The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas

Abstract: Background Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. Methods Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. Results In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. Conclusions Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. Virtual slides. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514

Purging e alte dosi di chemioterapia con reinfusione di cellule staminali autologhe in linfomi non Hodgkin follicolari resistenti/refrattari

In the era of monoclonal antibodies the role of autologous stem cell transplantation (ASCT) in the management of follicular lymphoma (FL) is still debated. To evaluate the safety and efficacy of myeloablative therapy with rescue of purged or unpurged harvests in FL pts. At our institution form 1997 to 2007 28 pts with refractory/resistant FL were eligible for ASCT. Before high dose therapy they received 2-3 cycles of CHOP-like regimen (ACOD), followed by Cyclophosphamide 4g/mq to mobilize the stem cells (SC). After SC collection the pts underwent 3 cycles of subcutaneous Cladribine at a daily dose of 0,14-0,10 mg/Kg for Day 1-5 every 3-4 weeks. The conditioning regimen was based on Mitoxantrone 60mg/mq + Melphalan 180 mg/mq, followed by SC re-infusion 24-hours later and G-CSF starting 24 hours after re-infusion. In 19 pts the SC underwent purging: in 10 harvests the CD34+ were selected by immunomagnetic beads, while in the other 9 pts, only Rituximab was used as “purging in vivo” agent. The remaining 9 pts received unpurged SC. Before ASCT 11 pts were in complete response (CR), 9 in partial response (PR) and 2 in stable disease. Two pts were not eligible for ASCT because of progressive disease (PD). The remaining 25 pts were eligible for ASCT. The engraftment was at a median of 11 days for leucocytes and 14 days for platelets (>20.000/mmc), with a delay of one day in the pts, who received purged SC. Grade 3-4 mucositis was described in 8 pts. During aplasia a 48% infection rate was reported, without differences between pts with purged or unpurged SC. One patient in CR presented myelodysplastic syndrome at 18 months from ASCT. After ASCT 22 pts were in CR, 2 in PR and one patient were not valuable, because died before response assessment. Nine pts in CR showed PD at a median time of 14 months from ASCT. With a median follow up of 5 years (range 2 months -10 years), 22 pts are alive and 11 (44%) in CR. Ten pts died, 5 for progressive disease and 5 for treatment-related causes; in particular 7 of them received in-vitro purged SC. Conclusions: Our chemotherapy regimen, which included the purine analogue Cladribine in the induction phase, seems safe and feasible. The high rate of CR reported and the sustained freedom from progression up to now, makes such modality of treatment a valid option principally in relapsing FL patients. In our experience, the addition of a monoclonal antibody as part of treatment confirms its role “in vivo purging” without observing an increased incidence of infection.

Il trapianto di midollo osseo aploidentico nelle patologie oncoematologiche: studio clinico

L’argomento della presente tesi di dottorato riguarda lo studio clinico del trapianto di cellule staminali emopoietiche aploidentiche nelle patologie oncoematologiche. Nel periodo di tempo compreso tra 1/12/2005 ed il 30/10/2007 sono stati arruolati 10 pazienti (6 LAM, 3 LAL, 1 LMC in crisi blastica mieloide) nell’ambito di uno studio clinico che prevedeva il trapianto di midollo osseo aploidentico per pazienti affetti da patologia oncoematologica in prima o successiva recidiva, per i quali non fosse disponibile un donatore di midollo osseo consanguineo o da banca. Lo schema di condizionamento al trapianto di midollo osseo utilizzato era il seguente: Fludarabina 150/m2, Busulfano orale 14mg/kg, Tiothepa 10mg/kg e Ciclofosfamide 160mg/kg. Per la profilassi della malattia da trapianto contro l’ospite è stata somministrata timoglobulina antilinfocitaria (ATG) al dosaggio complessivo di 12.5 mg/kg, short course metotrexate (+1, +3 e +11), cortisone e ciclosporina con tapering precoce al + 60. I pazienti hanno reinfuso una megadose di cellule CD34+ mediana pari a 12.8x106/kg. Tre pazienti non sono valutabili per l’attecchimento a causa di rigetto (1/3) o morte precoce (2/3). Sette pazienti sono valutabili per l’attecchimento; per questi pazienti il tempo mediano a 500 PMN/mmc e a 20 x 109/l piastrine è stato rispettivamente di 17 e 20 giorni. Quattro pazienti su 7 hanno svillupato una Graft versus Host Disease (GVHD) acuta di grado II-IV, mentre soltanto 1/7 ha sviluppato una GVHD cronica. Sette pazienti su 10 trapiantati hanno ottenuto una remissione completa successivamente al trapianto. Di questi, attualmente 2 pazienti sono vivi in remissione completa, mentre gli altri 5 sono ricaduti e successivamente deceduti. In conclusione, il trapianto aploidentico è una procedura fattibile ed efficace. Tale procedura è in grado di garantire un 20% di lungo sopravviventi in un setting di pazienti a prognosi estremamente infausta.

Resident angiogenic mesenchymal stem cells from multiorgan donor thoracic aortas

Stem cells are one of the most fascinating areas of biology today, and since the discover of an adult population, i.e., adult Stem Cells (aSCs), they have generated much interest especially for their application potential as a source for cell based regenerative medicine and tissue engineering. aSCs have been found in different tissues including bone marrow, skin, intestine, central nervous system, where they reside in a special microenviroment termed “niche” which regulate the homeostasis and repair of adult tissues. The arterial wall of the blood vessels is much more plastic than ever before believed. Several animal studies have demonstrated the presence of cells with stem cell characteristics within the adult vessels. Recently, it has been also hypothesized the presence of a “vasculogenic zone” in human adult arteries in which a complete hierarchy of resident stem cells and progenitors could be niched during lifetime. Accordingly, it can be speculated that in that location resident mesenchymal stem cells (MSCs) with the ability to differentiate in smooth muscle cells, surrounding pericytes and fibroblasts are present. The present research was aimed at identifying in situ and isolating MSCs from thoracic aortas of young and healthy heart-beating multiorgan donors. Immunohistochemistry performed on fresh and frozen human thoracic aortas demonstrated the presence of the vasculogenic zone between the media and the adventitial layers in which a well preserved plexus of CD34 positive cells was found. These cells expressed intensely HLA-I antigens both before and after cryopreservation and after 4 days of organ cultures remained viable. Following these preliminary results, we succeeded to isolate mesenchymal cells from multi-organ thoracic aortas using a mechanical and enzymatic combined procedure. Cells had phenotypic characteristics of MSC i.e., CD44+, CD90+, CD105+, CD166+, CD34low, CD45- and revealed a transcript expression of stem cell markers, e.g., OCT4, c-kit, BCRP-1, IL6 and BMI-1. As previously documented using bone marrow derived MSCs, resident vascular wall MSCs were able to differentiate in vitro into endothelial cells in the presence of low-serum supplemented with VEGF-A (50 ng/ml) for 7 days. Under the condition described above, cultured cells showed an increased expression of KDR and eNOS, down-regulation of the CD133 transcript, vWF expression as documented by flow cytometry, immunofluorescence, qPCR and TEM. Moreover, matrigel assay revealed that VEGF induced cells were able to form capillary-like structures within 6 hours of seeding. In summary, these findings indicate that thoracic aortas from heart-beating, multi-organ donors are highly suitable for obtaining MSCs with the ability to differentiate in vitro into endothelial cells. Even though their differentiating potential remains to be fully established, it is believed that their angiogenic ability could be a useful property for allogenic use. These cells can be expanded rapidly, providing numbers which are adequate for therapeutic neovascularization; furthermore they can be cryostored in appropriate cell banking facilities for later use.

Rag2-/-;gammac-/- immunodeficient mice, a new preclinical model to study antitumor approaches

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

Transformation, Progression und Zellinienspezifität der chronischen myeloischen Leukämie

Die Analyse der CML-Zellinie K562 mittels Fluoreszenz in situ Hybridisierung (FISH), Multiplex-FISH (M-FISH) und comparativer genomischer Hybridisierung (CGH) ergab einen hypotriploiden Karyotyp mit 67 Chromosomen und 21 verschiedenen Marker-Chromosomen. Das bei über 90% der CML-Patienten nachgewiesene Ph-Chromosom entsteht durch die reziproke Translokation t(9;22)(q34;q11). Bei 5 - 10% der Patienten resultiert das Ph-Chromosom aus varianten Translokation. Anhand der Untersuchung dreier varianter Translokation mittels Bruchpunkt-übergreifender FISH-Proben für die BCR- und ABL-Gene werden drei verschiedene Mechanismen der Entstehung komplexer Translokationen dargestellt. Das Auftreten sekundärer Aberrationen wurde in 15 CML-Blastenkrisen untersucht. Zudem wurde anhand der CGH-Analyse von CD34-positiven Zellen, Monozyten, Granulozyten und T-Zellen die Zellinienspezifität sekundärer Aberrationen untersucht. In einem Fall wurde eine sekundäre Aberration in allen vier Fraktionen gefunden. In zwei Fällen traten sekundäre Aberrationen in allen untersuchten Fraktionen mit Ausnahme der T-Zellen auf. Aufgrund dieser Ergebnisse lassen sich zwei alternative Modelle der Tumor-Progression der CML ableiten: 1. Sekundäre Mutatonen treten vor der Differenzierung der hämatopoetischen Stammzelle auf. 2. Sekundäre Mutationen treten in einer hämatopoetischen Vorläuferzelle nach der T-Zell-Differenzierung auf.

Impiego di cellule staminali in terapia veterinaria

Over the past few years, in veterinary medicine there has been an increased interest in understanding the biology of mesenchymal stem cells (MSCs). This interest comes from their potential clinical use especially in wound repair, tissue engineering and application in therapeutics fields, including regenerative surgery. MSCs can be isolated directly from bone marrow aspirates, adipose tissue, umbilical cord and various foetal tissues. In this study, mesenchymal stem cells were isolated from equine bone marrow, adipose tissue, cord blood, Wharton’s Jelly and, for the first time, amniotic fluid. All these cell lines underwent in vitro differentiation in chondrocytes, osteocytes and adipocytes. After molecular characterization, cells resulted positive for mesenchymal markers such as CD90, CD105, CD44 and negative for CD45, CD14, CD34 and CD73. Adipose tissue and bone marrow mesenchymal stem cells were successfully applied in the treatment of tendinitis in race horses. Furthermore, for the first time in the horse, skin wounds of septicemic foal, were treated applying amniotic stem cells. Finally, results never reported have been obtained in the present study, isolating mesenchymal stem cells from domestic cat foetal fluid and membranes. All cell lines underwent in vitro differentiation and expressed mesenchymal molecular markers.

Vascular wall stem cells. Selection and conditioning of progenitors useful for cell therapy. A pathological case study

The arterial wall contains MSCs with mesengenic and angiogenic abilities. These multipotent precursors have been isolated from variously-sized human adult segments, belying the notion that vessel wall is a relatively quiescent tissue. Recently, our group identified in normal human arteries a vasculogenic niche and subsequently isolated and characterized resident MSCs (VW-MSCs) with angiogenic ability and multilineage potential. To prove that VW-MSCs are involved in normal and pathological vascular remodeling, we used a long-term organ culture system; this method was of critical importance to follow spontaneous 3-D vascular remodeling without any influence of blood cells. Next we tried to identify and localize in situ the VW-MSCs and to understand their role in the vascular remodeling in failed arterial homografts. Subsequently, we isolated this cell population and tested in vitro their multilineage differentiation potential through immunohistochemical, immunofluorescence, RT-PCR and ultrastructural analysis. From 25-30cm2 of each vascular wall homograft sample, we isolated a cell population with MSCs properties; these cells expressed MSC lineage molecules (CD90, CD44, CD105, CD29, CD73), stemness (Notch-1, Oct-4, Sca-1, Stro-1) and pericyte markers (NG2) whilst were negative for hematopoietic and endothelial markers (CD34, CD133, CD45, KDR, CD146, CD31 and vWF). MSCs derived from failed homografts (H-MSCs) exhibited adipogenic, osteogenic and chondrogenic potential but scarce propensity to angiogenic and leiomyogenic differentiation. The present study demonstrates that failed homografts contain MSCs with morphological, phenotypic and functional MSCs properties; H-MSCs are long-lived in culture, highly proliferating and endowed with prompt ability to differentiate into adipocytes, osteocytes and chondrocytes; compared with VW-MSCs from normal arteries, H-MSCs show a failure in angiogenic and leiomyogenic differentiation. A switch in MSCs plasticity could be the basis of pathological remodeling and contribute to aneurysmal failure of arterial homografts. The study of VW-MSCs in a pathological setting indicate that additional mechanisms are involved in vascular diseases; their knowledge will be useful for opening new therapeutic options in cardiovascular diseases.

Mastocitosi cutanee: il mastocitoma in età pediatrica

Background: Mastocytosis is a rare disease involving mast cells (MC) and their CD34+ progenitors. According to the WHO consensus classification, cutaneous mastocytosis (CM) is considered a benign disease confined to the skin, preferentially seen in young children with a marked tendency to regress spontaneously. Aim of our study was the long-term assessment of the outcome of solitary (SM) and multiple (MM) mastocytomas in a pediatric population. Materials and methods: From January 1996 to December 2010, 241 pediatric patients with a diagnosis of CM were followed-up at the outpatient division of pediatric dermatology of the University of Bologna. We focused our retrospective evaluation on patients affected by SM or MM. We collected, through the analysis of medical records and with a telephone questionnaire for patients and their families, information on clinical aspects of the disease evolution and on the efficacy of topical steroid therapy. Results: Over the 241 considered patients we recorded: SM or MM in 176 (73%) pts., urticaria pigmentosa in 53 (22%) pts., telangiectasia macularis eruptiva perstans in 9 (4%) pts., diffuse CM in 2 (0,9%) pts. and polymorph CM in 1 (0,4%) pt. On 176 children affected by SM or MM (97 M vs. 79 F), 130 (74%) patients were followed-up with a mean of 56,3 (r. 4-142) months. A satisfactory outcome was recorded in 99 (76%) cases of whom 52 (53%) treated with topic steroids. Mean time to complete regression was 16.4 m. on treated patients vs. 34.7 m. on non treated patients (p=0,001). Conclusions: From our study emerged that resolution of the disease is independent from therapy, but the time to regression and to complete recovery of the coetaneous lesions is faster and favored by the application of topic steroid with an improvement of the quality of life for children and their families.

Alkylantien induzierte Zytotoxizität, DNA-schadensinduzierte Reparaturkapazität und Apoptoseinduktion von Immunzellen

Monozyten und Monozyten-abgeleitete Dendritische Zellen (DCs) spielen eine bedeutende Rolle im Immunsystem. Da DCs bei der Tumorabwehr mitwirken, ist es wichtig, dass Monozyten als auch DCs sich gegenüber zytotoxischen Agenzien aus der Chemotherapie wehren können. Chemotherapeutika reagieren mit der DNA, jedoch die DNA-Reparaturkapazität von Monozyten und DCs wurde noch nicht untersucht. Dazu wurde die Sensitivität in Monozyten und DCs gegenüber verschiedene genotoxische Agenzien untersucht. Dabei wurde herausgefunden, dass Monozyten sensitiv auf methylierende Agenzien (MNNG, MMS und Temozolomid) reagieren und ein verstärktes Zellsterben und Apoptoseinduktion zeigen. Im Vergleich zu weiteren Zytostatika wie Fotemustin, Mafosfamid und Cisplatin reagierten Monozyten und DCs gleich sensitiv. Diese Ergebnisse weisen auf einen Defekt in der Reparatur von DNA-Methylierungsschäden in Monozyten hin. Da die Expression des Reparaturproteins O6-Methylguanin-DNA Methyltransferase (MGMT) in Monozyten höher war als in DCs und deren Inhibierung durch O6-Benzylguanin keinen Effekt auf die Sensitivität von Monozyten hatte, wurde der Reparaturweg der Basenexzisionsreparatur untersucht. Im Vergleich zu DCs waren die Monozyten unfähig die BER durchzuführen, welche durch Einzelzellgelelektrophorese gemessen wurde. Expressionsuntersuchungen ergaben, dass in Monozyten XRCC1 und Ligase IIIα fehlen im Vergleich zu DCs, Makrophagen, hämatopoetische Stammzellen und Lymphozyten, welche diese Proteine exprimieren. Diese Ergebnisse zeigen einen spezifischen DNA-Reparaturdefekt in einer bestimmten Blutzellpopulation. Durch den BER Defekt in Monozyten kann es durch methylierende Tumorwirkstoffe während einer Chemotherapie zur Depletion und zu einer abgeschwächten Immunantwort kommen.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Evaluation der Leukämie-Reaktivität von PRAME- und p53-spezifischen T-Zellen bei lymphohämatopoetischen Neoplasien im Rahmen einer adoptiven Immuntherapie

Der Transplantat-gegen-Leukämie (GVL) Effekt als immuntherapeutisches Mittel bei der allogenen hämatopoetischen Stammzell Transplantation (HSZT) ist hauptsächlich durch Spender Lymphozyten vermittelt, welche hämatopoetische Minor-Histokompatibilitäts Antigene bzw. Leukämie-assoziierte Antigene (z. B.: PRAME, p53) erkennen. Der adoptive Transfer von Leukämie-spezifischen T-Zellen kann den GVL-Effekt, ohne ein Auftreten einer Transplantat-gegen-Wirt Erkrankung (GVHD), steigern. Unter Verwendung von HLA-A2 und human CD8 transgenen Mäusen (CD8yCyA2Kb) konnten in dieser Arbeit PRAME spezifische CD8+ zytotoxischen T-Zellen generiert werden. Diese zytotoxischen CD8+ T-Zellen zeigten in Chromfreisetzungsuntersuchungen lytische Aktivität gegen eine Vielzahl von Zelllinien, die PRAME endogen prozessieren sowie gegen das spezifische PRAME-Peptid. Des Weiteren wurden die hier generierten T-Zellen auf ihre zytotoxische Aktivität gegen akute myeloische Leukämie Blasten hin untersucht, und diese Untersuchungen zeigten AML-Reaktivität der PRAME-spezifischen sowie der als Vergleich genutzten p53- und HLA-A2-spezifischen T-Zellen. Das Potenzial der PRAME-spezifischen ZTL die GVL-Immunität in vivo zu erhöhen ohne das Vorkommen einer GVHD wurde in einem Tumor-Protektions-Model unter der Nutzung von NOD/SCIDgcnull Mäusen untersucht. Die PRA100- bzw. p53-ZTL wurden adoptiv in NOD/SCIDgcnull Rezipienten transferiert und gleichzeitig wurden die Tiere mit PRAME-, oder p53-exprimierende Tumorzelllinien inokuliert. Die Reduktion des Tumorwachstums bestätigte die Spezifität der T-Zellen auch in vivo. In weiteren in vivo Experimenten wurden NOD/SCIDgcnull Mäuse mit AML-Blasten rekonstituiert. Durch die Applikation von nur CD34 positiven Zellen aus einer AML-Probe, oder einer CD56 depletierten Probe, konnten Rekonstitutionen in 95 % aller Versuche erfolgreich beendet werden. Wurde eine Rekonstitution mittels PCR- und FACS-Analysen diagnostiziert, so folgten mehrere Applikationen der PRAME- oder p53-spezifischen ZTL. In diesen Untersuchungen konnten wir in einem therapeutischen AML-in vivo-Modell zeigen, dass die in diesen Untersuchungen generierten/verwandten ZTL in der Lage sind AML-Blasten in vivo zu bekämpfen und so die leukämische Last der Tiere im Blut sowie in der Milz auf unter 1 % zu regulieren. Der prozentuale Anteil humaner AML Zellen im Knochenmark konnte deutlich gesenkt werden (< 10 %). Zusammenfassend sind die von uns generierten PRAME-spezifischen T-Zellen in der Lage, in vitro und auch in vivo, endogen prozessiertes Protein auf Zelllinien und AML-Blasten zu erkennen und zu lysieren. Auch die p53-ZTL, welche als eine weitere Antigen-spezifische ZTL-Population in vivo getestet wurden, zeigten GVL-Effekte. Die Kenntnis von Tumor- bzw. Leukämie assoziierten Antigenen und die daraus erwachsene Möglichkeit der Generierung krankheitsspezifischer ZTL bietet die Grundlage für eine spezifische Immuntherapie maligner Erkrankungen.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

Leucemia Acuta Mieloide e signaling purinergico: possibili sviluppi terapeutici

I nucleotidi trifosfato sono, dal punto di vista evoluzionistico, tra le molecole più antiche e conservate tra le specie. Oltre al ruolo che ricoprono nella sintesi degli acidi nucleici e nel metabolismo energetico della cellula, negli ultimi anni è emerso sempre di più il loro coinvolgimento nella regolazione di numerose funzioni cellulari. Questi importanti mediatori cellulari sono presenti nel microambiente e cambiamenti nella loro concentrazione extracellulare possono modulare la funzionalità cellulare. I nucleotidi trifosfato ATP e UTP, presenti nel microambiente midollare, sono dei potenti stimolatori dei progenitori emopoietici. Essi stimolano la proliferazione e l’attecchimento delle cellule staminali emopoietiche, così come la loro capacità migratoria, attraverso l’attivazione di specifici recettori di membrana, i recettori purinergici (P2R). In questo studio abbiamo dimostrato che ATP e UTP esercitano un effetto opposto sul compartimento staminale leucemico di leucemia acuta mieloide (LAM). Abbiamo dimostrato che le cellule leucemiche esprimono i recettori P2 funzionalmente attivi. Studi di microarray hanno evidenziato che, a differenza di ciò che avviene nelle CD34+, la stimolazione di cellule leucemiche con ATP induce la down-regolazione dei geni coinvolti nella proliferazione e nella migrazione, mentre up-regola geni inibitori del ciclo cellulare. Abbiamo poi confermato a livello funzionale, mediante test in vitro, gli effetti osservati a livello molecolare. Studi di inibizione farmacologica, ci hanno permesso di capire che l’attività inibitoria dell’ATP sulla proliferazione si esplica attraverso l’attivazione del recettore P2X7, mentre i sottotipi recettoriali P2 prevalentemente coinvolti nella regolazione della migrazione sono i recettori P2Y2 e P2Y4. Esperimenti di xenotrapianto, hanno evidenziato che l’esposizione ad ATP e UTP sia dei blasti leucemici sia delle cellule staminali leucemiche CD38-CD34+ diminuisce la loro capacità di homing e di engraftment in vivo. Inoltre, il trattamento farmacologico con ATP, di topi ai quali è stata indotta una leucemia umana, ha diminuito lo sviluppo della leucemia in vivo.

The vulnerable carotid plaque. Identification of endothelial markers predictive of plaque weakness, rupture predisposition and neoangiogenesis

Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group demonstrated that intra-plaque neovessels show both a Nestin+/WT+ and a Nestin+/WT1- phenotype, the latter being correlated with complications and plaque instability. Aims. The aims of the present thesis are: (i) to confirm our previous results on Nestin/WT1 phenotype in a larger series of carotid atheromatous plaques, (ii) to evaluate the relationship between the Nestin+/WT1- neoangiogenesis phenotype and plaque morphology, (iii) to evaluate the relationship between the immunohistochemical and histopathological characteristics and the clinical instability of the plaques. Materials and Methods. Seventy-three patients (53 males, 20 females, mean age 71 years) were consecutively enrolled. Symptoms, brain CT scan, 14 histological variables, including intraplaque hemorrhage and diffuse calcifications, were collected. Immunohistochemistry for CD34, Nestin and WT1 was performed. RT-PCR was performed to evaluate Nestin and WT1 mRNA (including 5 healthy arteries as controls). Results. Diffusely calcified plaques (13 out of 73) were found predominantly in females (P=0.017), with a significantly lower incidence of symptoms (TIA/stroke) and brain focal lesions (P=0.019 and P=0.013 respectively) than not-calcified plaques, but with the same incidence of intraplaque complications (P=0.156). Accordingly, both calcified and not calcified plaques showed similar mean densities of positivity for CD34, Nestin and WT1. The density of Nestin and WT1 correlated with the occurrence of intra-plaque hemorrhage in all cases, while the density of CD34 correlated only in not-calcified plaques. Conclusions. We confirmed that the Nestin+/WT1- phenotype characterizes the neovessels of instable plaques, regardless the real amount of CD34-positive neoangiogenesis. The calcified plaques show the same incidence of histological complications, albeit they do not influence symptomatology and plaque vulnerability. Female patients show a much higher incidence of not-complicated or calcified plaques, receiving de facto a sort of protection compared to male patients.