Therapeutic potential of bleomycin plus suicide or interferon-β gene transfer combination for spontaneous feline and canine melanoma

We originated and characterized melanoma cell lines derived from tumors of two feline and two canine veterinary patients. These lines reestablished the morphology, physiology and cell heterogeneity of their respective parental tumors. We evaluated the cytotoxicity of bleomycin (BLM) alone, or combined with interferon-β (IFN-β) or HSVtk/GCV suicide gene (SG) lipofection on these cells. Although the four animals presented stage III disease (WHO system), SG treated feline tumors displayed stable disease in vivo, while the canine ones exhibited partial response. Their derived cell lines reflected this behavior. Feline were significantly more sensitive than canine cells to IFN-β gene transfer. BLM improved the antitumor effects of both genes. The higher levels of reactive oxygen species (ROS) significantly correlated with membrane and DNA damages, emphasizing ROS intervention in apoptotic and necrotic cell death. After 3 days of BLM alone or combined with gene treatments, the colony forming capacity of two canine and one feline treatments survivor cells almost disappeared. Taken together, these results suggest that the treatments eradicated tumor initiating cells and support the clinical potential of the tested combinations.Fil: Agnetti, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Fondello, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Villaverde, Marcela Solange. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentin

Therapeutic potential of gene therapy in combination with bleomycin for melanoma treatment

El melanoma es una forma de cáncer de piel extremadamente agresiva cuya incidencia continúa en aumento. Se establecieron y caracterizaron tres líneas celulares de melanoma humano. También, se utilizaron ocho líneas de melanoma canino, y dos de melanoma felino, derivadas de tumores de pacientes de nuestros protocolos clínicos veterinarios. A pesar de la heterogeneidad y plasticidad típicas del melanoma, todas las líneas evaluadas fueron sensibles a la bleomicina (BLM) co-administrada durante la transferencia del gen suicida (GS) o del gen del interferón-β (IFNβ). La magnitud de la respuesta a la terapia génica con IFNβ o GS no se vio limitada por la eficiencia de lipofección debido a un amplio efecto bystander. Se halló una correlación significativa entre el efecto citotóxico de la quimio-genoterapia y la fracción celular exhibiendo DNA subdiploide, altos niveles de especies reactivas del oxígeno (ROS) y pérdida del potencial mitocondrial (Δψm). Estos datos sugieren que la quimio-genoterapia induce una apoptosis/necrosis dependiente de la mitocondria. Durante el tratamiento de pacientes veterinarios con GS, se observaron aumentos transitorios del tamaño tumoral (pseudo-progresión) resultando, finalmente, en la reducción o estabilización tumoral. El índice proliferativo (IP, fracción de células en fases S, G2/M e hiperdiploide) podría reflejar la respuesta de las células tumorales. La respuesta al GS in vivo e in vitro correlacionó con el incremento del IP debido al GS (IPGS) de las líneas celulares derivadas de los tumores. Esto último, sumado a la disminución en la capacidad de formar colonias y melanoesferas, sugiere que la expresión sostenida de IFNβ y GS en combinación con BLM, reduce la fracción de células iniciadoras de tumor (TIC), al inducir una constante proliferación de las mismas. Todos estos resultados permiten vislumbrar el potencial clínico de esta estrategia.Malignant melanoma is an extremely aggressive cancer whose incidence increase worldwide. We established and characterized three human melanoma cell lines. We also used eight canine and two feline melanoma cell lines derived from tumors from patients of our veterinary clinical trials. Even though the melanoma displays typical heterogeneity, all the cell lines were sensitive to bleomycin (BLM) co-administrated with suicide- (SG) or interferon-β gene (IFNβ) transfer. Because of the bystander effect, the response magnitude of suicide- (SG) or interferon-β gene therapy was not limited by lipofection efficiency. A significant correlation was found between the cytotoxic effect of BLM and both genetic treatments and the fraction of cells exhibiting hypodiploid DNA content, high ROS levels and mitochondria membrane depolarization. These results suggest that these chemo-genetic treatments induced a mitochondria-dependent apoptosis/necrosis. During SG treatment of melanoma canine patients, we observed transient increases of tumor size (pseudo-progression) that finally resolved as tumor reduction. The proliferative index (IP, fraction of cells in S, G2/M and hiperdiploid phase) might reflect tumor cell response. The in vivo and in vitro GS response correlated with IP increment (IPGS) caused by GS treatment of the tumor derived cell lines. These results, and the significant reduction in the colony- and sphere-forming capacity, suggest that BLM and the sustained expression of IFNβ and SG, reduced the melanoma TIC compartment, by promoting long term TIC self-renewal repopulating activity. All these data support the clinical potential of this strategy.Fil: Fondello, Chiara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Mechanisms enhancing the cytotoxic effects of bleomycin plus suicide or interferon-β gene lipofection in metastatic human melanoma cells

Background: Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. Objective: To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-β gene (hIFNβ) lipofection in early passages of these melanoma cell lines. Methods: In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony forming capacity and mitochondrial membrane depolarization after treatments. Results: The three assayed cell lines displayed sensitivity to single and combined BLM/gene treatments. BLM improved the antitumor and anti-clonogenic effects of SG and hIFNβ genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFNβ gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFNβ gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFNβ gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. Conclusion: The presented results indicate that BLM improves the antitumor effects of SG and hIFNβ transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.Fil: Fondello, Chiara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Agnetti, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentin

Proliferation index and pseudoprogression as predictors of the therapeutic efficacy of suicide gene therapy for canine melanoma

In our veterinary clinical trials, the combination of systemic immunotherapy with local HSVtk/GCV suicide gene (SG) treatment induced tumor pseudo-progression as part of a strong local anti-tumor response. This phenomenon could be due to tumor inflammation, increased vascular permeability and to different tumor growth rates before, during and after SG therapy. The proliferation index (PI: the fraction of viable cells in S, G2/M and hyperdiploid phases) would reflect the in vivo and in vitro proportion of proliferating melanoma cells in the absence of treatment (PIB) or in response to SG (PISG).The extent of in vivo and in vitro melanoma cells response to SG exhibited a reverse correlation with PIB and a direct correlation with PISG. Then, the final SG outcome depended on the balance between PIB-dependent "re-growth resistance" vs. "re-growth sensitivity" to SG treatment (PIGS). In all the cell lines derived from canine tumors presenting partial responses to SG treatment, PISG prevailed over PIB. Conversely, as more aggressive was the tumor (greater PIB of the cell line) more the balance displacement towards re-growth resistance overcome SG re-growth sensitivity. All these parameters could have prognostic value for SG treatment response and allow us to glimpse the clinical benefit of this therapy.Fil: Fondello, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Agnetti, Lucrecia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Glikin, Gerardo Claudio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Combination of cytokine-enhanced vaccine and chemo-gene therapy as surgery adjuvant treatments for spontaneous canine melanoma

After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-β gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.Fil: Finocchiaro, Liliana Maria Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Agnetti, Lucrecia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fondello, Chiara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Glikin, Gerardo Claudio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Particulate mediators of the bystander effect linked to suicide and interferon-β transgene expression in melanoma cells

In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-â gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.Fil: Agnetti, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Fondello, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Arbe, María Florencia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Glikin, Gerardo Claudio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

The combination of bleomycin with suicide or interferon-B gene transfer is able to efficiently eliminate human melanoma tumor initiating cells

We explored the potential of a chemogene therapy combination to eradicate melanoma tumor initiating cells, key producers of recurrence and metastatic spread. Three new human melanoma cell lines, two obtained from lymph nodes and one from spleen metastasis were established and characterized. They were cultured as monolayers and spheroids and, in both spatial configurations they displayed sensitivity to single treatments with bleomycin (BLM) or human interferon-β (hIFNβ) gene or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) lipofection. However, the combination of bleomycin with SG or hIFNβ gene transfer displayed greater antitumor efficacy. The three cell lines exhibited a proliferative behavior consistent with melan A and gp100 melanoma antigens expression, and BRAF V600E mutation. BLM and both genetic treatments increased the fraction of more differentiated and treatment-sensitive cells. Simultaneously, they significantly decreased the sub-population of tumor initiating cells. There was a significant correlation between the cytotoxicity of treatments with BLM and gene transfer and the fraction of cells exhibiting (i) high proliferation index, and (ii) high intracellular levels of reactive oxygen species. Conversely, the fraction of cells surviving to our treatments closely paralleled their (i) colony and (ii) melanosphere forming capacity. A very significant finding was that the combination of BLM with SG or hIFNβ gene almost abrogated the clonogenic capacity of the surviving cells. Altogether, the results presented here suggest that the combined chemo-gene treatments are able to eradicate tumor initiating cells, encouraging further studies aimed to apply this strategy in the clinic.Fil: Fondello, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Agnetti, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Simian, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad Nacional de San Martin. Instituto de Nanosistemas; ArgentinaFil: Villaverde, Marcela Solange. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentin

Inhibition of bioenergetic metabolism by the combination of metformin and 2-deoxyglucose highly decreases viability of feline mammary carcinoma cells

Feline mammary carcinoma (FMC) is a highly aggressive pathology that has been proposed as an interesting model of breast cancer disease, especially for the hormone refractory subgroup. Recently, cancer cell metabolism has been described as a hallmark of cancer cells. Here, we investigate the effects and mechanism of metabolic modulation by metformin (MET, anti-diabetic drug), 2-deoxyglucose (2DG, hexokinase inhibitor) or a combination of both drugs, MET/2DG on two established FMC cells lines: AlRB (HER2 (3 +) and Ki67 15%). We found that treatments significantly decreased both FMC cells viability by up to 80%. AlRB resulted more sensitive to 2DG than AlRATN (IC50: 3.15 vs 6.32 mM, respectively). The combination of MET/2DG potentiated the effects of the individually added drugs on FMC cells. In addition, MET/2DG caused an increased in intracellular oxidants, autophagic vesicles and completely inhibited colony formation.Conversely, only MET significantly altered plasma membrane integrity, presented late apoptotic/necrotic cells and increased both glucose consumption and lactate concentration. Our results support further studies to investigate the potential use of this metabolic modulation approach in a clinical veterinary setting.Fil: Arbe, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Fondello, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Agnetti, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Alvarez, Gabriel Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Área de Química Biológica; ArgentinaFil: Tellado, Matías Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Área de Química Biológica; ArgentinaFil: Glikin, Gerardo Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Finocchiaro, Liliana Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Villaverde, Marcela Solange. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Cytokine-Enhanced Vaccine and Interferon-β plus Suicide Gene Therapy as Surgery Adjuvant Treatments for Spontaneous Canine Melanoma

We present here a nonviral immunogene therapy trial for canine malignant melanoma, an aggressive disease displaying significant clinical and histopathological overlapping with human melanoma. As a surgery adjuvant approach, it comprised the co-injection of lipoplexes bearing herpes simplex virus thymidine kinase and canine interferon-β genes at the time of surgery, combined with the periodic administration of a subcutaneous genetic vaccine composed of tumor extracts and lipoplexes carrying the genes of human interleukin-2 and human granulocyte-macrophage colony-stimulating factor. Following complete surgery (CS), the combined treatment (CT) significantly raised the portion of local disease-free canine patients from 11% to 83% and distant metastases-free (M0) from 44% to 89%, as compared with surgery-only-treated controls (ST). Even after partial surgery (PS), CT better controlled the systemic disease (M0: 82%) than ST (M0: 48%). Moreover, compared with ST, CT caused a significant 7-fold (CS) and 4-fold (PS) rise of overall survival, and >17-fold (CS) and >13-fold (PS) rise of metastasis-free survival. The dramatic increase of PS metastasis-free survival (>1321 days) and CS recurrence- and metastasis-free survival (both >2251 days) demonstrated that CT was shifting a rapidly lethal disease into a chronic one. In conclusion, this surgery adjuvant CT was able of significantly delaying or preventing postsurgical recurrence and distant metastasis, increasing disease-free and overall survival, and maintaining the quality of life. The high number of canine patients involved in CT (301) and the extensive follow-up (>6 years) with minimal or absent toxicity warrant the long-term safety and efficacy of this treatment. This successful clinical outcome justifies attempting a similar scheme for human melanoma.Fil: Finocchiaro, Liliana Maria Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fondello, Chiara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gil Cardeza, Maria Lourdes. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossi, Ursula Amaranta. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Villaverde, Marcela Solange. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Riveros, Maria D.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; ArgentinaFil: Glikin, Gerardo Claudio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin