Maria Teresa Del Vecchio

Chemo-Immunotherapy of Metastatic Colorectal Carcinoma With Gemcitabine Plus FOLFOX 4 Followed by Subcutaneous Granulocyte Macrophage Colony-Stimulating Factor and Interleukin-2 Induces Strong Immunologic and Antitumor Activity in Metastatic Colon Cancer Patients

PURPOSE Tumor cell killing by anticancer drugs may be supported by their immuno- and pharmacologic effects. Chemotherapy is in fact able to (A) upregulate tumor-associated antigen expression, including carcinoembryonic antigen (CEA) or other target molecules such as thymidylate synthase (TS); and (B) downregulate tumor cell resistance to the death signals induced by tumor antigen-specific cytotoxic T lymphocytes. This provides the rationale for combining chemo- and immunotherapy. MATERIALS AND METHODS We describe the results of a translational phase II trial designed to evaluate the toxicity, antitumor activity and immunologic effects of gemcitabine + FOLFOX-4 (oxaliplatin, fluorouracil, and folinic acid) polychemotherapy followed by the subcutaneous administration of granulocyte macrophage colony-stimulating factor and low-dose interleukin-2 in colorectal carcinoma patients. The study involved 29 patients (16 males and 13 females with a mean age of 69 years), 21 of whom had received a previous line of treatment, and 19 had liver involvement. RESULTS The treatment was well tolerated and induced very high objective response (68.9%) and disease control rates (96.5%), with an average time to progression of 12.5 months. An immunologic study of peripheral blood mononuclear cells (PBMCs) taken from 20 patients showed an enhanced proliferative response to colon carcinoma antigen and a significant reduction in suppressive regulatory T lymphocytes (CD4+CD25T-reg+). A cytofluorimetric study of the PBMCs of five HLA-A(*)02.01+ patients who achieved an objective response showed an increased frequency of cytolytic T lymphocyte precursors specific for known CEA- and TS-derived epitopes. CONCLUSION The results show that our regimen has strong immunologic and antitumor activity in colorectal cancer patients and deserves to be investigated in phase III trials.

Regulatory (foxp3+) T-cell Tumor Infiltration Is a Favorable Prognostic Factor in Advanced Colon Cancer Patients Undergoing Chemo or Chemoimmunotherapy

Antitumor immune response and chemotherapy-induced immunomodulation in colon cancer patients represented the rationale to design new strategies, like GOLFIG chemoimmunotherapy (gemcitabine, oxaliplatin, 5-fluorouracil/folinic acid, granulocyte macrophage colony-stimulating factor, and aldesleukine), that resulted a safe and very active regimen. Antitumor activity and immunity feedback to GOLFIG were strictly correlated with the best outcome observed in patients with autoimmunity signs, increase of central memory T cells, and decrease of regulatory T cells (Treg) in the peripheral blood. We thus investigated a potential correlation between the Treg tumor infiltration at diagnosis and the clinical outcome in a current randomized phase 3 trial aimed to compare the GOLFIG regimen with the standard FOLFOX chemotherapy (GOLFIG-2). An immunohistochemistry study was carried out to quantify the infiltration of Treg/FoxP3+ T lymphocytes in tumor samples of 57 patients enrolled in the GOLFIG-2 trial. Treg tumor infiltration scores were correlated with overall survival, treatment-relative survival, and progression-free survival (PFS). Higher Treg tumor infiltration scores were associated with a better prognosis in the whole series (Treg high score vs. low score: overall survival=mean 43.2 mo vs. 28.6 mo, P=0.0005) and a better outcome after treatment (Treg high score vs. low score: PFS=mean 15.8 mo vs. 8.8 mo, P=0.0009; treatment-relative survival=mean 23.1 mo vs. 18.2 mo, P=0.004). PFS was significantly longer in GOLFIG high versus all other subgroups (mean 18.1 mo vs. 9.9 mo, P=0.01). Our results suggest that a higher FoxP3+ T-lymphocyte tumor infiltration score is a favorable prognostic factor in colon cancer patients undergoing chemo or chemoimmunotherapy.

Dendritic Cell-Mediated Cross-Presentation of Antigens Derived from Colon Carcinoma Cells Exposed to a Highly Cytotoxic Multidrug Regimen with Gemcitabine, Oxaliplatin, 5-Fluorouracil, and Leucovorin, Elicits a Powerful Human Antigen-Specific CTL Response with Antitumor Activity in Vitro

Gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF) is a novel multidrug regimen inducing high levels of necrosis and apoptosis in colon carcinoma cells. This regimen is also able to promote a process of Ag remodeling including up-regulation of immunotherapy targets like carcinoembryonic Ag (CEA), thymidylate synthase (TS). We have conducted a preclinical study aimed to investigate whether these drug-induced modifications would also enhance colon cancer cell immunogenicity. Several CTL lines were thus generated by in vitro stimulating human HLA-A(*)02.01+ PBMCs, from normal donors and colon cancer patients, with autologous dendritic cells cross-primed with cell lysates of colon cancer cells untreated, irradiated, or previously exposed to different drug treatments including the GOLF regimen. Class I HLA-restricted cytolytic activity of these CTL lines was tested against colon cancer cells and CEA and TS gene transfected target cells. These experiments revealed that CTLs sensitized with GOLF-treated cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs.

Macrophage migration inhibitory factor in prostatic adenocarcinoma: correlation with tumor grading and combination endocrine treatment-related changes.

Macrophage migration inhibitory factor (MIF) is a ubiquitary cytokine whose expression has been investigated in tumors, showing a correlation between tumor aggressiveness and production of this protein by neoplastic cells. The aim of our study was to correlate MIF expression with tumor grade (Gleason scoring system) and histopathological changes after combined endocrine treatment (CET) of prostate adenocarcinoma.

Immunity feedback and clinical outcome in colon cancer patients undergoing chemoimmunotherapy with gemcitabine + FOLFOX followed by subcutaneous granulocyte macrophage colony-stimulating factor and aldesleukin (GOLFIG-1 Trial).

Purpose: GOLFIG chemoimmunotherapy regimen proved to be a safe and very active chemoimmunotherapy regimen in advanced colon cancer patients. We have thus investigated the immunobiological feedback to the treatment and its possible correlation with the clinical outcome of these patients. Experimental Design: This clinical and immunologic study involved 46 patients, 27 males and 19 females, enrolled in the GOLFIG-1 phase II trial who received gemcitabine (1,000 mg/m2 on days 1 and 15), oxaliplatin (85 mg/m2 on days 2 and 16), levofolinic acid (100 mg/m2 on days 1, 2, 15, and 16), and 5-fluorouracil (400 mg/m2 as a bolus, and 800 mg/m2 as a 24-hour infusion on days 1, 2, 15, and 16) followed by s.c. granulocyte macrophage colony-stimulating factor (100 μg, on days 3-7) and interleukin 2 (0.5 × 106 IU twice a day on days 8-14 and 17-29). Results: The regimen was confirmed to be safe and very active in pretreated patients with metastatic colorectal cancer. A subgroup analysis of these patients revealed a prolonged time to progression and survival in six patients who developed late signs of autoimmunity. A multivariate analysis validated the occurrence of autoimmunity signs as an independent predictor of favorable outcome. A parallel immunologic study detected in the peripheral blood mononuclear cells of these patients a progressive increase in lymphocyte and eosinophil counts, amplification in central memory, a marked depletion of immunosuppressive regulatory T cells, and activation of colon cancer–specific cytotoxic T cells. Conclusions: Our results suggest that immunity feedback to GOLFIG regimen and its antitumor activity are tightly correlated.

Cytotoxic drugs up-regulate epidermal growth factor receptor (EGFR) expression in colon cancer cells and enhance their susceptibility to EGFR-targeted antibody-dependent cell-mediated-cytotoxicity (ADCC).

Cetuximab is a human-murine chimeric IgG1 monoclonal antibody to epidermal growth factor-receptor (EGFR) which exerts synergistic antitumour interactions with several cytotoxic drugs. Therefore, it is presently recommended in combination with chemotherapy in the treatment of colon, head and neck and non-small cell lung cancer. Cetuximab has been designed to inhibit EGFR signalling; however, preclinical evidence suggests that its anti-cancer effects in vivo are also related to the ability of its human IgG1 backbone to trigger immunological mechanisms. Here we have investigated whether the exposure to different cytotoxic drugs may affect the susceptibility of colon cancer cells in vitro to cetuximab immuno-targeting and related lymphokine-activated killer (LAK)-mediated antibody-dependent cell cytotoxicity (ADCC). Five colon cancer cell lines expressing a different k-ras mutational status were evaluated for: (i) EGFR-expression, (ii) susceptibility to LAK cells and (iii) cetuximab-mediated ADCC, before and after exposure to 5-flurouracil (5-FU), gemcitabine (Gem), irinotecan (Iri) alone or in multiple two/three drug combinations. These drugs were able to up-regulate EGFR expression on the surface of all the colon cancer cell lines with a maximal effect observed few hours after the exposure to GILF regimen (Gem, Iri, Levofolinic acid and 5-FU). Chemotherapy was able to greatly enhance the sensitivity to either LAK cells or cetuximab-mediated ADCC in all the colon cancer cell lines with a mechanism independent from k-ras status. The results of our study suggest that chemotherapy may enhance cetuximab-mediated immuno-targeting and ADCC thus providing the rationale to design novel immuno-biochemotherapy regimens.

Chemotherapeutic drugs may be used to enhance the killing efficacy of human tumor antigen peptide-specific CTLs.

The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of Tregs, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.

Fractal Analysis in Human Pathology

Living structures may be described as being in a self-organizing, fluctuating steady-state far from equilibrium.1 Self-organization and a state far from equilibrium are characteristics of chaotic structures. Chaotic structures present fractal geometry, so is not too astonishing that the branching pattern of the airways in the lung or the arterial vascular pattern of the cardiovascular system have been described with fractal properties.2–4 Like coastlines, a tumor examined by light microscopy has a complex, irregular border and retains a similar level of complexity over a range of magnifications.5–7 Euclidean morphometric measurements were found to be invalid outside precisely defined conditions of resolution and magnification.8 In our Institute we are applying fractal dimension analysis to study human tumors at light and ultrastructural levels. Here, we present data obtained studying the epithelial–connective tissue interface in basal cell carcinoma of the skin, the boundaries of invasive bladder carcinomas (urothelial neoplasia), and the lymphocytic nuclear membrane in mycosis fungoides and chronic dermatitis.

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer‐related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone‐sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non‐raft into the raft compartment where it colocalized with the γ‐secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

Macrophage migration inhibitory factor in the human prostate: identification and immunocytochemical localization.

Macrophage migration inhibitory factor (MIF) is a lymphokine originally identified for its capacity to inhibit the random migration of macrophages. Recent data have further extended knowledge of the physiological role of this protein, showing that MIF is produced by several human organs and tissues. The present study was intended to evaluate the expression and tissutal localization of MIF in the human prostate.