G Martinelli

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.

Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4–11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34+ cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K–Akt survival pathway.

Neoplastic involvement of nipple-areolar complex in invasive breast cancer

The neoplastic involvement of the nipple-areolar complex was histologically studied in 1,291 available consecutive mastectomy specimens with primary invasive breast carcinoma. Tumor involvement of the nipple-areolar complex was found in 150 specimens (12 percent) and was not suspected on gross examination in 99 patients (8 percent). A significant finding of our study was the relatively high rate of tumor foci in the nipple-areolar complex (7 percent) in those patients with early invasive stage I or II breast carcinoma eligible for conservative therapy. Analysis of nipple-areolar complex involvement with consideration of different clinico-morphologic variables indicates that it was directly associated with tumor size. No significant correlation was found with axillary metastases, tumor histologic type, or with the presence of noninvasive cancer in the vicinity of the dominant tumor. Our estimate of the significant change of finding tumor in the nipple-areolar complex, especially in the patient group eligible for conservative therapy, underlines the need for postoperative radiation.

Fludarabine-containing regimens severely impair peripheral blood stem cells mobilization and collection in acute myeloid leukaemia patients

We studied the effects of an intensified induction/consolidation treatment containing fludarabine (ICE/FLAN/FLAN) on the mobilization and collection of peripheral blood stem cells (PBSC) in 31 consecutive untreated acute myeloid leukaemia (AML) patients. The complete remission (CR) rate was comparable to classic inductions (68% after ICE; 84% after ICE‐FLAN I). To mobilize PBSC, 19 patients received 10 μg/kg/d of granulocyte‐colony stimulating factor (G‐CSF) starting at day 13 after FLAN, 13 (69%) of whom were found to be nonmobilizers. When a second G‐CSF administration was performed in 10/13 patients mobilization was either not achieved (8/10) or was considered insufficient (<1 × 106 CD34+ cells/kg) (2/10) and all 13 were subsequently submitted to bone marrow harvest. The harvest was considered adequate in 12/13 (92%) patients and autologous BMT (ABMT) has so far been performed in 10/12 cases with a mean of 8.6 × 108/kg nucleated reinfused cells. The median times to neutrophil and platelet recovery after ABMT did not significantly differ from those of two previous series of patients treated with ABMT without fludarabine‐containing regimens. Adequate amounts of PBSC were obtained in 6/19 (31%) patients, who were then reinfused. Median times for platelet recovery were significantly longer than in a previous series of 26 AML cases reinfused with PBSC after treatment with the ICE‐NOVIA induction/consolidation regimen (125 v 20 d to 20 × 109 plt/l, P < 0.02; 218 v 37 d to 50 × 109 plt/l, P < 0.02). In addition, times for platelet recovery after ICE/FLAN/FLAN were not significantly different from those in a previous group treated with ABMT performed after ICE/NOVIA,without fludarabine. We conclude that fludarabine‐containing regimens severely impair mobilization and collection of PBSC in AML patients and seem unsuitable when PBSC autotransplantation is programmed.

The prognostic value of cytogenetics is reinforced by the kind of induction/consolidation therapy in influencing the outcome of acute myeloid leukemia - Analysis of 848 patients

We studied the impact of cytogenetics and kind of induction/consolidation therapy on 848 adult acute myeloid leukemia (AML) patients (age 15–83). The patients received three types of induction/consolidation regimen: standard (daunorubicin and cytosine arabinoside (3/7); two cycles); intensive (idarubicin, cytosine arabinoside and etoposide (ICE), plus mitoxantrone and intermediate-dose Ara-C (NOVIA)); and low-dose (low-dose cytosine arabinoside). CR patients under 60 years of age, if an HLA-identical donor was available received allogeneic stem cell transplantation (allo-SCT); otherwise, as part of the program, they underwent autologous (auto)-SCT. CR rates significantly associated with ‘favorable’ (inv(16), t(8;21)), ‘intermediate’ (‘no abnormality’, abn(11q23), +8, del(7q)) and ‘unfavorable’ (del (5q), −7, abn(3)(q21q26), t(6;9), ‘complex’ (more than three unrelated cytogenetic abnormalities)) karyotypes (88% vs65% vs 36%, respectively; P = 0.0001). these trends were confirmed in all age groups. on therapeutic grounds, intensive induction did not determine significant increases of cr rates in any of the considered groups, with respect to standard induction. low-dose induction was associated with significantly lower cr rates. considering disease-free survival (dfs), multivariate analysis of the factors examined (including karyotype grouping) showed that only age >60 years significantly affected outcome. However, in cases where intensive induction was adopted, ‘favorable’ karyotype was significantly related to longer DFS (P = 0.04). This was mainly due to the favorable outcome of t(8;21) patients treated with intensive induction. Patients receiving allo-SCT had significantly longer DFS (P = 0.005); in particular, allo-SCT significantly improved DFS in the ‘favorable’ and ‘intermediate’ groups (P = 0.04 and P = 0.048, respectively). In conclusion our study could provide some guidelines for AML therapy: (1) patients in the ‘favorable’ karyotype group seem to have a longer DFS when treated with an intensive induction/consolidation regimen, adopted before auto-SCT instead of standard induction; this underlines the importance of reinforcement of chemotherapy, not necessarily based on repeated high-dose AraC cycles. Allo-SCT, independently of induction/consolidation therapy, should be considered an alternative treatment; (2) patients in the ‘intermediate’ karyotype group should receive allo-SCT; (3) patients in the ‘unfavorable’ karyotype group should be treated using investigational chemotherapy, considering that even allo-SCT cannot provide a significantly longer DFS, but only a trend to a better prognosis.

Dual tyrosine kinase inhibitors in chronic myeloid leukemia

The Bcr-Abl inhibitor imatinib mesylate induces complete hematologic and cytogenetic remissions in most newly diagnosed chronic myeloid leukemia (CML) patients, but relatively few of them achieve molecular remission. In addition, imatinib is much less effective in advanced phase-CML as well as in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), mainly due to the development of drug resistance. The challenge for the future is to improve current clinical results with kinase inhibitors in CML, developing strategies that can eradicate residual disease and overcome or prevent resistance. ‘Dual’ Src and Abl kinase inhibitors are an attractive class of compounds, since (a) these molecules are able to bind Bcr-Abl with less stringent conformational requirements with respect to imatinib, therefore allowing for efficient inhibition of several, resistance-associated mutant forms of Bcr-Abl; (b) Src kinases have been shown to be involved in Bcr-Abl-mediated leukemogenesis as well as upregulated in some patients resistant to imatinib. Here, we review the development, the mode of action and the preclinical or early clinical evaluation of several novel dual Src and Abl kinase inhibitors.

Clinical efficacy and antiangiogenic activity of thalidomide in myelofibrosis with myeloid metaplasia. A pilot study

Increased neoangiogenesis has been reported in myelofibrosis with myeloid metaplasia (MMM). Thus we studied the effects of thalidomide, an antiangiogenic drug, in 12 MMM patients. Before treatment, all the cases showed a significantly increased micro-vessel density (MVD); in all eight tested cases bFGF and VEGF plasma levels were higher than controls. All patients presented disease progression in the last 3 months with standard therapy, regarding splenomegaly, anemia and/or thrombocytopenia and/or hyperleukocytosis. Thalidomide was administered at daily doses increasing from 100 to 600 mg. Eleven out of 12 patients were evaluable. No progression of disease was seen during the treatment in any case. In particular, spleen size decreased in 7/11 patients, anemia improved in 3/4 (two are now transfusion independent), thrombocytopenia in 2/2 and hyperleukocytosis in 2/5 patients. Side-effects were frequent, although not severe. After treatment, VEGF and bFGF plasma levels varied widely and in selected cases decreased. In particular, VEGF and/or bFGF decreased in 4/5 responders and in 1/3 non-responders. Moreover, MVD significantly decreased in all the responders evaluated after treatment. We conclude that thalidomide is a feasible therapy in MMM patients and looks promising at least to control the growth progression of disease.

Atypical juvenile polyposis

Two cases of atypical juvenile polyposis are described in males of 9 months and 25 years‐of‐age. The first was associated with congenital megacolon and presented as juvenile polyps with features suggesting mild dysplasia. In the second case six histological lesions are found: 1 hyperplastic polyps; 2 juvenile polyps; 3 hyperplastic polyps with adenomatous areas; 4 juvenile polyps with areas of dysplastic epithelium; 5 adenomas; and 6 adenocarcinomas. On the basis of the morphological features we propose a pathogenetic sequence of focal mucosal hyperplasia to adenoma and carcinoma through stages of non‐neoplastic and non premalignant polyps. Finally, the possibility that hyperplastic epithelium can in some circumstances have a greater dysplastic potential than normal colorectal mucosa is raised.

Demonstration of cytokeratin intermediate filaments in oocytes of the developing and adult human ovary

The intermediate filaments (IF) present in the various cells of human ovaries were studied by immunolocalization using antibodies to cytokeratins (CKs), vimentin, desmin and alpha-smooth muscle (α-SM) actin. Oocytes revealed a single paranuclear aggregate, which reacted with antibodies to CKs 8, 18 and 19 both in adult and fetal ovaries. The existence of this aggregate was also documented by electron microscopy. Ovarian surface epithelium and granulosa cells consistently coexpressed CKs 8, 18, 19 and vimentin. During follicle maturation vimentin remained unchanged in the granulosa layer while CKs content decreased, showing variation in the amount and distribution of the different CK-types. Thecal cells of secondary and mature follicles showed α-SM actin positivity. These contractile fibres increased in mature follicles. Ordinary fibrous stromal cells showed isolated cells which were desmin and α-SM actin positive. A similar pattern of IF expression and distribution existed in all stages of development in fetal and embryonic ovaries. These results indicate that CKs are present in human oocytes and that the coexpression of vimentin and CKs can be regarded as a peculiar feature of all ovarian cell types except oocytes and ordinary stromal cells. Contractile properties have been documented associated with a modification in expression of IF proteins. This is likely to represent an integral part of folliculogenesis along with the functional hormone-dependent changes.

Incidence, Etiology, Histologic Findings, and Course of Thoracic Inflammatory Aortopathies

BACKGROUND The aims of this study were to detect the incidence of thoracic histologically proven aortitis in a large series of 788 patients operated on for thoracic aortic disease, to describe the surgical and histologic features of inflammatory thoracic aortopathies, and to evaluate the frequency of postsurgical complications and mortality. METHODS Thirty-nine patients (4.9%) were affected by aortitis (mean age, 72.6 +/- 9.6). There were 24 women (61.5%). Thirty-four (87.2%) were operated on because of aneurysms and 5 because of dissection. In all cases the diagnosis of aortitis was incidental and was made on the basis of histopathologic findings. RESULTS Histologically, there were 30 cases of giant cell aortitis (76.9%), 3 inflammatory aneurysms (7.7%), 2 cases of aspecific lymphoplasmacellular aortitis (5.1%), 1 of Takayasu aortitis, 1 of systemic erythematosus lupus-associated aortitis, and 1 of Behçets disease-associated aortitis. The only case of infectious aortitis was a syphilitic aortitis. In 79.5% of cases, inflammatory infiltrates were moderate to severe in degree; the most widespread inflammation was seen in Takayasu aortitis, systemic erythematosus lupus-associated aortitis, and in Behçets disease. The overall in-hospital mortality was 10.3% (4 of 39 patients). Neurologic complications occurred in 4 patients (10.3%). CONCLUSIONS During surgery of the thoracic aorta, an inflammatory etiology of aneurysms is found in almost 5% of cases. The inflammatory process is in a histologically advanced phase, often with systemic development. Surgery can be associated with high morbidity and mortality.