1 and IGF2. CONCLUSIONS: The expression of IGF1 and IGF2 seems to predict relapse in GIST patients.

KIT and PDGFRalpha mutations in 104 patients with gastrointestinal stromal tumors (GISTs): a population-based study.

BACKGROUND: The prognostic significance of KIT or platelet-derived growth factor receptor alpha (PDGFRalpha) mutations in gastrointestinal stromal tumors (GISTs) is still controversial. PATIENTS AND METHODS: In all, 104 patients were diagnosed with GISTs by KIT immunoreactivity; tumor DNA was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17 and in PDGFRalpha exons 12 and 18. Disease-free survival (DFS) was analyzed in 85 radically resected patients. RESULTS: KIT mutations occurred in exon 11 (69), in exon 9 (11) and in exon 17 (1). PDGFRalpha mutations were detected in exon 18 (10) and in exon 12 (3). Ten GISTs were wild type. Exon 11 mutations were as follows: deletions in 42 cases and point mutations in 20 cases and insertions and duplications, respectively, in 2 and 5 cases. A better trend in DFS was evident for duplicated and point-mutated exon 11 KIT GISTs. There was a significant association between PDGFRalpha mutations, gastric location and lower mitotic index. Moreover, PDGFRalpha-mutated GISTs seemed to have a better outcome. CONCLUSIONS: Point mutations and duplications in KIT exon 11 are associated with a better clinical trend in DFS. PDGFRalpha-mutated GISTs are preferentially localized in the stomach and seem to have a favorable clinical behavior.

Information to cancer patients: a questionnaire survey in three different geographical areas in Italy.

GOALS OF WORK: The way to inform cancer patients varies widely among different countries. In most Mediterranean countries, the traditional approach has been of partial disclosure of the truth. The intent of our work was to find if the attitude in Italy has changed in recent years and if differences still exist among different geographical regions in our country. Both patients' and physicians' attitudes vary in Italy depending on geographical area. In the South, the focus is on traditional values and full involvement of patients' family, with limited communication to (and limited autonomy of) the patient. Such attitude is less pronounced in Central Italy, whereas the North is more oriented to open communication and full decisional autonomy of the patient. MATERIALS AND METHODS: Approximately 600 consecutive patients in three different centres in Northern, Central and Southern Italy (respectively, Udine, Ancona and Catanzaro) were asked to answer a 26-item questionnaire on communication aspects to investigate the quality of the information given and potential differences between geographical areas. RESULTS: Questionnaires were completed by 587 patients (median age 60 years, 57% women), mainly with gastrointestinal (32%) or breast cancer (30%). About 370 patients (63%) had active disease at time of interview. A high proportion of patients were correctly informed on diagnosis (86%) and therapy (84%). On the contrary, patients fully aware of their prognosis were only about 43%. Nevertheless, most patients (60%) stated they were completely satisfied with the information received. There were differences between geographical areas on various information aspects, with patients from Southern Italy being, in general, less informed. CONCLUSIONS: In Italy, the cultural attitude towards communication in oncology is changing on both the physician and the patient side. There are still significant geographical differences, but there is a general trend suggesting improved awareness about diagnosis and treatment, with the notable exception of prognosis.

Effectiveness of the CRCAPRO program in identifying patients suspected for HNPCC.

Subjects affected by hereditary non-polyposis colorectal cancer exhibit a high susceptibility to colon and extracolonic tumours, due to MMR gene defects. Revised Bethesda criteria are used to select patients as candidates for genetic tests. Recently, the CRCAPRO model has been developed, based on family history of colorectal and endometrial cancers. Our study aims to evaluate the reliability of CRCAPRO in identifying mutation carriers. We used the CRCAPRO program to evaluate carrier probability risk in 99 patients fulfilling Amsterdam or Bethesda guidelines. MLH1 and MSH2 were studied by direct sequencing in all the 99 patients, and the study of microsatellite instability and of MMR proteins expression was performed. Nine MLH1 and nine MSH2 germline mutations were identified. Five out of the nine patients with MLH1 mutation showed a CRCAPRO risk evaluation of less than 20%. The same happened for four out of nine patients with MSH2 mutation. Of the 17 patients with an estimated risk of more than 80%, only four harboured a mutation, all in the MSH2 gene. The highest risk calculated by the CRCAPRO system in the nine carriers of a MLH1 mutation has been 31.7%. In our experience, the CRCAPRO program sensitivity and specificity appears to be low but needs to be further evaluated in larger samples.

[Clinical guidelines for the management of gastrointestinal stromal tumors]. / Raccomandazioni cliniche per la diagnosi, la terapia ed il follow-up dei tumori stromali gastrointestinali.

Treatment of gastrointestinal stromal tumors (GIST) has been revolutioned by the recently discovered molecular mechanism responsible for the oncogenesis of this disease. In addition, due to the rapid progress at molecular and clinical level observed in the last few years, there is a need to review the current state of the art in order to delineate appropriate guidelines for the optimal management of these tumors. A panel of experts from several specialities, including medical oncology, surgery, pathology, molecular biology and imaging, were invited to participate in a meeting to present and discuss a number of pre-selected questions, and to achieve a consensus according to the categories of the National Comprehensive Cancer Network (NCCN) and the Standard Options Recommandations (SOR) of the French Federation of Cancer Centers. Generally, consensus points were from categories 2A of the NCCN and B2 of the SOR. Conventional histologic examination with immunohistochemistry for CD117, CD34, SMA, S-100 and desmin is considered standard. Molecular analysis for the identification of KIT and PDGFRA mutation may be indicated in CD117-negative GIST. Complete tumor resection with negative margins is the optimal surgical treatment. Adjuvant imatinib should be considered an experimental approach. Neoadjuvant imatinib is also experimental, although its use may be justified in unresectable or marginally resectable GIST. Imatinib should be started in metastatic or recurrent disease, and should be continued until progressive disease or drug intolerance. In these cases, sunitinib can be used. The optimal criteria for the assessment and monitoring of GIST undergoing imatinib therapy are not well known, but they should include reduction in tumor size and disease stabilization, as well as reduction of tumor density on CT scan and metabolic activity on PET scan.

MSH2 splice site mutation and endometrial cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited syndrome of cancer susceptibility caused by germ line mutations of genes participating in mismatch repair (MMR). Carriers of MMR gene mutations have an increased risk of colorectal cancers and cancer of other organs. Tumors of the endometrium represent the most frequent extracolonic malignancies in HNPCC. It has been suggested that women harboring MMR gene mutations have a higher risk of endometrial cancer than of colon cancer. Here, we describe an HNPCC patient with early-onset endometrial cancer and a strong familial history of endometrial tumors who harbored a germ line MSH2 splice site mutation (IVS9_2A>G). This mutation was responsible for abnormal messenger RNA processing, leading to the introduction of a premature stop signal and to the expression of a truncated MSH2 protein. In addition, the same mutation was associated with loss of MSH2 protein expression, high microsatellite instability, and PTEN inactivation. Although a direct relationship between the endometrial cancer susceptibility and the MSH2 mutation we found cannot be established, our observations, consistent with the work of other authors, suggest the involvement of germ line MSH2 abnormalities in endometrial tumor development and support the case for endometrial cancer screening in women from HNPCC families.

Oxidant injury in neonatal erythrocytes during the perinatal period.

UNLABELLED: It has been known for many decades that oxidative stress leads to oxidation of hemoglobin and damage to the erythrocyte membrane. More recently, the factors involved in denaturating of membrane proteins and lipid peroxidation have been investigated in detail, as well as the mechanism of reactive oxygen species formation in red cells. Oxidative stress depletes adenosine triphosphate (ATP) and adenine nucleotides, whereas adenosine monophosphate (AMP) deaminase seems to depress energy metabolism by blocking the salvage pathway of purine nucleotides. Depletion of ATP and activation of AMP deaminase are related to calcium ion concentrations. Denaturating of membrane proteins generally precedes lipid peroxidation and consequent phagocytosis due to caspase activation. Extensive investigations demonstrated the key role of oxidative stress and iron release in a reactive form causing membrane protein damage via the Fenton reaction and hydroxyl radical production. In the absence of efficient protection by antioxidant factors and other molecules such as flavonoids, oxidative stress is responsible for the release of iron in reactive form, predisposing red cells to hemolysis through the formation of senescence antigen. Other well-known sources of oxidative stress in red cells are free radical production outside the red cell by activated phagocytes, endothelial metabolism, hyperoxia, ischemia-reperfusion and the arachidonic acid cascade. CONCLUSION: The recent insight into the mechanism of oxidative injury of red cells and evidence of relationships between erythrocyte oxidative stress and hypoxia suggest that increased hemolysis is induced by severe hypoxia and acidosis in the fetus as well as the newborn.

New biomarkers of fetal-neonatal hypoxic stress.

UNLABELLED: The complex pathophysiological mechanisms underlying perinatal hypoxia make it difficult to define early markers of severe hypoxia-ischemia encephalopathy. However, as progress in the development of neuroprotective therapeutic measures continues, the early identification of neonates at risk of severe hypoxic-ischemic encephalopathy is an important goal for appropriate decision making. Although the timing of perinatal hypoxic brain damage may vary and is sometimes unknown, high levels of non-protein-bound iron and high nucleated red blood cell counts in cord blood indicate an antepartum origin of neurological impairment, because they can occur only as a consequence of a pre-existing asphyxic event. CONCLUSION: The combined assessment of nucleated red blood cells and non-protein-bound iron at birth seems extremely useful for the early identification of newborns at high risk of brain damage. Activin A also seems to be a reliable marker of perinatal hypoxia. Prospective long-term follow-up studies are needed to verify their predictive role.

Free radicals and brain damage in the newborn.

Newborns and particularly preterm infants are at high risk of oxidative stress and they are very susceptible to free radical oxidative damage. Indeed, there is evidence of an imbalance between antioxidant- and oxidant-generating systems which causes oxidative damage. The brain may be especially at risk of free radical-mediated injury because neuronal membranes are rich in polyunsaturated fatty acids and because the human newborn has a relative deficiency of brain superoxide dismutase and glutathione peroxidase. The brain of the term fetus is at higher risk of oxidative stress than that of the preterm fetus, as a consequence of its higher concentration of polyunsaturated fatty acids and the maturity of the N-methyl-D-aspartate receptor system at term. There seems to be a maturation-dependent window of vulnerability to free radical attack during oligodendrocyte development. Early in its differentiation, the oligodendrocyte may be vulnerable because of active acquisition of iron for differentiation at a time of relative delay in the development of certain key antioxidant defenses in the brain. Excess free iron and deficient iron-binding and -metabolizing capacity are additional features favoring oxidant stress in premature infants. Free radicals may be generated by different mechanisms, such as ischemia-reperfusion, neutrophil and macrophage activation, Fenton chemistry, endothelial cell xanthine oxidase, free fatty acid and prostaglandin metabolism and hypoxia. Reactive oxidant production by these different mechanisms contributes in a piecewise manner to the pathogenesis of perinatal brain injury.

Red blood cell involvement in fetal/neonatal hypoxia.

Free radical release plays an important role in the development of brain injury following hypoxic-ischemic encephalopathy. It causes endothelial cell damage and anomalies in NMDA receptors, synaptosome structure and astrocyte function. Mitochondrial dysfunctions caused by asphyxia, reperfusion after ischemia, arachidonic acid cascade, catecholamine metabolism and phagocyte activation are known sources of reactive oxygen species, particularly the superoxide anion (O2(-)). O2(-) mainly induces peroxidation by the Fenton/Haber Weiss reaction or via iron-oxygen complexes. Since both reactions require reactive heavy metals, non-protein-bound iron (NPBI) is essential for the induction of lipid peroxidation. Experimental studies have demonstrated the neurotoxicity of iron in ischemia-reperfusion. Normal axonal transport of brain iron is also reported to be disrupted in hypoxia-ischemia, leading to a buildup of iron in the white matter. The free iron content of erythrocytes (ICRBC) is considered a marker of oxidative stress. Free iron release is accompanied by the oxidation of membrane proteins and the appearance of senescent antigen, as measured by autologous IgG binding. Our preliminary results suggest a significant positive correlation between plasma free iron and the number of nucleated red cells in cord blood, currently considered a reliable index of lasting intrauterine asphyxia but also possessing a high predictive value for poor neurodevelopmental outcome. The rate of erythropoiesis and the entity of ICRBC are related to the degree of asphyxia and the probability of neurological impairment. Since even an increase in NPBI during asphyxia is related to a poor outcome, iron released by red cells could possibly also contribute to NPBI levels.

Early neonatal brain injury in histologic chorioamnionitis.

The relation between clinical or histologic chorioamnionitis and early neonatal adverse neurologic outcome was investigated (n = 483). Histologic, but not clinical, evidence of chorioamnionitis was found to be a significant predictor of periventricular echodensity (odds ratio, 2.4; 95% CI, 1.8-3.2), echolucency (3.3; 1.9-5.6), ventriculomegaly (2.7; 1.8-4.2), intraventricular hemorrhage > or =3 (3.5; 2.4-5.2), and seizures (2.3; 1.4-3.7).

Cisplatin, epirubicin and cyclophosphamide (PEC) in the treatment of advanced ovarian cancer.

We report the long-term results of a series of patients affected by advanced epithelial ovarian cancer treated with the PEC combination (cisplatin 60 mg/m2, epirubicin 60 mg/m2 and cyclophosphamide 750 mg/m2, all at day 1, every 21 days). Response was evaluated after three cycles, and treatment continued in responsive patients. A total of 80 patients with a median follow-up of 55 months were studied. Fifty-eight patients with stage III ovarian cancer and 22 patients with stage IV received PEC as primary treatment (41 patients), or for residual disease after surgery (37 patients), or for relapsed disease after primary surgery (2 patients). The overall response rate was 67.5% (20.0% complete response, 47.5% partial response), with 22.5% stable disease and 3.7% progressive disease. Median progression free survival was 13.0 months, and median survival was 25 months. Grade III-IV toxicity was moderate: leukopenia 20.0% of patients, thrombocytopenia 5.0%, anemia 16.2%. No cardiac toxicity was observed. In conclusion, the PEC combination, an anthracycline-containing platinum-based regimen, proved to be effective in advanced ovarian cancer, in terms of response rate and overall survival. The regimen was devoid of significant toxicity and in particular of cardiac toxicity.

Total hydroperoxide and advanced oxidation protein products in preterm hypoxic babies.

Previous studies have shown that plasma lipoproteins are a common target of free radical-induced oxidative stress in hypoxic newborn infants. In contrast to lipids, the reaction of proteins with various oxidants during hypoxia has not been extensively studied. We tested the hypothesis that tissue hypoxia results in increased production of protein oxidation in cord blood of preterm newborns. Heparinized blood samples of 39 hypoxic and 16 control preterm newborns were obtained from the umbilical vein, after cord clamping immediately after delivery. Plasma levels of total hydroperoxide (TH), advanced oxidation protein products (AOPP), hypoxanthine (Hx), xanthine (Xa), and uric acid (UA) were measured. Higher Hx, Xa, UA, TH, and AOPP levels were found in hypoxic newborn infants than in controls. Statistically significant correlations were observed between: TH and Hx (r = 0.54, p = 0.003, n = 28), AOPP and Hx (r = 0.64, p = 0.0001, n = 27), and TH and AOPP plasma levels (r = 0.50, p = 0.02, n = 21). In summary, TH, AOPP, Hx, Xa, and UA production is increased in fetal blood during hypoxia. The more severe the hypoxia, the higher the lipid and protein damage by free radicals.

Nucleated red blood cell count at birth as an index of perinatal brain damage.

OBJECTIVE: The prognostic value of the nucleated red blood cell count at birth with respect to perinatal brain damage and neonatal outcome was assessed in infants at high risk of having neurologic damage. STUDY DESIGN: The nucleated red blood cell count at birth, pulsed Doppler ultrasonography in the cerebral arteries, cranial fontanelle sonograms, and neurodevelopmental status were evaluated in 337 newborn infants. RESULTS: The nucleated red blood cell count at birth was significantly higher (1) in neonates with abnormal Doppler ultrasonographic parameters for the cerebral arteries at 48 to 72 hours after birth than in healthy neonates, (2) in 6-month-old infants with sequelae of hypoxic-ischemic encephalopathy than in healthy infants, and (3) in 3-year-old children with abnormal developmental status than in those with no abnormalities at follow-up. Significant correlations were observed between the nucleated red blood cell count and gestational age, Apgar score at 1 and 5 minutes, pH, base deficit, fraction of inspired oxygen, blood oxygen content, and birth weight. CONCLUSIONS: The nucleated red blood cell count at birth not only reflects a response of the infant to perinatal hypoxia but is also a reliable index of perinatal brain damage.