Tumor infiltrating T lymphocytes expressing FoxP3, CCR7 or PD-1 predict the outcome of prostate cancer patients subjected to salvage radiotherapy after biochemical relapse.

Tumor immunologic microenvironment is strongly involved in tumor progression and the presence of tumor infiltrating lymphocytes (TIL) with different phenotypes has been demonstrated to be of prognostic relevance in different malignancies. We investigated whether TIL infiltration of tumor tissues could also predict the outcome of prostate cancer patients. To this end, we carried out a retrospective analysis correlating the outcome of locally advanced prostate cancer patients undergone salvage radiotherapy upon relapse after radical surgery with the infiltration by different TIL populations. Twenty-two patients with resectable prostate cancer, with a mean age of 67 (+/-3.93) years, who received salvage radiotherapy with a mean of 69.66 (+/- 3.178) Gy in 8 weeks, between June 1999 and January 2009 and with a median follow up of 123 (+/- 55.82) months, were enrolled in this study. We evaluated, by immunohistochemistry, the intratumoral (t) and peripheral stroma (p) infiltration by CD45, CD3, CD4, CD8, CCR7, FoxP3 or PD-1-positive cells on tumor samples taken at the diagnosis (d) and relapse times (R). We correlated these variables with patients' biochemical progression free survival (bPFS), post-radiotherapy progression free survival (PFS), and overall survival (OS). Substantial changes in the rate of TIL subsets were found between the first and the second biopsy with progressive increase in CD4, CCR7, FoxP3, PD-1+ cells. Our analysis revealed that higher CD8p,R+ and lower PD-1R+ TIL scores correlated to a longer bPFS. Higher CD8p,R+ and CCR7t,R+ TIL scores and lower CD45p,R+ and FoxP3p,R+ TIL scores correlated to a prolonged PFS and OS. These results suggest that the immunological microenvironment of primary tumor is strictly correlated with patient outcome and provide the rationale for immunological treatment of prostate cancer.

Carbonic anhydrase IX is a marker of hypoxia and correlates with higher Gleason scores and ISUP grading in prostate cancer.

BACKGROUND: Carbonic anhydrase IX is a member of α-carbonic anhydrases that is preferentially expressed in solid tumors. It enables bicarbonate transport across the plasma membrane, neutralizing intracellular pH and conferring to cancer cells a survival advantage in hypoxic/acidic microenvironments. Overexpression of carbonic anhydrase IX in cancer tissues is regulated by hypoxia inducible factor 1α - mediated transcription and the enzyme is considered a marker of tumor hypoxia and poor outcome. The role of carbonic anhydrase IX in prostate cancer has not been fully clarified and controversy has arisen on whether this enzyme is overexpressed in hypoxic prostate cancer tissues. METHODS: We analyzed the expression of carbonic anhydrase IX and hypoxia inducible factor 1α in two prostate cancer cell lines, LNCaP and PC-3, and in 110 cancer biopsies, by western blotting and immunocyto/histochemistry. RESULTS: In LNCaP and PC-3 cells, carbonic anhydrase IX was mostly cytoplasmic/nuclear, with very limited membrane localization. Nuclear staining became stronger under hypoxia. When we analyzed carbonic anhydrase IX expression in human prostate cancer biopsies, we found that protein staining positively correlated with hypoxia inducible factor 1α and with Gleason pattern and score, as well as with the novel grading system proposed by the International Society of Urological Pathology for prostate cancer. Once more, carbonic anhydrase IX was mainly cytoplasmic in low grade carcinomas, whereas in high grade tumors was strongly expressed in the nucleus of the neoplastic cell. An association between carbonic anhydrase IX expression level and the main clinic-pathological features involved in prostate cancer aggressiveness was identified. CONCLUSIONS: There was a statistically significant association between carbonic anhydrase IX and hypoxia inducible factor 1α in prostate cancer tissues, that identifies the enzyme as a reliable marker of tumor hypoxia. In addition, carbonic anhydrase IX expression positively correlated with prostate cancer grading and staging, and with outcome, suggesting that the protein may be an independent prognosticator for the disease. The nuclear translocation of the enzyme in hypoxic cancer cells may epitomize a biological switch of the tumor towards a less favorable phenotype.

Emerging Targets For Prostate Adenocarcinoma Therapy: How Molecular Biology May Drive Towards a More Tailored Approach.

Prostate adenocarcinoma is the most diagnosed male cancer in the Western world and the second leading cause of cancer-related mortality. Albeit most of the patients with prostate adenocarcinoma are currently treated by surgery and/or radiation therapy, more than 30-40% of affected subjects will eventually progress and develop advanced disease. To date, management decisions depend on the clinical stage of the patient and the histological diagnosis which unfortunately often lack to predict the real prognosis. Current therapies have shown to be insufficient, mainly in the metastatic disease. For clear-cut diagnosis and follow-up, we promptly need molecular markers also useful in predicting patient's outcome. Advances in cancer genomics have led to a plethora of biomarkers, which must now to be rigorously validated in the clinical setting. Recent insights on prostate adenocarcinoma biology which unveiled some of the biological mechanisms leading to this tumour, have managed in devising novel strategies for therapy. Immunotherapeutic agents, selective adrenal inhibitors, anti-angiogenic molecules, newly engineered androgen receptor inhibitors, compounds targeting the bone microenvironment are demonstrated to limit cancer growth by blocking specific signaling pathways. Such strategies can be complemented to existing therapeutic paradigm in improving beneficial outcome. Moreover, other emerging pharmacological compounds have shown encouraging results and several clinical trials are ongoing. This review summarizes the developing targeted therapies for prostate adenocarcinoma and discuss their potential benefit mainly in the castration- resistant forms.

Translationally controlled tumor protein in prostatic adenocarcinoma: correlation with tumor grading and treatment-related changes.

Prostate cancer is the second leading cause of cancer-related death. The androgen deprivation therapy is the standard treatment for advanced stages. Unfortunately, virtually all tumors become resistant to androgen withdrawal. The progression to castration-resistance is not fully understood, although a recent paper has suggested translationally controlled tumor protein to be implicated in the process. The present study was designed to investigate the role of this protein in prostate cancer, focusing on the correlation between its expression level with tumor differentiation and response to treatment. We retrieved 292 prostatic cancer specimens; of these 153 had been treated only by radical prostatectomy and 139 had undergone radical prostatectomy after neoadjuvant treatment with combined androgen blockade therapy. Non-neoplastic controls were represented by 102 prostatic peripheral zone specimens. In untreated patients, the expression of the protein, evaluated by RT-qPCR and immunohistochemistry, was significantly higher in tumor specimens than in non-neoplastic control, increasing as Gleason pattern and score progressed. In treated prostates, the staining was correlated with the response to treatment. An association between protein expression and the main clinicopathological factors involved in prostate cancer aggressiveness was identified. These findings suggest that the protein may be a promising prognostic factor and a target for therapy.

CT perfusion in the characterisation of renal lesions: an added value to multiphasic CT.

OBJECTIVE: To prospectively evaluate if computed tomography perfusion (CTp) could be a useful tool in addition to multiphasic CT in renal lesion characterisation. MATERIALS AND METHODS: Fifty-eight patients that were scheduled for surgical resection of a renal mass with a suspicion of renal cell carcinoma (RCC) were enrolled. Forty-one out of 58 patients underwent total or partial nephrectomy after CTp examination, and a pathological analysis was obtained for a total of 49 renal lesions. Perfusion parameters and attenuation values at multiphasic CT for both lesion and normal cortex were analysed. All the results were compared with the histological data obtained following surgery. RESULTS: PS and MTT values were significantly lower in malignant lesions than in the normal cortex (P < 0.001 and P = 0.011, resp.); PS, MTT, and BF values were also statistically different between oncocytomas and malignant lesions. According to ROC analysis, the accuracy, sensitivity, and specificity to predict RCC were 95.92%, 100%, and 66.7%, respectively, for CTp whereas they were 89.80%, 93.35%, and 50%, respectively, for multiphasic CT. CONCLUSION: A significant difference between renal cortex and tumour CTp parameter values may suggest a malignant renal lesion. CTp could represent an added value to multiphasic CT in differentiating renal cells carcinoma from oncocytoma.

Effect of chocolate and Propolfenol on rabbit spermatogenesis and sperm quality following bacterial lipopolysaccharide treatment.

The aims of the study were to evaluate the effects of chocolate and propolis-enriched diets on rabbit spermatogenesis, sperm motility, and ultrastructure following bacterial lipopolysaccharide (LPS) treatment. Thirty-two New Zealand White rabbits were divided into four groups. The LPS-Propolfenol(®) group received propolis (500 mg/kg/day) in their diet for 15 days, while the LPS-chocolate group was fed 70% cacao chocolate (1 g/1 kg/day) for the same period. Following the diet treatments, rabbits in the LPS-Propolfenol(®) and LPS-chocolate groups, and an LPS group received a single intraperitoneal dose of 50 µg/kg LPS, and the control group received only saline. Kinematic sperm traits were evaluated with a computer assisted sperm analyzer (CASA) system, and ultrastructural characteristics were examined by transmission electron microscopy (TEM). Testicular and epididymal tissues were observed by light microscopy and TEM and multiplex real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect and quantify toll-like receptor-4 (TLR-4) gene expression. The values of the analyzed semen parameters of rabbits treated with LPS-Propolfenol(®) and LPS-chocolate did not show any variations compared with the control group, but they were lower in rabbits treated only with LPS. Alterations observed in the testicular tissue of LPS treated-rabbits were not detected in specimens from the LPS-chocolate and LPS-Propolfenol(®) groups, which showed normal spermatogenesis. The TLR-4 mRNA expression was similar in controls, in LPS treated, and in LPS-chocolate groups, but it was significantly (p < 0.01) decreased in LPS-Propolfenol(®) rabbits. In conclusion, a chocolate and propolis-enriched diet showed a protective effect on the spermatogenetic process of buck rabbits following LPS treatment.

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.

Tumor infiltration by chemokine receptor 7 (CCR7)(+) T-lymphocytes is a favorable prognostic factor in metastatic colorectal cancer.

The immune interactions occurring within the tumor microenvironment have a critical role in determining the outcome of colorectal cancer patients. We carried-out an immunohistochemical analysis of tumor infiltrating T-lymphocytes expressing chemokine receptor 7 (CCR7) in a series of colorectal cancer patients enrolled in a prospective clinical trial. We demonstrated that a high tumor infiltration score of this lymphocyte subset is predictive of longer progression free survival and overall survival.

Tumor infiltration by T lymphocytes expressing chemokine receptor 7 (CCR7) is predictive of favorable outcome in patients with advanced colorectal carcinoma.

PURPOSE: An efficient adaptive immunity is critical for a longer survival in cancer. We investigated the prognostic value of tumor infiltration by CD8(+) T cells expressing the chemokine-receptor-7 (T(ccr7)) and the correlation between tumor infiltration by T(ccr7) and regulatory CD4(+)FoxP3(+) T cells (T(reg)) in 76 metastatic colorectal cancer (mCRC) patients enrolled in a phase III trial. EXPERIMENTAL DESIGN: T(ccr7) and T(reg) cell infiltration in tumor samples was quantified by immunohistochemistry. The correlation among T(ccr7), T(reg) tumor infiltration, and patients' outcome was evaluated. RESULTS: High T(ccr7) tumor infiltration was predictive of prolonged OS [high vs. low T(ccr7) score: median 38 months (95% CI: 24.5-51.4) vs. 20 months (95% CI: 11.4-28.5); HR = 0.48 (95% CI: 0.24-0.96); P = 0.03] and prolonged progression-free survival [PFS; high vs. low T(ccr7) score: median 12 months (95% CI: 7.7-16.2) vs. 7 months (95% CI: 5.2-8.7); HR = 0.54 (95% CI: 0.28-1.01); P = 0.01] after front-line chemotherapy. Regression analysis did not show correlation between T(ccr7) and T(reg) infiltration levels. However, the cluster of patients showing concomitant high infiltration by both T(ccr7) and T(reg) disclosed a favorable outcome [double high vs. double low tumor infiltration score: median OS = 35 months (95% CI: 20.8-49.1) vs. 17 months (95% CI: 4.6-29.3); HR = 0.32 (95% CI: 0.12-0.87); P = 0.02 and median PFS = 11 months (95% CI: 9.4-12.5) vs. 5 months (95% CI: 2.2-7.7); HR = 0.43 (95% CI: 0.17-1.06); P = 0.01]. CONCLUSIONS: High T(ccr7) tumor infiltration score is a favorable prognostic factor for mCRC. Our findings underline the relevance of microenvironment-related immunologic events for patient outcome.

Analysis of the IgVH genes in T cell-mediated and antibody-mediated rejection of the kidney graft.

AIMS: Grafts have been shown to be sites where the alloimmune response develops in a direct interaction between the targeted tissue and the immune effectors. An important issue in renal rejection is B cell infiltrate that may contribute to the development or persistence of rejection. Analysis of gene-expression patterns also provides a window on the biology and pathogenesis of renal allograft rejection. METHODS: To better understand the role exerted by B cells in a renal acute rejection, the authors analysed the IgVH gene repertoire in six cases of transplanted kidneys with acute T cell-mediated rejection (TCMR), three of which were associated with antibody-mediated rejection (ABMR). RESULTS: The authors found mutated and unmutated sequences, without any evidence of clonal relationships, in all patients with TCMR alone and in two of the three cases with both acute TCMR and ABMR. The remaining patient showed glomerular inflammation and thrombosis, with diffuse C4d glomerular and peritubular capillary deposition, and hypermutated V region genes. CONCLUSIONS: These results suggest that there is more than one pathway to the onset and perpetuation of CD20 (+) B cells infiltration in acute rejection; furthermore, the CD20 (+) B cells' clonal expansion may be responsible for a more severe pattern of ABMR, through immune-mediated tissue damage.

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.

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.

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/m(2) on days 1 and 15), oxaliplatin (85 mg/m(2) on days 2 and 16), levofolinic acid (100 mg/m(2) on days 1, 2, 15, and 16), and 5-fluorouracil (400 mg/m(2) as a bolus, and 800 mg/m(2) as a 24-hour infusion on days 1, 2, 15, and 16) followed by s.c. granulocyte macrophage colony-stimulating factor (100 mug, on days 3-7) and interleukin 2 (0.5 x 10(6) 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.

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 T(reg)s, 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.

Anti-angiogenetic effects of immune-reconstituted influenza virosomes assembled with parathyroid hormone-related protein derived peptide vaccine.

BACKGROUND: C-IRIV/PTR-4 is a novel anticancer vaccine construct composed of immune-reconstituted influenza virosomes (IRIV) assembled with the PTH-rP derived peptide (PTR)-4, a synthetic CTL epitope with HLA-A(*)02.01 amino acid binding motifs. This peptide is able to generate a human PTH-rP specific CTL response with anti-tumor activity in vitro and in mice. MATERIALS AND METHODS: We have investigated the immunological and preventive anti-tumor activity of C-IRIV/PTR-4 compared with the soluble PTR-4 peptide, in HHD mice inoculated with autologous PTH-rP+ tumor cells. RESULTS: Peptide vaccination with either a soluble and an IRIV formulation showed similar immunological activity and the ability to purge the tumor tissue of tumor cell clones able to produce the target antigen (PTR-rP). The most efficient protection from tumor growth was however observed in animals vaccinated with C-IRIV/PTR-4 in which an additional IRIV related anti-angiogenetic effect was detected in the tumor tissue. CONCLUSIONS: These results confirm the immunological activity of PTR-4 vaccination and suggest a more efficacious therapeutic potential of C-IRIV/PTR-4 against bone metastases and malignancies like breast, prostate and lung which very often over-express PTH-rP.

[Urachus' carcinoma: a case report]. / Carcinoma dell'uraco. Case report.

Urachus' carcinoma represents a rare oncologic disease with an unfavourable prognosis due to the usual delay of correct diagnosis for its anatomical localization. Its surgical treatment varies from radical cystectomy to segmentary resection of the bladder with pelvic lymphadenectomy. We report a case occurred in a ninety years old female, in which the diagnosis was achieved only at laparotomy. The patient was submitted to surgery with the only generic diagnosis of "lower abdominal mass", and treated with segmentary resection.

Chemo-immunotherapy of colorectal carcinoma: preclinical rationale and clinical experience.

Advanced colorectal cancer is a common disease with an high mortality rate. For four decades, pharmacological treatment of the advanced disease was based on the use of 5-fluorouracil alone or in combination with biomodulators such as folinic acid and intereferon alpha. In the last 5 years, response to therapy has been considerably ameliorated thanks to the discovery of new drugs such as oxaliplatin and CPT-11. These agents, in combination with 5-fluorouracil, according to various schedules of treatment, have reached a significant improvement of palliation, response rate and survival. Immunotherapy is an uprising modality of treatment for human cancer including colorectal carcinoma. Its rationale is based on the knowledge that tumour cells are genetically unstable and produce molecular structures which allow their recognition and destruction by the immune-surveillance system. Therefore, humoral as well as cellular compartments of the immune system can be utilized according to a "passive" strategy (e.g. monoclonal antibody administration and adoptive immunotherapy) or an "active" approach, by using different modalities of vaccine therapy. In this context, monoclonal antibodies (mAbs) and cancer vaccines are being tested for the treatment of advanced colorectal cancer. Due to their genetic instability and extraordinary adaptative potential, tumour cells may acquire resistance to the immune effectors and mAbs exactly as they do for cytotoxic drugs. To improve the results of both immunological and chemical modality of cancer treatment, an increasing number of authors is starting to combine chemo and immunotherapy in the attempt to circumvent the limitations of both strategies. This report tries to review the possible rationale of the chemo-immunotherapy combination, illustrating preliminary results of preclinical and clinical studies.

5-fluorouracil-based chemotherapy enhances the antitumor activity of a thymidylate synthase-directed polyepitopic peptide vaccine.

BACKGROUND: Thymidylate synthase (TS), a key enzyme in DNA synthesis, is often overexpressed in cancer cells. Some chemotherapeutic agents, such as 5-fluorouracil (5-FU), act by inhibiting TS expression. We evaluated whether a novel 28-amino acid multiepitope peptide, TS/PP, that contains the sequences of three TS-derived epitopes with binding motifs for HLA-A(*)02.01 could induce a TS-directed cytotoxic T-lymphocyte (CTL) response with antitumor activity. METHODS: TS/PP peptide immunologic activity in CTL lines derived from human leukocyte antigen (HLA)-A(*)02.01+ peripheral blood mononuclear cells (PBMCs) was tested in the presence of interleukin-2 and autologous TS/PP peptide-loaded dendritic cells. Immunologic and antitumor activities of TS/PP and its toxicity were also evaluated in vivo in HLA-A(*)02.01 transgenic (HHD) mice that were vaccinated with TS/PP, control, or TS-peptide cocktail and treated with or without 5-FU chemotherapy. The mice were also inoculated subcutaneously with TS-expressing EL-4/HHD lymphoma cells to assess immune response against these tumor cells. RESULTS: TS/PP-specific CTL lines showed a TS-multiepitopic specificity and were able to kill TS+/HLA-A(*)02.01+ breast and colon carcinoma cells. The killing ability against target cells previously exposed to sublethal doses of 5-FU was statistically significantly greater than against untreated target cells (43.5% versus 26.5% at 25/1 effector to target ratio [Difference {diff} = 17.0]; 95% confidence interval [CI] = 12.6 to 20.4) for MDA-MB-231 breast carcinoma cells and 73.5 versus 48.5 (diff = 25.0; 95% CI = 16.2 to 33.8) for the SW-1463 colon carcinoma cells. HHD mice vaccinated with TS/PP manifested a TS-peptide-specific CTL response with no sign of autoimmunity or toxicity. Furthermore, treatment of these mice with 5-FU delayed or prevented the occurrence of tumors formed by inoculation with autologous (TS+)EL-4/HHD lymphoma cells. CONCLUSIONS: The multiepitopic TS/PP vaccine induces a tumor-specific immune response in mice and is especially potent when used in combination with 5-FU-based chemotherapy.

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.

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.