Cisplatin plus paclitaxel and maintenance of bevacizumab on tumour progression, dissemination, and survival of ovarian carcinoma xenograft models.

BACKGROUND: Bevacizumab is being incorporated as first-line therapy with standard-of-care chemotherapy on epithelial ovarian carcinoma (EOC). We investigated bevacizumab combined with chemotherapy on tumour progression and mouse survival in EOC xenograft models. METHODS: Bevacizumab was administered concomitantly with cisplatin plus paclitaxel (DDP+PTX), continued after induction (maintenance) or started after chemotherapy. The effect on tumour progression was monitored by bioluminescence imaging (BLI) (1A9-luc xenograft). Tumour dissemination into the peritoneal organs and ascites formation (HOC22 xenograft) was evaluated by histological analysis at the end of treatment (interim) and at euthanasia (survival). The effects on overall survival (OS) were investigated in both EOC models. RESULTS: Bevacizumab with PTX+DDP delayed tumour progression in mice bearing EOC xenografts. OS was significantly extended, with complete responses, by bevacizumab continued after stopping chemotherapy in the HOC22 xenograft. Bevacizumab alone inhibited ascites formation, with only limited effect on tumour burden, but combined with PTX+DDP reduced ascites and metastases. Bevacizumab started after induction with PTX+DDP and maintained was equally effective on tumour progression and survival on 1A9-luc xenograft. CONCLUSION: Bevacizumab combined with chemotherapy not only affected tumour progression, but when administered as maintenance regimen significantly prolonged survival, reducing ascites, and tumour dissemination. We believe our findings are consistent with the clinical results and shed light on the potential effects of this kind of treatment on tumour progression.

[Robotic rectal resection in rectal cancer: short term results in a monocentric prospective study]. / Trattamento chirurgico del cancro del retto con tecnica robotica: risultati a breve termine di uno studio prospettico monocentrico.

AIM: The aim of this study was to evaluate technical feasibility, oncological safety and short-term clinical results of robotic rectal resection for cancer. METHODS: From January 2008 to July 2010, 46 patients (27 males and 19 females, median age 69 years, median BMI 24.6 kg/m2) with histologically-proven adenocarcinoma of medium and distal rectum were enrolled in a prospective database. Preoperative assessment was performed with colonoscopy with biopsies, thoraco-abdominal CT scan, pelvic MRI and endorectal-ultrasound (ERUS). In the case of locally advanced non metastatic disease (T3/4 or N1/2), patients received preoperative radiotherapy (45 Grays in 5 weeks) and chemotherapy (oral Capecitabine). The robotic system was a four-arms Da Vinci® (Intuitive Surgical, Sunnyvale, CA, USA); arms position is not modified during the entire surgical procedure. RESULTS: Twenty-five patients received a preoperative radio-chemotherapy. Surgical procedure was an abdomino-perineal amputation in nine patients and an anterior resection in the remaining 37, with temporary ileostomy in 16 cases and a laparoscopic mobilization of splenic flexure in 25. Median operative time was 251 minutes, median time of first bowel movements 1.7 days and median hospital stay 6.7 days. Major complications requiring reoperation verified in 2 patients, while overall complication rate is 15.2%. Median number of harvested lymph nodes per patient was 18; median distance of the tumour from distal resection margin was 2 cm; distance of the tumour from circumferential margin was superior to 1 mm in all of the patients. At a median follow up of 11 months, all patients are alive and disease-free. CONCLUSION: Robotic rectal resection is a feasible technique which can provide good oncological and short-term clinical results.

GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease.

BACKGROUND: Periodontal disease is a degenerative illness that leads to resorption of the alveolar bone. Mesenchymal stromal cells (MSC) represent a novel tool for the production of biologic constructs for the treatment of degenerative bone diseases. The preparation of MSC differentiated into osteogenic lineage for clinical use requires the fulfillment of strict good manufacturing practice (GMP) procedures. METHODS: MSC were isolated from BM samples and then cultured under GMP conditions. MSC were characterized phenotypically and for their differentiative potential. Cells were seeded onto collagen scaffolds (Gingistat) and induced to differentiate into osteogenic lineages using clinical grade drugs compared with standard osteogenic supplements. Alizarin Red S stain was used to test the deposition of the mineral matrix. Standard microbiologic analysis was performed to verify the product sterility. RESULTS: The resulting MSC were negative for CD33, CD34 and HLA-DR but showed high expression of CD90, CD105 and HLA-ABC (average expressions of 94.3%, 75.8% and 94.2%, respectively). Chondrogenic, osteogenic and adipogenic differentiation potential was demonstrated. The MSC retained their ability to differentiate into osteogenic lineage when seeded onto collagen scaffolds after exposure to a clinical grade medium. Cell numbers and cell viability were adequate for clinical use, and microbiologic assays demonstrated the absence of any contamination. DISCUSSION: In the specific context of a degenerative bone disease with limited involvement of skeletal tissue, the combined use of MSC, exposed to an osteogenic clinical grade medium, and biomimetic biodegradable scaffolds offers the possibility of producing adequate numbers of biologic tissue-engineered cell-based constructs for use in clinical trials.

Successful treatment with T-depleted autologous peripheral blood stem cell transplantation of refractory chronic autoimmune thrombocytopenic purpura.

Autoimmune thrombocytopenia (AITP) is a disorder due to specific platelet auto-antibodies directed against platelet surface glycoproteins. AITP in adults is usually chronic, idiopathic and frequently refractory to conventional treatments. Myelo- and immuno-suppressive chemotherapy followed by autologous peripheral blood stem cell (PBSC) transplantation is an experimental approach for severe chronic refractory AITP. We report a case of a woman with AITP, refractory to the conventional therapy, submitted to T-cell-depleted autologous PBSC transplantation, which obtained long term stable response on platelet count. We deem that the positive outcome of our patient depends on T-cells depletion of the graft, which reduces autoreactive T clones.

Low efficiency of a newly introduced high-density microparticles method for B cell depletion in multiple myeloma patients undergoing autologous hematopoietic stem cell transplantation.

Autologous peripheral blood stem cell (PBSC) transplantation proved to increase complete remission (CR) and DFS in multiple myeloma (MM) patients. CD34(+) cell selection has been used to reduce possible myeloma cell contamination in the graft, but it has not been showed to offer substantial advantages when compared to unpurged grafts; on the contrary, an increase of infectious complications was observed. We investigated the feasibility of a new negative-selection method in this setting. B cell negative selection was performed by using Eligix B cell HDM method. B cell contamination in the yield and in the final product was investigated by flow cytometry. Three patients with newly diagnosed MM entered the study. CD34(+) cell recovery in the three procedures was 73, 97, and 106%, and CD3(+) cell recovery was 88, 86, and 102%, respectively. CD20(+) cell depletion was 100% in all procedures, while CD19(+) cell depletion was 0.37, 1.21, and 0.07 log, respectively. We found an unexpected unreliability and a low efficiency in this B cell depletion method and suggest the need for further extensive testing before its introduction in the preclinical and clinical settings, at least in MM patients. In fact, reasons of such unsatisfactory results are still controversial: platelet contamination/activation in the preselection product, plasma protein interference, reduced CD19 antigen expression on immature B cells, lack of specificity of anti-CD19 monoclonal antibodies, instable binding between anti-CD19-coated high-density microparticles (HDM) and CD19 antigen may, alone or in combination, be involved in the system's low performance.

Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma.

We measured the levels of the vascular endothelial growth factor (VEGF), matrix metalloproteinases type 2 and type 9 (MMP-2 and MMP-9) and tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the plasma of patients with ovarian carcinoma (n=40), in other gynaecological pathologies (n=30) and in the plasma of healthy volunteers (n=26). MMP-2 and MMP-9 (pro and active forms) gelatinolytic activity was measured by zymography. Enzyme-linked immunosorbent assays (ELISA) were used to assay soluble VEGF and TIMPs. Preoperative plasma VEGF levels were significantly higher in patients with ovarian cancer than in healthy volunteers (P<0.0001) or patients with a benign gynaecological pathology (P<0.0001). The expression of pro-MMP-9 was higher in the plasma of ovarian cancer patients than in the plasma of women with non-malignant disease (P=0.01) or healthy women (P<0.0002). Pro-MMP-2 was detected in the plasma of ovarian cancer patients, but levels did not differ from those in non-malignant disease or healthy donor samples. Plasma TIMP-1 and TIMP-2 levels were significantly higher in patients with ovarian carcinomas than in healthy volunteers (P<0.0001 and P=0.006, respectively) or in the patients with a non-malignant pathology (P<0.0001 and P=0.002, respectively). Sub-group analysis showed that VEGF and pro-MMP-9 were higher in the plasma of patients with serous carcinomas than other histological types. Furthermore, plasma VEGF and pro-MMP-9 levels were higher in the plasma of cancer patients with thrombocytosis. Throughout the study, and in the univariate analysis, no correlation was found between the VEGF, MMP and TIMP levels. Only TIMP-1 was associated with a poor survival and mortality risk.

A short synthesis of argatroban. a potent selective thrombin inhibitor.

Argatroban was synthesized in seven steps from 4-methylpiperidine. The condensation of (+/-)-trans-benzyl 4-methylpipecolic acid ester with N(alpha)-Boc-N(omega)-nitro-L-arginine led to two diastereomers that were separated. One of them is the precursor of argatroban.

P-Glycoprotein Expression in Acute Myeloid Leukaemia Cells at Diagnosis: Its relationship to Daunorubicin or Idarubicin Induction Therapy and Survival; Malignancy.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR). We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells. Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005). The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005). Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs. 90% with IDR); Group 2 obtained 40% of CR with DNR vs. 70% with IDR (p < 0.005). The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

P-Glycoprotein Expression in Acute Myeloid Leukaemia Cells at Diagnosis: Its relationship to Daunorubicin or Idarubicin Induction Therapy and Survival.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR). We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells. Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p lt; 0.005). The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005). Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs. 90% with IDR); Group 2 obtained 40% of CR with DNR vs. 70% with IDR (p < 0.005). The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

HOXC5 and HOXC8 expression are selectively turned on in human cervical cancer cells compared to normal keratinocytes.

A growing number of data have sustained the involvement of homeobox genes expression deregulation in cancer. In this study, we have performed an exhaustive survey of the expression of the 39 class I HOX genes expressed in normal and malignant human cervix keratinocytes. Using RT-PCR, we observed that the vast majority (34/39) of HOX genes are expressed in normal keratinocytes. Only HOXA2, HOXA7, HOXC5, HOXC8 and HOXD12 were found to be silent. Interestingly, this pattern is conserved in the transformed keratinocytes (SiHa cells) except for the appearance of HOXC5 and HOXC8 mRNA. The HOXC5 and HOXC8 expression was also observed in two other transformed keratinocytes cell lines of independent origins, Eil-8 and 18-11S3, and confirmed by in situ hybridization. Our data add weight to the body of evidence attributing to a specific adult tissue a particular combination of expressed HOX genes and suggest that HOXC5 and/or HOXC8 could be involved in the process leading to the transformation of cervical keratinocytes.

MMP inhibitors: experimental and clinical studies.

Matrix metalloproteases (MMPs) are a family of structurally related enzymes that are capable of degrading proteins of the extracellular matrix. These enzymes play a role in tissue remodelling associated with both physiological and pathogenic processes. A high expression of MMPs is associated with cancer malignancy: it is related to the tumor's ability to metastasize and to the process of angiogenesis. Treatment with MMP inhibitors alone or in combination with cytotoxic therapy is an interesting novel approach to control tumor progression. The expected mechanism of action of these compounds and the difference in side effects compared to cytotoxic drugs make the definition of endpoints and the assessment of response difficult. Furthermore, it is not yet clear whether tumor vascularization or, more specifically, MMP expression/activation should be a criterion of eligibility for this kind of treatment. This review provides an overview of the characteristics of MMPs and their role in tumor progression, metastasis and angiogenesis. Preclinical and clinical studies with synthetic MMP inhibitors are described. The presence of MMPs in biological fluids of patients and their use in prognostic evaluation and in determining the efficacy of treatment with MMP inhibitors is discussed.

Expression and modulation of homeobox genes from cluster B in endothelial cells.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

The 140-kilodalton antiangiogenic fragment of thrombospondin-1 binds to basic fibroblast growth factor.

Thrombospondin-1 (TSP) inhibits the angiogenic activity of basic fibroblast growth factor (bFGF). Here we address the hypothesis of a direct interaction between TSP and bFGF. Gel permeation chromatography and cross-linking experiments demonstrated that bFGF binds to TSP in solution. bFGF also bound to immobilized TSP in a solid-phase assay. Binding was dose-dependent, with a Kd in the nanomolar range, and was inhibited by anti-TSP antibodies. The 140-kDa carboxyl-terminal fragment of TSP, but not the 25-kDa heparin-binding fragment, fully retained the bFGF binding capacity. Accordingly, binding was inhibited by monoclonal antibodies directed against this fragment. Heparin completely blocked bFGF binding to TSP and to the 140-kDa fragment. TSP and its 140-kDa fragment inhibited the binding of bFGF to endothelial cells at concentrations (> or = 100 nM) that inhibited endothelial cell proliferation but not motility. Low-affinity binding was inhibited more than high-affinity binding (up to 76 and 41% inhibition, respectively), and the inhibition was reversed by anti-TSP antibodies. Vitronectin and transforming growth factor beta, potentially associated with TSP, did not affect bFGF binding to endothelial cells. Although TSP did not affect the activation of the high-affinity receptors, it reduced the long-term internalization of bFGF. We conclude that TSP binds to bFGF through a domain within its 140-kDa fragment, a mechanism that might affect bFGF interaction with endothelial cells, activity, and association with the extracellular matrix.

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations

The microtubule-affecting drug paclitaxel has antiangiogenic activity.

Endothelial cell migration is a critical event during angiogenesis, and inhibitors of cell motility can affect the angiogenic process. Paclitaxel (Taxol(R)), a microtubule-stabilizing antineoplastic cytotoxic drug, inhibits motility and invasiveness of several cell types. The aim of this study was to investigate the effect of paclitaxel on endothelial cell functions and on angiogenesis. In vivo, paclitaxel (20-28 mg/kg i.v.) significantly inhibited the angiogenic response induced by tumor cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel) injected s.c. into C57BL/6N mice. In vitro, paclitaxel inhibited endothelial cell proliferation, motility, invasiveness, and cord formation on Matrigel in a dose-dependent manner. The antiangiogenic activity of paclitaxel was not linked to its cytotoxicity, since inhibition of endothelial cell chemotaxis and invasiveness occurred at drug concentrations which did not affect endothelial cell proliferation. Another cytotoxic drug, cisplatin, that inhibited endothelial cell proliferation in vitro, did not affect angiogenesis in vivo. These data indicate that paclitaxel has a strong antiangiogenic activity, a property that might contribute to its antineoplastic activity in vivo.

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells.

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.

Isolation from a multigene family of the active human gene of the metastasis-associated multifunctional protein 37LRP/p40 at chromosome 3p21.3.

The 37 kD precursor of the 67 kD laminin receptor (37LRP) is a polypeptide whose expression is consistently upregulated in aggressive carcinoma. Interestingly, the 37LRP appears to be a multifunctional protein involved in the translational machinery and has also been identified as p40 ribosome-associated protein. Although highly conserved cDNAs corresponding to this polypeptide have been isolated from several species including vertebrates, invertebrates, plants and prokaryotes, characterization of any of the corresponding active genes has never been reported. In this study, we have cloned an intron-containing fragment which permitted us to isolate the active 37LRP/p40 human gene. This gene contains seven exons and six introns. Ribonuclease protection experiments suggest multiple transcription start sites. The promoter area does not bear a TATA box but contains four Sp1 sites. The first intron is also GC rich containing five Sp1 sites. Intron 4 contains the full sequence of the small nuclear RNA E2 and two Alu sequences are found in intron 3. Fluorescent in situ hybridization localized the 37LRP/p40 active gene on chromosome 3 in the locus 3p21.3 which, interestingly, is a hot spot for genetic alterations in several cancers and particularly in small cell lung carcinoma.

Expression of the 67 kD laminin receptor in human ovarian carcinomas as defined by a monoclonal antibody, MLuC5.

Previous immunohistochemical data from our laboratory have demonstrated that expression of the 67 kD laminin receptor (67LR), a cancer-associated, high-affinity laminin-binding protein, is upregulated in ovarian carcinoma cells compared with normal serosal cells, and that this increased expression in cancer cells could be related to patient outcome. The aim of this study was to validate MLuC5, a monoclonal antibody that recognises the 67LR, as a tool to perform future immunohistochemical studies on larger populations of ovarian carcinoma patients. Expression of the 67LR was determined in 51 primary human ovarian carcinoma samples using immunohistochemistry and MLuC5. The 67LR was detected in ovarian carcinoma cell clusters of variable extent. Analysis of the data determined that 67LR expression was significantly increased in the samples from patients with disease progression, compared with those with no evidence of disease after completion of primary therapy, and in pooled grade 2 and 3 tumours compared to borderline and grade 1 tumours (P < 0.05, chi-squared test). No other significant correlation between 67LR expression and other clinicopathological parameters could be established. These data suggest that the 67LR is correlated to ovarian tumour progression. Detection of the 67LR using this monoclonal antibody could constitute an interesting parameter in prognosis determination of ovarian cancer.