In vitro efficacy of ARQ 092, an allosteric AKT inhibitor, on primary fibroblast cells derived from patients with PIK3CA-related overgrowth spectrum (PROS).

Postzygotic mutations of the PIK3CA [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha] gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the "clean" cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples (i.e., primary fibroblasts) were derived from cultured tissues obtained from six PROS patients [3 boys, 3 girls; aged 2 to 17 years] whose spectrum of PIK3A-related overgrowth included HHML [hemihyperplasia multiple lipomatosis; n = 1], CLOVES [congenital lipomatosis, overgrowth, vascular malformations, epidermal nevi, spinal/skeletal anomalies, scoliosis; n = 1], and MCAP [megalencephaly capillary malformation syndrome; n = 4]. We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients' derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets [pAKTS1 (Thr246), pRPS6 (Ser235/236), and pRPS6Kß1 (Ser371)]. The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors [i.e., wortmannin, LY249002, and rapamycin] in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity [ARQ 092 at 0.5, 1, and 2.5 µM blunted phosphorylation of AKT and its downstream targets (in the presence or absence of serum) and inhibited proliferation after 72 h; rapamycin at 100 nM did not decrease AKT phosphorylation] and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies.

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Nonhormonal management of postmenopausal women: effects of a red clover based isoflavones supplementation on climacteric syndrome and cardiovascular risk serum profile.

PURPOSE OF INVESTIGATION: The aim of this prospective randomized study was to evaluate a red clover based isoflavones supplementation in the treatment of climacteric syndrome and its effects on cardiovascular risk serum profile. MATERIALS AND METHODS: The study included 150 healthy postmenopausal women that were randomly assigned to receive phytoestrogens tablets, amounting in a total daily intake of 60.8 mg red clover isoflavones plus 19.2 mg soy isoflavones (n = 75), or placebo (n = 75). The authors evaluated teh following: daily number of hot flushes and Kupperman Index at baseline and after one and three months; serum total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, and antithrombin III (ATIII) at baseline and after three and six months. RESULTS: One hundred twenty-eight patients completed the study: 67 in the active group and 61 in the placebo group. The treatment led to a progressive significant reduction (p < 0.05) of the number of hot flushes in the active group compared to placebo already after one month, while Kupperman Index was statistically reduced after three months. No significant variation in total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, PT, PTT, fibrinogen, and ATIII were found. CONCLUSION: The present findings suggest that a red clover based isoflavones supplementation in healthy postmenopausal women is promptly effective on climacteric syndrome, improves neurovegetative symptoms, safe on cardiovascular risk serum profile, and does not modify lipids and coagulation.

Low-dose estrogen and drospirenone combination: effects on metabolism and endothelial function in postmenopausal women with metabolic syndrome.

PURPOSE: Metabolic syndrome and endothelial dysfunction play a relevant role in the cardiovascular risk in post-menopause. The aim of the study was to assess the effects of a low-dose hemihydrate estradiol and drospirenone combination on cardiovascular risk parameters in postmenopausal women with metabolic syndrome. MATERIALS AND METHODS: Twenty-eight healthy women (group A) and 28 women with metabolic syndrome (group B) were treated with hemihydrate estradiol one mg + drospirenone two mg. At recruitment and after six months, clinical and laboratory parameters of metabolic syndrome were evaluated. Endothelial function was assessed measuring the flow-mediated dilatation of the brachial artery and the intima-media thickness of the common carotid artery. RESULTS: After six months an overall improvement of metabolism was observed in both groups reaching statistical significance for triglycerides, total cholesterolemia, and systolic pressure in group B. A trend to lower baseline flow-mediated dilatation was also found in group B. CONCLUSIONS: Drospirenone improves cardiovascular risk factors and does not impair endothelial function in menopausal women with metabolic syndrome.

Arterial hypertension and female sexual dysfunction in postmenopausal women.

PURPOSE: To evaluate female sexual dysfunction in hypertensive postmenopausal women and the effects of antihypertensive therapy. MATERIALS AND METHODS: Female sexual dysfunction was assessed by the Female Sexual Function Index (FSFI) in three groups of postmenopausal patients: normotensive women (group A: 240 women), hypertensive women without therapy (group B: 220 women), hypertensive women on therapy (group C: 80 women). RESULTS: The incidence of female sexual dysfunction was increased in group B compared to groups A and C. Healthy patients showed higher FSFI scores compared to hypertensive patients (groups B and C). Hypertensive-treated patients accounted for higher scores in all items compared to hypertensive patients without therapy. CONCLUSIONS: Essential hypertension significantly affects female sexual function. Physicians should recognize and properly manage FSD in hypertensive women.

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets.

Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.

Anti-angiogenic activity of carebastine: a plausible mechanism affecting airway remodelling.

Ebastine is a well-known selective second-generation histamine H(1) receptor antagonist, which is used for seasonal and perennial allergic rhinitis and chronic urticaria. Angiogenesis plays a crucial role in the development of airway inflammation and remodelling in allergic rhinitis and asthmatic patients, in whom, indeed, the mucosa displays increased vascularity and overexpression of vascular endothelial growth factor (VEGF). The aim of the present study was to evaluate the anti-angiogenic properties of carebastine, the active metabolite of ebastine. The effects of carebastine on human umbilical vein endothelial cell (EC) (HUVEC) and human pulmonary artery EC (HPAEC) proliferation, migration and capillary-like tube formation were investigated in vitro, and in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Moreover, the effect of carebastine on phosphorylation of the cell VEGF receptor fetal liver kinase-1, or VEGF receptor 2 (VEGFR-2), and Akt kinase (Akt) was evaluated by Western blotting. Carebastine inhibited VEGF-induced HUVEC and HPAEC proliferation, migration and angiogenesis in a dose-dependent manner in vitro. Cell proliferation was inhibited by 42 and 64% in HUVECs and 62 and 75% in HPAECs upon exposure for 48 and 72 h, respectively, to 20 microM carebastine (p < or = 0.03), and even more with 30 microM carebastine. Cell migration was inhibited by 37 and 70% in HUVECs (p < or = 0.03) and 60 and 78% in HPAECs (p < or = 0.01) in the presence of 10 and 30 microM carebastine, respectively. Carebastine (20 microM) caused a significant reduction (70-86%; p<0.01) in topological parameters of the capillary network produced in vitro by both EC lines on a basement membrane extract. Carebastine (30 and 50 microM) inhibited the VEGF-induced angiogenesis in the CAM assay in vivo two- and three-fold, respectively (p<0.001). Finally, both EC lines, on exposure to 10 and 20 microM carebastine, showed a four- to six-fold reduction (p < or = 0.01) in both VEGF- and H1 receptor-induced VEGFR-2 and Akt phosphorylation. Overall, these data provide the first evidence regarding the anti-angiogenic activity of ebastine, and suggest its potential use as an anti-angiogenic molecule, besides its antihistaminic activity for the treatment of allergic diseases in which angiogenesis takes place.

Cell proliferation and outcome following doxorubicin plus CMF regimens in node-positive breast cancer.

At the Istituto Nazionale Tumori of Milan, a randomised adjuvant chemotherapy trial was carried out from 1982 to 1990 to compare alternating with sequential regimens of doxorubicin and CMF in 403 patients with more than 3 positive axillary nodes. Tumour proliferative activity was determined in 71% (285 cases) of women entering the clinical study. We investigated the relation between proliferative rate, determined as the [(3)H]thymidine labelling index (TLI) on tumour specimens obtained at diagnostic surgery, and clinical outcome following the 2 regimens, in which the same drugs were administered at the same dose intensity but with a different schedule. A high TLI was significantly associated with 12-year overall relapse (P = 0.009), distant metastasis (P = 0.001), and death (P = 0.002), even in the presence of information provided by tumour size, lymph node involvement, oestrogen receptors, and treatment regimen. The highest relapse-free survival (RFS) probability (45%, 95% CI 34-55%) was observed for patients with tumour TLI <5% and subjected to the sequential treatment. The lowest RFS probability (11%, 95% CI 0-26%) was observed for patients with tumour TLI >9% following the alternating regimen. Intermediate RFS probabilities, ranging from 23% to 34%, were observed for the other kinetic subgroups following the 2 treatment regimens. The benefit of sequential administration of doxorubicin and CMF was evident mainly in patients with tumours at low to intermediate proliferation.

Biomarkers and outcome after tamoxifen treatment in node-positive breast cancers from elderly women.

The predictive role of tumour proliferative rate and expression of p53, bcl-2 and bax proteins, alone and in association with tumour size, nodal involvement and oestrogen receptors (ER), was analysed on 145 elderly patients (> or =70 years of age) with histologically assessed node-positive breast cancers treated with radical or conservative surgery plus radiotherapy followed by adjuvant tamoxifen for at least 1 year. The 7-year probability of relapse was significantly higher for patients with tumours rapidly proliferating (hazard ratio (HR) = 2.0, P = 0.01), overexpressing p53 (HR = 4.4, P = 0.0001), weakly or not exhibiting bcl-2 (HR = 1.9, P = 0.02), without ERs (HR = 3.4, P = 0.0001) or with > or = 4 positive lymph nodes (HR = 2.3, P = 0.003) than for patients with tumours expressing the opposite patho-biological profile. Conversely, tumour size and bax expression failed to influence relapse-free survival. Adjustment for the duration of tamoxifen treatment did not change these findings. Oestrogen receptors, cell proliferation, p53 accumulation and bcl-2 expression were also predictive for overall survival. Within ER-positive tumours, cell proliferation, p53 accumulation, bcl-2 expression and lymph node involvement provided significant and independent information for relapse and, in association, identified subgroups of patients with relapse probabilities of 20% (low-risk group, exhibiting only one unfavourable factor) to 90% (high-risk group, exhibiting three unfavourable factors). Such data could represent the initial framework for a biologically tailored therapy even for elderly patients and highlight the importance of a patho-biological characterization of their breast cancers.

Clinical studies of Bcl-2 and treatment benefit in breast cancer patients.

Interest in translational studies aimed at investigating the role of biologic markers in predicting clinical outcome of breast cancer patients and, in particular, response to specific treatments, has progressively increased. Among biologic variables presently under investigation, apoptosis markers, in particular Bcl-2 and Bax expression, are receiving much attention for their relationship with the cellular response to genotoxic damage in experimental tumors. Retrospective, independent studies were carried out by several research groups on about 5000 patients with breast cancer at different stages and with an adequate follow-up. The outcome of separate analyses as a function of treatment generally demonstrated that Bcl-2 overexpression, which correlates with biologic features of a differentiated phenotype (slow proliferation, high steroid receptor levels, absence of p53 and c-erB-2 expression), is associated with a favorable outcome. Such a finding is mainly evident following surgery as well as endocrine treatment. Conversely, no or weak Bcl-2 expression, alone or in association with bax overexpression, appears indicative of a radiation response, and preliminary emerging evidence supports the involvement of such an association of apoptosis-related markers even as predictors of long-term response to neoadjuvant cytotoxic treatment. Although the findings of an involvement of Bcl-2 and Bax as determinants of treatment response should be confirmed within the context of randomized clinical trials, they indicate a combined consideration of proteins that negatively and positively regulate apoptosis in translational studies on the effect of chemical and physical agents at a cellular level.

Thrombolysis in acute ischemic stroke.

The administration of rt-PA to patients with acute ischemic stroke can result in improved functional outcomes. The safe and effective use of rt-PA in routine medical practice requires that patients seek help early, have a well-defined onset of their symptoms, be carefully examined for contraindications to rt-PA, receive a CT scan and interpretation to exclude hemorrhage, and receive the drug within a 3-hour period (Table 5). Intravenous rt-PA is given in a dosage of 0.9 mg/kg (up to a maximum of 90 mg) with 10 percent of the dose administered as a bolus followed by a 60-minute infusion within 3 hours of the onset of symptoms. If these conditions cannot be achieved, the drug should not be administered. Although most patients will not meet the criteria of the NINDS trial, rt-PA is an important advance in the treatment of acute ischemic stroke.

Expression of p53, glutathione S-transferase-pi, and Bcl-2 proteins and benefit from adjuvant radiotherapy in breast cancer.

BACKGROUND: In clinical breast cancer research, the utility of certain biomarkers as predictors of response to surgery, chemotherapy, or hormonal therapy has been studied intensively. Much less research has been done on the relevance of biologic predictors of response to radiotherapy, which represents an effective local-regional treatment for breast cancer. PURPOSE: The utility of biomarkers involved in DNA damage repair (p53 protein), control of programmed cell death (p53 and Bcl-2 proteins), and cellular detoxification (glutathione S-transferase-pi [GST-pi] enzyme) in predicting local breast cancer recurrence was analyzed retrospectively in two cohorts of breast cancer patients. These patients had had no detectable metastases in the axillary lymph nodes (i.e., node-negative) or in distant sites and had had similar distributions of clinicopathologic and biologic prognostic features. They had been treated by conservative surgery alone (139 case patients) or by conservative surgery followed by adjuvant radiotherapy (496 case patients) during the period from 1984 through 1990. METHODS: The expression of the p53, GST-pi, and Bcl-2 proteins in the specimens of primary breast tumor obtained from these patients was determined by use of immunohistochemistry; cell proliferation activity and levels of steroid receptors were determined by use of a [3H]thymidine-labeling index assay and the dextran-coated charcoal technique, respectively. The median time of follow-up of patients was 6 years. In the analyses of patient outcomes, only local failures that presented as first events were considered. RESULTS: After surgery alone, the risk of local recurrence at 6 years was higher for patients with tumors exhibiting elevated levels of p53 and GST-pi protein expression than for patients with low levels (hazard ratio [HR] = 3.1, 95% confidence interval [CI] = 1.3-7.7, two-sided P = .012; HR = 2.7, 95% CI = 1.1-6.4, two-sided P = .026, respectively). Weak or no observable expression of Bcl-2 protein was only suggestive of a higher frequency of local failures. Adjustment for patient age, tumor size, cell proliferation, and estrogen receptor status did not change these findings. Conversely, in the series of patients given conservative surgery followed by radiotherapy, there was no difference in local tumor recurrence between patients with tumors expressing or not expressing each of the three markers. CONCLUSIONS: Our study provides indirect evidence of a benefit from radiation therapy in preventing local breast cancer relapse, particularly among node-negative patients with tumors that express elevated levels of the p53 or GST-pi proteins or that express little or no Bcl-2 protein.

Cell proliferation in 3,800 node-negative breast cancers: consistency over time of biological and clinical information provided by 3H-thymidine labelling index.

For breast cancer, many prognostic markers that initially appeared promising have failed to maintain their clinical predictive value. Few reports have analyzed the consistency over time of biological and clinical information provided by biomarkers. Tumour cell proliferation has acquired relevance as an indicator of prognosis and of response to treatment. Since its clinical role has been investigated for some decades, cell proliferation represents an ideal marker for an over-time validation. In 3,800 node-negative breast cancers recruited between 1972 and 1991, the consistency of information provided by the 3H-thymidine labelling index (TLI), in terms of basic relations with other clinico-pathological and biological variables and clinical predictivity, was evaluated using a combined analysis of results previously published by our group for distinct series of patients. Clinical predictivity was analyzed on a subset of 2,067 patients given local-regional therapy until relapse and followed for a median time from 6 to 10 years. Over the entire period TLI maintained a weak direct relation with tumour size and an inverse strong relation with steroid receptors. An increase in TLI was observed for tumours in post-menopausal patients up to the mid 1980s. During 3 different accrual periods (1972-1983; 1984-1987; 1988-1991), TLI was a consistent and independent predictor of relapse-free time, distant metastasis and overall survival, regardless of its consideration as a continuous variable or with a cutoff value of 3%. The reproducibility of our results over time provides support to the consistency of the methodology used and of the biological and clinical information obtained when using TLI as an indicator of breast cancer cell proliferation.

Contribution of ploidy and cell kinetics from fine-needle aspirates for the diagnosis of breast lesions: study of 606 consecutive cases.

A total of 606 consecutive patients with palpable breast lesions underwent physical examination, mammography, and fine-needle aspiration for cytologic and flow-cytometric (FCM) DNA analyses. FCM determinations included DNA ploidy and the fraction of cells in S + G2 + M phases. Aneuploid clones were considered indicative of malignancy; diploid, rapidly proliferating (S+G2+M>12%) clones were considered suggestive of malignancy; and diploid, slowly proliferating clones were considered indicative of benignancy. Sensitivity, specificity, and the predictive accuracy for positive or negative results of FCM information were 82%, 90%, 93.1% and 69.8%, respectively. These values were lower than those observed for the three conventional diagnostic assays owing to the presence of false-positive results (in 23 cases) or unassessable DNA plots (in 90 cases). FCM information alone detected five cases in which the other tests gave inconclusive results and in association to the conventional diagnostic triple test, increased the incidence of positive conclusive cases from 97.9% to 99.2%. However, further refinements are needed before this approach can be used as a routine diagnostic tool.

Changes in biological markers after primary chemotherapy for breast cancers.

The profiles of functional (proliferative rate and cell distribution in the cell cycle) and phenotypic (nuclear DNA content and hormone receptor status) biological markers and the expression of P53 and Bcl-2 proteins were prospectively evaluated in breast cancers before and after different regimens of primary chemotherapy. Overall, changes induced on the 2 proliferation indices (3H-thymidine labelling index, 3H-dT LI, and flow-cytometric S-phase fraction, FCM-S) mainly consisted of a decrease for rapidly proliferating tumours and an increase or no change for slowly proliferating tumours. However, when considered as a function of treatment type, changes of 3H-dT LI and FCM-S were superimposable in rapidly proliferating tumours, regardless of the type of treatment, and in slowly proliferating tumours only after anthracycline-including regimens. Conversely, following CMF, FCM-S was increased in 90% of the cases and 3H-dT LI in only 50%. Our data imply that the 2 proliferation indices could reflect different phenomena: an actual variation of proliferative activity by 3H-dT LI and an accumulation of cells in the S-phase by FCM-S. In addition, a higher accumulation of cells in G2-M phases could be detected by FCM after anthracycline-including regimens than after CMF. The fraction of P53-positive cells was reduced by primary chemotherapy in about 50% of P53-positive tumours, whereas Bcl-2 expression was only marginally affected. DNA ploidy and hormone receptor status did not change in about 75% of cases, regardless of the chemotherapeutic regimen.

Biologic and clinicopathologic factors as indicators of specific relapse types in node-negative breast cancer.

PURPOSE AND METHODS: We evaluated, in 1,800 patients with node-negative tumors treated with locoregional therapy until relapse, the competitive risks for different types of metastasis by cell proliferation (3H-thymidine labeling index [3H-dT LI]), estrogen receptors (ERs), and progesterone receptors (PgRs), and by the integration of biologic and clinicopathologic information. RESULTS: Hormone receptor status and proliferative activity of the primary tumor were not indicative of contralateral failures. Hormone receptors failed to predict the 8-year incidence of locoregional recurrence, but they were significant indicators of distant metastasis and overall survival. The latter finding was confirmed even in multivariate analysis. Conversely, cell proliferation predicted both locoregional and distant metastases and survival, regardless of patient age, tumor size, and ER and PgR status. Recursive partitioning and amalgamation analysis ascribed to cell proliferation an important prognostic role for locoregional recurrence together with patient age and tumor size. CONCLUSION: Biologic markers, in particular cell proliferation, provide information for the different types of relapse and could complement the predictive role of pathologic staging.