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Oltre la chemioterapia

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1 Oltre la chemioterapia
prof. Giuseppe Naso ONCOLOGIA MEDICA POLICLINICO UMBERTO I

2 Recent gene-profiling studies have nicely confirmed that ER-
positive and ER- negative breast cancer are essentially two different disease M.J.Piccart-Gebhart: JCO vol.21, 8: 2003

3 ER+ ER- HER-2+ ?

4

5 The EGFR (ErbB) family and ligands
TGFa Amphiregulin b-cellulin HB-EGF Epiregulin NRG2 NRG3 Heregulins b-cellulin Heregulins Cysteine-rich domains 100 44 82 33 36 59 24 48 79 28 Tyrosine kinase domain Her 2: Recettore di membrana ad attività tirosinochinasi appartenente alla famiglia di EGFR. Condivide con gli altri membri della famiglia dei recettori tirosin-chinasi di tipo I, la struttura generale in cui si riconoscono una parte esterna glicosilata, un singolo elemento idrofobico ad alfa elica che attraversa la membrana cellulare e una parte intracitoplasmatica contenente un sito di fosforilazione con funzione di regolazione negativa. Rispetto agli altri membri della famiglia non è stato identificato alcun ligando che interagisca direttamente con erbB-2. C-terminus ErbB-1 Her1 EGFR ErbB-2 Her2 neu ErbB-3 Her3 ErbB-4 Her4

6 Cell surface K R R K

7 Cell surface R R K K

8 Cell surface Intra-Cellular Signaling
PI3K Shc Grb2 Ras Sos Raf MEK1/2 Akt MAPK PTEN GSK-3 mTOR FKHR Bad Intra-Cellular Signaling NF- [PI4P,PI4,5 P2] [PI3,4P2 PI3,4,5 P3] K K

9 Cell Cycle Progression
Cell surface R R PI3K Shc Grb2 Ras Sos Raf MEK1/2 Akt MAPK PTEN GSK-3 mTOR FKHR Bad Intra-Cellular Signaling NF- [PI4P,PI4,5 P2] [PI3,4P2 PI3,4,5 P3] K K p27 Gene Transcription/ Cell Cycle Progression

10 Cell Cycle Progression
Cell surface R R PI3K Shc Grb2 Ras Sos Raf MEK1/2 Akt MAPK PTEN GSK-3 mTOR FKHR Bad Intra-Cellular Signaling NF- [PI4P,PI4,5 P2] [PI3,4P2 PI3,4,5 P3] K K rac rho p27 Gene Transcription/ Cell Cycle Progression Survival Proliferation Angiogenesis Metastasis Cellular Responses

11 Anti ErbB1-2 receptors Mabs
(Trastuzumab, 2C4, C225) HER1,HER2,HER4, Tyrosine Kinase Inhibitors (ZD 1839, OSI 774, EKB 559, GW 2016, CI 1033) K K SHC GRB2 PI3K SOS Ras farnesyl transferase Inhibitors (BMS , R115777) RAS PTEN AKT RAF RAF inhibithors mTOR inhibitors (CCI 779) Mek inhibitors (CI 1040) MEK 1/2 mTOR FKHR GSK-3 BAD MAP Cell cicle progression Survival

12 Meccanismo d’azione di Estradiolo e Tam
a confronto AF1 ERE ATTIVAZIONE COMPLETA DELLA TRASCRIZIONE ESTRADIOLO ER ERE AF2 ERE ATTIVAZIONE PARZIALE DELLA TRASCRIZIONE (solo AF1) AF1 TAMOXIFEN ER AF2

13 Es A.I. FASLODEX AF1 ERE ATTIVAZIONE COMPLETA DELLA TRASCRIZIONE
ESTRADIOLO ER ERE AF2 AF1 A.I. FASLODEX Es BLOCCO COMPLETO DELLA TRASCRIZIONE ER AF2

14 In most cases AF-1/AF-2 are present in resistant tumours,
and in many of these its activity continues to regulate tumour growth

15 La cascata delle chinasi della via ras
ras-GDP ras-GTP Tyrosine Kinase SOS raf GRB2 forma inattiva Forma attivata ATP MEK ATP MAPK MAP ATP fos myc jun

16 Hormone-dependent growth
La cascata delle chinasi della via ras ras-GDP ras-GTP Tyrosine Kinase SOS raf GRB2 forma inattiva Forma attivata ATP MEK ATP MAPK AKT MAP ATP COREPRESSORE fos myc jun AF1 MAP COATTIVATORE ATP Hormone-dependent growth

17 Che succede se si utilizzano contemporaneamente
un antiestrogeno e un inibitore della TK attivata dagli EGFRs ?

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22 Effects of ER and EGFR blockade are:
Reproducible: Total cell kill has been achieved in 5/6 experiments by 4 months using Tamoxifen and Iressa Seen with other anti-hormonal drugs: Faslodex plus Iressa achieved a total cell kill by 3 months Observed in other cell lines: T47D cells show equivalent early responses

23 Role of HER2 in breast cancer
HER2 gene amplification or receptor overexpression occurs in approximately 25 % of breast cancers HER2-positive tumours are associated with poor prognosis L’overspressione di Her-2 è presente in circa il 30% di carcinoma mammario ed è associato a una prognosi peggiore

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25 HER2 status as a prognostic indicator
Infatti come fattore prognostico, Her-2, in un’analisi uni e multivariata, è secondo solo allo stato linfonoale. n=86 node-positive patients/Southern blot assay/median follow-up 46 months ER = oestrogen receptor PR = progesterone receptor Slamon DJ et al. Science 1987; 235: 177–182

26 HER2 status as a prognostic indicator: Disease-free survival of node-positive breast cancer patients related to HER2 status 100 80 60 40 20 HER2 gene <3 copies Disease-free survival probability HER2 gene ³3 copies Differenza delle curve di Disease Free Survival in pazienti N+ in rapporto alla overspressione di Her2 Log rank p=0.001 Time (months) Seshadri R et al. J Clin Oncol 1993; 11: 1936–1942

27 HER2 status as a prognostic indicator: Survival of node-negative breast cancer patients related to HER2 status 1.00 0.75 0.50 0.25 Not amplified Cumulative probability Amplified Amplified: >10 copies/nucleus Not amplified: <3 copies/nucleus Borderline: excluded Curve di sopravvivenza in pazienti con N negativi, statisticamente significative. Log rank p<0.001 Time to death (months) Ross JS, Fletcher JA. Stem Cells 1998; 16: 413–428

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29 HER hierarchy

30 Bivalence of EGF-like ligands

31 Relative potency of HER dimers

32 HER2-promoted recycling of HER1
cbl

33 Herceptin® in breast cancer: what evidence do we have?
Metastatic breast cancer erbB2 overexpression No prior chemotherapy for MBC Measurable disease KPS ≥60% Trastuzumab + AC (n=143) No prior anthracyclines AC (n=138) Eligible patients (n=469) Trastuzumab + Paclitaxel (n=92) Questo è lo studio registrativo nel quale pazienti affette da carcinoma mammario sono state randomizzate a trattamento chemioterapico con o senza herceptin Vedi dettagli nella diapo. Prior anthracyclines Paclitaxel (n=96) Slamon et Al N Engl J Med, March 2001

34 os = p 0.05 Slamon DJ at al. 2001

35 Herceptin + Taxani (+/- Platino) TTP (3 studi randomizzati)
Slamon NEJM 2001 Robert SABCS 2002 Marty ECCO 2003

36 Herceptin + Taxani (+/- Platino) OS (3 studi randomizzati)
p=NR Slamon NEJM 2001 Marty ECCO 2003 Robert SABCS 2002

37 Goal of the second-generation HER2 programme
Develop an agent with clinical activity superior to Herceptin® For example Better response rate Longer duration of response More clinical benefit in tumours that express intermediate (2+ or 1+) or even low levels (0) of HER2

38 Pertuximab (2C4)

39 Anti-HER2 epitope map Extracellular domain 7C2 2C4 Pertuximab CRD-1
(aa 23–53) 2C4 Pertuximab (aa 22–584) CRD-1 CRD-2 Herceptin® (aa 529–625) Intracellular domain TK CT Bald L, Fendly B

40 Inhibition of heregulin binding by 2C4, a monoclonal antibody to HER2
HER2:HER3 HER3 0.10 0.08 0.06 0.04 0.02 0.00 Bound/total 2C4 (nM)

41 Inhibition of ligand binding by 2C4 to HER2-containing receptor complexes
100 80 60 40 20 Control 2C4 EGF Relative ligand binding (%) HRG HRG EGFR/HER2 HER3/HER2 HER4/HER2 Receptor combination

42 Effect of Herceptin® or 2C4 on ligand-mediated MAP kinase activation
EGF – – – – – – – TGFa – – – – – – – HRG – – – – – – – + + + 2C4 – – + – – + – – + – Herceptin® – – – + – – + – – +

43 Summary In contrast to Herceptin®, 2C4 is very effective in
Blocking HER2 association with other HER family members Inhibiting HER2 activation and subsequent downstream signalling suppressing the growth of tumours with ligand-activated HER2

44 NC TC METASTASYS CELL GROWTH (Apoptotic blockade) (HIF-1 HIF-2)
ONCOGENES ACTIVATION ONCO-SUPPRESSOR GENES INACTIVATION MSR – NER – BER - OSR TC CELL GROWTH (Apoptotic blockade) CELL GROWTH VEGF-A VEGF-B VEGF-C VEGF-D neo-ANGIOGENESIS METASTASYS HYPOSSIA (HIF-1 HIF-2) neo-LYNPHOGENESIS

45 Angiogenesis is required for solid tumor growth
INTRODUCTION Angiogenesis is required for solid tumor growth The switch to an angiogenic phenotype represent a pivotal step in the multistage process toward malignancy (Folkman 1996) For solid tumors to grow beyond 1-2 mm3 they require increased access to oxygen and nutrients from the blood supply (Folkman 1971) 1-2 mm3

46 Solid tumors stimulate angiogenesis by secreting angiogenic factors
INTRODUCTION Solid tumors stimulate angiogenesis by secreting angiogenic factors Tumor angiogenesis begins when growing tumors, in response to hypoxia or other stimuli, secrete angiogenic factors Vascular endotelial growth factor is one of the most potent promoters of angiogenesis and has been identified as a fundamental regulator of tumor neovascolarizzazion

47 INTRODUCTION Tumor angiogenesis promotes tumor growth and metastasis
The creation of new blood vessels and lymphatic tissue by solid tumors allows them to enlarge and to metastasize to distal sites Inhibition of tumor angiogenesis, therefore, has the potential to inhibit tumor growth and spread VEGF levels apper to have prognostic significance in human tumors ( LAM, breast cancer, colon cancer,hepatocellular cancer, NSCLC and ovarian cancer)

48 The VEGF family are critical tumor-secreted angiogenic factors
INTRODUCTION The VEGF family are critical tumor-secreted angiogenic factors The vascular endothelial growth factor (VEGF) family are critical tumor secreted signaling molecules that stimulate angiogenesis and lymphangiogenesis There are five members of the VEGF family (VEGF-A, placental growth factor, VEGF-B, VEGF-C, VEGF-D, VEGF-E) VEGF is a diffusabile endothelian cell-specific mitogen pro-angiogenic factor that increases vascular permeability VEGF-B VEGF-A VEGF-E VEGF-C VEGF-D

49 Different VEGF family members bind and activate different VEGF receptors on endothelial cells
VEGFR-1 is activated by VEGF-A and VEGF-B VEGFR-2 is activated by VEGF-A, VEGF-C, VEGF-D, and VEGF-E VEGFR-3 is activated by VEGF-C and VEGF-D

50 BEVACIZUMAB (avastin; Genentech)
Monoclonal antibody, specific for VEGF A Derived from the murine antibody A4.6.1, comprises 93% human antibody and 7% murine derived antigen-binding-protein, the humanization proving a longer half-life and less immunogenicity Able to neutralize the biological properties of human VEGF, including endothelian cell mitogenic activity, vascular permeability-enhancing activity and angiogenic properties( Kim K.1992) The antibody did not recognize the other growth factor tested including FGF, EGF, PDGF

51 BEVACIZUMAB: Preclinical Studies
Extensive preclinical studies have demonstrated the efficacy in different cell lines subcutaneously injcect into nude mice ( Kim KJ 1993) while did not reveal a direct effect on the growth of tumor cells (Warren RS 1995) TUMOR SUPPRESSION IS MEDIATED THROUGH INHIBITION OF NEOVASCULARIZZATION Warren and coworkers demonstrated that treatment with anti-VEGF ab was effective in suppressing primary tumor growth as well liver metastatis growth in a murine model of Colorectal cancer

52 BEVACIZUMAB: Clinical Studies
More than 30 clinical trials with Bevacizumab in solid tumors and hematologic malignancies are planned, ongoing or recenttly completed In general, the therapeutic strategies being evaluated can be diveded into the following categories: Bevacizumab monotherapy Bevacizumab in combination with chemotherapy or radiotherapy Bevacizumab in combination with other putative anti-angiogenic strategies ( low-dose INF, thalidomide..) Bevacizumab in combination with tumor-target therapies ( erlotinib, trastuzumab, imatinib) Bevacizumab in combination with immunotherapy

53 Bevacizumab phase I-II clinical trial efficacy results in breast cancer
3mg/kg mg/Kg mg/Kg Total N° of Pts Objective Response Rate (n/%) (5.6%) (7.3%) (6.3%) (6.7) Median Response Duration (months) Stable disease at 154 days (11%) (17%) (19%) (16%) Median Survaival (months) Sledge GW et al. JCO 2003

54 BEVACIZUMAB:Summary of trials in solid tumor

55 Inhibition of Ubiquitin-Proteasome pathway
Bortezomib

56 L’emivita di una proteina è determinata in gran parte dal
suo residuo terminale Residui altamente stabilizzanti (t1/2 >20 ore) Ala Cys Gly Met Pro Ser Thr Val Residui intrinsecamente destabilizzanti (t1/2= da 2 a 390 min.) Arg His He Leu Lys Phe Trp Tyr Residui destabilizzanti solo dopo modificazione chimica Asn Asp Gln Glu

57 Degrades more than 80% proteins
The Proteasome: Enzyme with Important Impact on Multiple Regulatory Pathways Is found in all eukariotic cells, from yeast to man Is present in the cytoplasm and nucleus Degrades more than 80% proteins

58 Tumor microenvironment
Extracellular matrix

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70 Inibitori di traduzione del segnale:
Ras e FT-I

71 Neoplastic Transformation Immortalization Inactivation of Rb and p53
telomerase activation Neoplastic Transformation Activation of ras-pathway Weinberg, A. 2003

72 Farnesyl-transferase inhibitors
The ras gene is mutated in one fifth of all human cancers. I geni RAS sono una famiglia di geni altamente conservati identificati in organismi evolutivamente distanti quali lieviti, drosofila, nematodi, xenpus e mammiferi. I prodotti funzionali dei geni RAS presentano un’alta omologia strutturale e funzionale. Nell’uomo e roditori vi sono 3 geni RAS: H-RAS, K-RAS e N-RAS. Le proteine RAS di mammifero sono 4 in quanto KRAS dà origine per splicing alternativo a 2 due diversi prodotti, e hanno un ruolo chiave nella trasduzione del segnale, nella proliferazione cellulare e nella trasformazione cellulare; infatti, ognuna delle proteine RAS espressa dalle cellule di mammifero può funzionare come oncogene in seguito a mutazioni attivanti. La potenzialità trasformante degli oncogeni RAS è dovuta a sostituzioni di singoli aminoacidi in posizioni specifiche caratterizzando circa un quinto dei tumori umani e il 50% dei tumori del colon.

73 RAS

74 ras (p21) G-Protein (intrinseca attività GTP-asica)
ras agisce come un interruttore molecolare che si alterna da una forma inattiva (ras-GDP) ad una forma attiva (ras-GTP)

75 The switch function of RAS

76 La cascata delle chinasi della via ras
ras-GDP ras-GTP Tyrosine Kinase SOS raf GRB2 forma inattiva Forma attivata ATP MEK ATP MAPK MAP ATP fos myc jun

77 Prenilazione

78 Carbossi-terminale (C-terminale)
Prenilazione Carbossi-terminale (C-terminale) CAAX CC – CXC - CCXX Farnesilazione es.: ras,rho Geranil-geranilazione es.:rab Gruppo Farnesile (C15) Gruppo Geranilgeranile (C20) C=cisteina A=residui alifatici X=qualunque aminoacido

79 C15 affinchè acquisisca azione trasformante, la proteina RAS, occorre che vada incontro a una modificazione post-trasduzionale che prevede tra l’altro l’attacco con un gruppo a 5 atomi di carbonio (farnesile), che risulta essenziale per l’ancoraggio delle proteine RAS alla membrana plasmatica. Partendo da questa osservazione sono stati sviluppati gli inibitori della farnesil trasferasi che bloccano questo passaggio.

80 Classes of FTase inhibitors
Peptide CAAX peptidomimetics : L-778,123 Nonpeptide CAAX peptidomimetics : Tipifarnib, Sarosat Bisubstrate Inhibitors : BMS Inhibitors classes I Inhibitors classes II

81 Peptide CAAX peptidomimetics : L-778,123
affinchè acquisisca azione trasformante, la proteina RAS, occorre che vada incontro a una modificazione post-trasduzionale che prevede tra l’altro l’attacco con un gruppo a 5 atomi di carbonio (farnesile), che risulta essenziale per l’ancoraggio delle proteine RAS alla membrana plasmatica. Partendo da questa osservazione sono stati sviluppati gli inibitori della farnesil trasferasi che bloccano questo passaggio. Peptide CAAX peptidomimetics : L-778,123 Nonpeptide CAAX peptidomimetics : Tipifarnib, Sarosat Bisubstrate Inhibitors : BMS

82 Phase I-II studies with farnesyl-trasferase inhibitors in clinical development
Drug Company Route RD DLT Activity R Janssen/ oral mg bid, Myelosuppression, PR NSCLC Zarnestra Ortho-Biotech fatigue, Tipifarnib neurotoxicity,skin SCH Schering – Plough oral mg bid, Nausea, vomiting, 1 PR NSCLC Sarosar diarrhea, fatigue BMS Bristol Meyer iv mg/m2 24 h liver, neuropathy Squibb c.i weekly L-778,123 Merck iv 560 mg/m2 c.i Myelosuppression, sonnolence, fatigue Ad oggi gli inibitori delle farnesiltrasferasi in fase di studio sono 4. Studi di fase I con gli inibitori delle farnesiltrasferasi, in fase di sviluppo clinico.

83 Phase II clinical trials
Tipifarnib (R115777): Phase II clinical trials Although breast tumors show a low incidence of ras mutations, tipifarnib showed preclinical activity in some cell lines. A phase 2 study was conducted in 42 patients with breast cancer who had failed 2 lines of chemotherapy and/or 2 lines of endocrine therapy. Most patients received doses of tipifarnib at 300 mg twice daily. Partial responses (median time, 26 weeks) were seen in 10% of the patients, with 15% demonstrating stable disease for 6 months or longer for a combined clinical benefit seen in 25% of patients. Disease regression was documented in metastases to liver, lung, lymph nodes, and skin; the median time to progression was 13 weeks. The antitumor effect was not related to estrogen receptor, HER-2/neu, or ras mutation status. Johnston SR.: SABCS 2000

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85 Combination of 2 selective inhibitors approach

86 Combined blockade of EGFR and VEGF
Erlotinib Cetuximab, etc Bevacizumab etc. EGFR Endothelial cells Cancer cells VEGF Angiogenesis Cell Proliferation Tortora 2004

87 Combined blockade of EGFR and VEGF
Jung YD et al, Eur J Cancer 2002; 38: Ciardiello F et al. Clin Cancer Res 2000; 6:

88 ZD6474 Inhibits KDR and EGFR
TGF EGFR ZD6474 KDR Endothelial cells Cancer cells VEGF Angiogenesis Cell Proliferation Tortora & Ciardiello, 2002

89 ZD6474: Phase I Studies ZD6474 is a potent orally-active inhibitor of VEGF-signalling Inhibits VEGF-induced hypotension, vascular permeability and angiogenesis in vivo Broad-spectrum anti-tumour activity in mouse models consistent with an anti-angiogenic mechanism Dosing for > 100 days has been feasible in man Phase I adverse events generally mild Anti-VEGF activity suggested by hypertension and delay in dermal wound angiogenesis

90 ZD6474 inibisce la crescita di tumori divenuti resistenti a Gefitinib o Cetuximab
controllo controllo Cetuximab Gefitinib >ZD6474 Cetuximab > ZD6474 Ciardiello et al., 2004

91 Agents affecting all VEGFRs
AZD2171 Enzastaurin

92 AZD2171 AZD2171 VEGFR3 (Flt-4) VEGFR1 (Flt-1) VEGFR2 (KDR) AZD2171 is an oral therapy with potential application in multiple tumor types AZD2171 has activity against VEGF receptors 1, 2 & 3- No activity on EGFR Phase I clinical studies in refractory solid tumors underway Manageable toxicity in early phase I

93 Enzastaurin Potent oral selective inhibitor of PKC (IC50 = 0.006 M)
Is an acyclic indolylmaleimide that competes with the ATP binding site preventing substrate phosphorylation Inactive against other kinases Antiangiogenic activity in preclinical models. Well tolerated in preclinical toxicology studies. Administered once daily PO. Normal volunteer and Phase I studies completed. Phase II studies ongoing. MW = Company Confidential Copyright © 2004 Eli Lilly and Company

94 PKC-b and the Proposed Action of Enzastaurin on Angiogenesis and Apoptosis
VEGF ENZASTAURIN Receptor DAG Tumor invasion Angiogenesis IP3/Ca2+ Apoptosis PKC-b AKT COX2 GSK3b Caspase 9 IL-8 IL-6 Protein translation Activation mRNA Company Confidential Copyright © 2004 Eli Lilly and Company

95 Blood vessels are not only endothelial cells (L.M. Ellis)
Pericytes protects endothelial cells from apoptosis and overexpress PDGF-R PDGF-R is overexpressed by many tumors PDGF-R and VEGF cooperate Improving Anti-VEGF therapy by expanding the targets

96 Multiple target agents affecting
VEGFRs (and PDGF-R) PTK787 SU11248

97 PTK/ZK: A multi-VEGF receptor tyrosine kinase inhibitor
Formula: C24H21N4Cl MW = Complete inhibitor of the VEGF receptor tyrosine kinases VEGFR1(FLT-1), VEGFR2 (KDR) and VEGFR3 (FLT-4). It also inhibits PDGF-R. Well tolerated up to 1250 mg/day (phase III dose, used up to 15 mo) Rapidly absorbed (1 to 2.5 hours), T1/2: 3-6 hrs Renal metabolism The purpose of this slide is to introduce PTK/ZK as a multi-VEGF receptor inhibitor PTK/ZK is multi-VEGF receptor inhibitor that completely inhibits the activation of VEGF receptors: VEGFR-1, VEGFR-2, and VEGFR-31,2 PTK/ZK also inhibits the PDGF receptor which plays a role in blood vessel stabilization PTK/ZK is being codeveloped by Novartis Pharmaceuticals Corporation AG and Schering AG Berlin, Germany 1. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK , a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 2000;60: 2. Bold G, Altmann KH, Frei J, et al. New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis. J Med Chem. 2000;43(12):

98

99 Conclusioni (I)

100 Genes changing in association with response
Good responders Poor responders 256 genes 49 genes 14 genes shared biologically responsive biologically refractory

101 Response in patients treated with FAC/paclitaxel preoperative chemotherapy
150 Genes pCR NoPCR Hortobagyi GN ASCO 2002

102 Conclusioni (II)

103 “…A targeted therapy only works when the target is present and plays an important role in the cancer…”

104 IRESSA and NSCLC: a very important lesson No target % of Response Target % of Response

105 dott.ssa A. De Benedetto dott. A. Lugini
Prof. Enrico Cortesi dott.ssa A. De Benedetto dott. A. Lugini dott. A. Padovani dott.ssa R. Ferraldeschi dott.ssa G. D’auria dott. P. Ceratti dott.ssa N. Pizzardi dott.ssa L. Mentuccia dott.ssa M. Mazzoli dr. V. Picone Oncologia Medica B Univ. Di Roma “La Sapienza”


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