p53 and HER2/neu expression in relation to chemotherapy response in patients with non-small cell lung cancer

The aim of the study was to investigate a relation between p53 and HER2/neu expression in resected lung tumors and the response of those tumors to neoadjuvant chemotherapy. The study population included 67 consecutive patients with non-small cell lung cancer (NSCLC) in stage II or III who were operated on at the Institute of Tuberculosis, Warsaw, Poland, between 20 April 2001 and 10 March 2003. All patients received two cycles of chemotherapy consisting of cisplatin and vinorelbine prior to the operation. The response to therapy was assessed as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD), on the basis of CT scans performed before and after neoadjuvant chemotherapy. p53 and HER2/neu protein expression were evaluated by immunohistochemistry (IHC) using antibodies against p53 (clone PAb 1801, Novocastra) and against HER2/neu (Dako) in paraffin-embedded specimens of tumors. A response to therapy (CR+PR) was observed in 27 patients, while 40 patients (SD+PD) were regarded as resistant to therapy. Resistance was observed significantly more often in tumors above 3 cm in diameter. p53 expression was found in 16 tumors (23.9%) and HER2/neu in 26 tumors (38.8%). We observed a nonsignificant tendency to chemoresistance in tumors with HER-2/neu overexpression and also in tumors with p53 overexpression. If we consider HER-2/neu and p53 together, chemoresistance was observed statistically significantly more often when one or both markers were positive (p<0.05). This significance was independent of tumor size.     

Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine.

Patients with metastatic melanoma were immunized with an immunodominant peptide derived from the gp100 melanoma-melanocyte differentiation Ag that was modified to increase binding to HLA-A+0201. A total of 10 of 11 patients who received the g209-2M peptide alone developed precursors reactive with the native g209 peptide, compared with only 5 of 16 patients who received g209-2M peptide plus IL-2 (p2 = 0.005). Peptide reactivity closely correlated with the recognition of HLA-A+0201 melanoma cells (p < 0. 001). The decrease in immune reactivity when peptide was administered with IL-2 appeared specific for the immunizing peptide, since reactivity to an influenza peptide resulting from prior exposure was not affected. Preexisting antitumor precursors did not decrease when peptide plus IL-2 was administered. The administration of GM-CSF or IL-12 also resulted in a decrease in circulating precursors compared with the administration of peptide alone, though not as great a decrease as that seen with IL-2. Immunization with peptide plus IL-2 did, however, appear to have clinical impact since 6 of the 16 patients (38%) that received peptide plus IL-2 had objective cancer regressions. It thus appeared possible that immunization with peptide plus IL-2 resulted in sequestering or apoptotic destruction of newly activated immune cells at the tumor site. These represent the first detailed studies of the impact of immunization with tumor peptides in conjunction with a variety of cytokines in patients with metastatic cancer.     

A phase II study of combined administration of dacarbazine and carboplatin with home therapy of recombinant interleukin-2 and interferon-α2a in patients with advanced malignant melanoma

Chemotherapy and interleukin-2 (IL-2) and/or interferon (IFN) produce objective responses in a proportion of patients with advanced malignant melanoma. The duration of response to chemotherapy is usually less than 4 months, and immunotherapy has resulted in longlasting remissions in a small number of patients with metastatic melanoma. The current study was conducted to improve the antitumor efficacy and the interactions between recombinant (r) IL-2, rIFN2a and chemotherapy. A total of 16 evaluable patients with metastatic malignant melanoma were entered into a phase-II study designed to assess the response rate and therapeutic efficacy of dacarbazine and carboplatin followed by rIL-2 and rIFN2a. Patients received 750 mg/m² dacarbazine with 400 mg/m² carboplatin each by intravenous bolus on days 1 and 22. Recombinant IL-2 and IFN2a were administered on an outpatient basis (home therapy) subcutaneously for 6 consecutive weeks: 4.8×10⁶ IU/m² rIL-2 daily, 5 days a week; 6.0×10⁶ IU/m² rIFN2a thrice weekly. There were responses in 6 of the 16 enrolled patients with an overall response rate of 37.5% (95% confidence interval: 14%–61%). All responding patients had partial responses. The median survival time of the responding patients was significantly better than that of patients with progressive and stable disease (P=0.03). The median duration of response was 11 months (range 2–24 months). Responses in lung, liver, soft tissue and lymph-node sites were noted.     

Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low‐molecular‐weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high‐grade STS sera using hydrogel core‐shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty‐two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non‐metastatic tumors. Cox‘s regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H‐related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.     

A phase II trial of intraperitoneal interleukin-2 in patients with platinum-resistant or platinum-refractory ovarian cancer

Ovarian cancer patients with persistent (platinum-resistant) or progressive (platinum-refractory) disease respond poorly to second line chemotherapy and have low survival expectancy. New and improved therapeutic approaches are needed and immune biologics are one possibility. Interleukin-2 (IL-2) is a T-cell growth factor believed to be important in anti-tumor immunity. We performed a phase II clinical trial with intraperitoneal (IP) recombinant IL-2 administered in weekly infusions of 6 × 10⁵ IU/m². Thirty-one subjects were sequentially entered into the study and clinical responses were surgically confirmed in 24 patients. The primary end point of this study was clinical response with immunologic measurements as secondary end points. The IP regimen was generally well tolerated. Of the 24 patients assessed for response, there were 6 (4 complete, 2 partial) responses for an overall response rate of 25.0% [95% confidence interval (CI) of 11–45]. The median survival of the 31 patient cohort was 2.1 years (95% CI of 1.3–4.4), but for the 6 patients with responses the median survival has not been reached (range 24–120+ months). Eosinophil and lymphocyte numbers were continuously monitored during treatment. Peripheral blood eosinophils were markedly increased at the completion of treatment (p < 0.0001) and associated with increased circulating eotaxin (p = 0.03). We also found significant associations between changes in CD3 counts and survival (p = 0.05) and between IFNγ-secreting CD8 T cells at early time points and survival (p = 0.04). This study provides important evidence for IP IL-2 in platinum-resistant ovarian cancer and identifies several immune correlates of survival.     

Sequential administration of interferon γ and interleukin-2 in metastatic renal cell carcinoma: results of a phase II trial

Background: Because of the known efficacy of several cytokines in the treatment of advanced renal cell cancer (RCC), we have conducted a phase II trial of the efficacy and toxicity of subcutaneous interferon γ (IFNγ) and interleukin-2 (IL-2). Methods: 63 patients with progressive metastatic RCC were treated with 100 μg recombinant IFNγ1b administered three times weekly during weeks 1 and 2 and with 4.5 MU recombinant IL-2 administered on 4 consecutive days during weeks 3 and 4, every 6 weeks. Results: 11% of patients had an objective response (CR: 3%, PR: 8%), 33% had SD. Toxicity was generally mild. The median duration of remissions (CR + PR) was 9.6 months; the median duration of SD 8 months. A significant survival benefit was evident at a median observation time of 51 months for patients (44%) responding to therapy (P < 0.0001). Conclusions: we conclude that sequential treatment with IFNγ and IL-2 may prolong survival in patients with metastatic RCC responding to therapy. Received: 2 April 2000 / Accepted: 21 April 2000     

Continuous infusion of interleukin-2 and cyclophosphamide as treatment of advanced cancers: a National Biotherapy Study Group Trial

We have evaluated the effect of Interleukin-2 [IL-2] after Cyclophosphamide (C) chemotherapy in 41 patients with metastatic cancer. IL-2 was given as a continuous infusion priming cycle 36 hours after C at 1 gm/m2 intravenously. In 39 evaluable patients, there were no complete remissions [CR], 2 partial remissions [PR], and 1 had a minor response [MR]. Stable disease for 30 days was seen in 16 patients whereas 20 progressed. The durations of partial and minor responses were brief, ranging from 1-6 months. Grade 3-4 neutropenia was seen in 41%. This was more severe than seen with IL-2 alone or IL-2 combined with lower doses of C. The marrow suppression was due to the chemotherapy. This combination of IL-2 and C appears to be reasonably well tolerated by patients, but toxicity is greater and the response rate is no better than results achieved by IL-2 alone. Responses of 26 patients with renal cancer appear to be inferior to our historical data using IL-2/LAK cells without C. Immune monitoring demonstrated changes expected with C chemotherapy (i.e., a non-selective decline in immune function). C induced no further differences in IL-2 induced changes in immune function.     

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.     

Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (DeltaPsim) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and DeltaPsim disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and DeltaPsim disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or DeltaPsim disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.     

Prospective analysis of KRAS wild-type patients with metastatic colorectal cancer using cetuximab plus FOLFIRI or FOLFOX4 treatment regimens

Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor, has proven to be efficient in the treatment of metastatic colorectal cancer. We made a prospective study of the efficacy and toxicities of cetuximab-combination first-line (FOLFOX4) versus second/third-line (FOLFIRI) chemotherapy in 98 KRAS wild-type patients who had metastatic colorectal cancer. Wild-type KRAS had been identified by direct sequencing. Associations between clinical response/progression-free survival/overall survival/toxicities and cetuximab-combination chemotherapy timing were evaluated. The overall response rate was significantly higher for first-line treatment than for second/third-line treatment (relative risk = 1.707, 95% confidence interval = 1.121-2.598). Both progression-free survival and overall survival indicated significantly longer survival of first-line treatment than second/third-line treatment patients. This study is a validation of a molecular analysis of KRAS wild-type status for the prediction of response to cetuximab-combination chemotherapy for metastatic colorectal cancer patients; its predictive role was less prominent in the second/third-line than in the first-line treatment patients.     

Pre-treatment prediction of chemoresistance in second-line chemotherapy of ovarian carcinoma: value of serological tumor marker determination (tetranectin, YKL-40, CASA, CA 125)

OBJECTIVE: To examine if the determination of the levels of serological tumor markers at time of relapse had any predictive value for chemoresistance in the second-line treatment of ovarian cancer patients. METHODS: From a registry of consecutive single-institution patients with epithelial ovarian carcinoma pretreated with paclitaxel plus platinum, we selected 82 patients with (a) solid tumor recurrence, and (b) second-line chemotherapy consisting of topotecan (platinum-resistant disease) or paclitaxel plus carboplatin (platinum-sensitive disease). Stored serum samples were analyzed for the biochemical tumor markers tetranectin, YKL-40, CASA (cancer-associated serum antigen), and CA 125. The serum tumor marker levels at time of relapse were correlated with response status at landmark time after 4 cycles of second-line chemotherapy. Univariate and multivariate logistic regression analyses (chemoresistant vs non-chemoresistant disease) were performed. RESULTS: At landmark time, 26% of patients had progression according to the GCIG (Gynecologic Cancer Intergroup) progression criteria. In univariate logistic regression analysis, the tumor markers tetranectin (OR 0.4; 95% CI: 0.2-0.8; p=0.008), YKL-40 (OR 1.8; 95% CI: 1.0-3.3; p=0.045), and CASA (OR 1.8; 95% CI: 1.2-2.7; p=0.007) had predictive value for second-line chemoresistance, whereas serum CA 125 had no predictive value. In a multivariate logistic regression analysis, serum tetranectin and CASA both had independent predictive value for chemoresistance. The combined determination of tetranectin and CASA had a specificity of 90% with 33% sensitivity for the prediction of chemoresistance (area under the receiver operating characteristic curve = 0.78; 95% CI: 0.66-0.91; p=0.001). CONCLUSION: Low serum levels of tetranectin, or high serum levels of CASA or YKL-40, are associated with increased risk of second-line chemoresistance in patients with ovarian cancer.     

KIN enhances stem cell-like properties to promote chemoresistance in colorectal carcinoma

Chemotherapy is widely used in colorectal cancer (CRC) treatment, especially in advanced stage patients. However, it is inevitable to develop chemoresistance. Recently, cancer cells acquired stem cell-like properties or cancer stem cells (CSC) were proved to attribute to chemoresistance. Here, we found that KIN protein was elevated in CRC cell lines and tissue specimens as compared to normal controls. Upregulation of KIN positively correlates with the metastatic status of CRC patients. Patients with high KIN expression showed poor prognosis and were with a short survival time. Overexpression of KIN enhanced, while silencing KIN impaired, chemoresistance to oxaliplatin (Ox) or 5-fluorouracil (5-FU) in CRC cell lines. Further investigation demonstrated that overexpression of KIN rendered CRC cells enriching CSC markers and CSC phenotype, and silencing KIN reduced CSC markers and CSC phenotype. Our findings suggest that the KIN level may be a suitable marker for predicting chemotherapy response in CRC, and silencing KIN plus chemotherapy may be a novel therapy for CRC treatment.     

Serum concentrations of interleukin-18 in early and advanced cancer patients: enhanced secretion in metastatic disease

The recent availability of adequate methods for cytokine measurement could contribute to better understanding the immunophysiopathology of neoplastic disease. Unfortunately, very little data is available about cytokine secretion in cancer patients. At present, IL-2, IL-12 and IL-15 represent the major antitumor cytokines in humans. Preliminary clinical studies have shown a progressive decline in IL-2 levels with cancer progression, whereas IL-12 seems to increase in the advanced disease. IL-18 is the latest cytokine discovered by potential anticancer and anti-angiogenetic activity, and it has effects similar to those of IL-12. This preliminary study was carried out to analyze IL-18 secretion in early or advanced cancer patients. The study included 40 cancer patients (lung cancer, 21; gastrointestinal tumors, 19), 17 of whom had metastatic disease, and 50 healthy controls. Serum levels of IL-18 were measured by ELISA. No significant difference in IL-18 mean levels was seen between controls and non-metastatic patients. In contrast, metastatic patients showed significantly higher IL-18 mean values with respect to both healthy controls and non-metastatic patients. This preliminary study seems to suggest that metastatic disease may be characterized by enhanced IL-18 secretion the biological and prognostic significance to be established by successive clinical investigation.     

Phase I/II study of sequential chemoimmunotherapy (SCIT) for metastatic melanoma: outpatient treatment with dacarbazine,granulocyte-macrophage colony-stimulating factor,low-dose interleukin-2, and interferon-alpha

A regimen of sequential chemoimmunotherapy (SCIT) was studied in a phase I/II study to analyze toxicity, anti-tumor and immunomodulatory effects in patients with previously untreated metastatic cutaneous melanoma. Treatment consisted of dacarbazine (DTIC) 800 mg/m2 administered intravenously (i.v.) on day 1, followed by subcutaneous (s.c.) molgramostim (GM-CSF), 10 times 2.5 micro g/kg on days 2 to 12, s.c. low-dose interleukin-2 (IL-2), 10 times 1.8 MU on days 8 to 18, and s.c. interferon-alpha-2b (IFN-alpha), 5 times 6 MU on days 15 to 20. Dosages were not escalated. Therapy was given in the form of outpatient treatment. Changes in T-lymphocyte phenotype and in soluble mediators were monitored during treatment. A total of 32 patients with stage IV melanoma were enrolled in the study. Treatment was well tolerated, without serious toxicity. In all cases, it could be given as outpatient treatment. Ten subjects out of the 31 patients evaluated showed an objective response, with 4 complete responses (CR) and 6 partial responses (PR); the response rate amounted to 32% (95% CI: 16-49%). Median survival of all patients was 8 months, with those patients who responded to treatment living longer than the non-responding group. Survival rate at 1 year was 22%. Monitoring of the effects of treatment revealed increased numbers of activated T-lymphocytes, both in the CD4 and in the CD8 subsets. The levels of soluble mediators such as sIL-2R and sCD8 were also increased. Changes were noted as early as the GM-CSF treatment period, and were observed to a further extent during IL-2 treatment. In the present study, it was found that this sequential chemoimmunotherapy regimen consisting of 4 agents (DTIC, GM-CSF, IL-2, IFN-alpha) has acceptable toxicity, can be administered on an outpatient basis, results in increased numbers of activated T-lymphocytes, and induces activity against metastatic melanoma that warrants further investigation.     

miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma

Although many researches have been undertaken to disclose the mechanisms of chemoresistance, the mechanisms remain unclear. The aim of this study is to elucidate the role of miR-181a–Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma (TSCC). We found that cisplatin-induced chemoresistance in TSCC cell lines underwent EMT (epithelial–mesenchymal transition) and was accompanied by enhancing metastatic potential (migration and invasion in vitro), miR-181a downregulation and Twist1 upregulation. Functional analyses indicated that miR-181a reversed chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Twist1 was confirmed as a direct miR-181a target gene by luciferase reporter gene assays. Twist1 knockdown by siRNA led to a reversal of the chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Our study demonstrates that miR-181a–Twist1 pathway may play an important role in the development of cisplatin-chemoresistance, with EMT and an increase the metastatic potential of TSCC cells.     

Regulation of estrogen activity in human endometrium: effect of IL-1beta on steroid sulfatase activity in human endometrial stromal cells

We investigated the effect of interleukin 1beta (IL-1beta) on steroid sulfatase (STS) activity and the expression of STS mRNA in human endometrial stromal cells. Endometrial tissue samples were obtained from patients undergoing hysterectomy to remove uterine fibroids. Stromal cells were isolated from the tissue preparation and cultured. IL-lbeta (1 approximately 100 ng/ml) was added into the culture medium and incubated for 24 h. The expression of STS mRNA was measured by competitive RT-PCR. The addition of IL-lbeta at 10 and 100 ng/ml suppressed STS mRNA expression to 55.2 +/- 12.8% and 25.1 +/- 10.9%, respectively, of the control sample to which no IL-lbeta had been added. STS activity was measured by radiolabelled steroid metabolite using thin layer chromatography, and this activity was also significantly suppressed in response to the administration of IL-lbeta in a dose-dependent manner. When IL-1 receptor antagonist (IL-1ra) was added together with IL-1beta to the culture medium, mRNA expression and STS activity were recovered. The present study is the first to demonstrate IL-1beta regulation of STS activity locally in human endometrium. IL-1beta suppressed mRNA and activity of STS in stromal cell culture. This initial demonstration of IL-1beta regulation of STS implies that IL-1beta may control the steroid microenvironment in human uterine endometrium by reducing biologic action of estrogen.     

Cancer chemotherapy-induced lymphocytosis: a revolutionary discovery in the medical oncology

The recent advances in the investigation of tumor immunobiology have suggested that cancer chemotherapy, in addition to its well known cytotoxic activity, may play modulatory effects on the endogenous production of cytokines involved in the control of both tumor angiogenesis and antitumor immunity. Cancer chemotherapy constantly acts with inhibitory effects on anti-bacterial, anti-viral and anti- mycotic immune responses, whereas its action on anticancer immunity, which is mainly mediated by lymphocytes, has still to be better investigated and defined. The present study was carried out to evaluate the influence of chemotherapy on lymphocyte count and its relation to the clinical response in cancer patients suffering from the most commonly frequent tumor histotypes, including lung, colorectal, breast and prostate carcinomas. The study included 144 consecutive metastatic solid tumor patients. Lung cancer patients were treated with cisplatin plus gemcitabine, colorectal cancer patients received oxaliplatin plus 5-fluorouracil, while those affected by breast cancer or prostate carcinoma were treated with taxotere alone. An objective tumor regression was achieved in 66 out of 144 (46 percent) patients, whereas the remaining 78 patients had only a stable disease (SD)or a progressive disease. Independently of tumor histotype and chemotherapeutic regimen, a lymphocytosis occurred in patients who achieved an objective tumor regression in response to chemotherapy, and lymphocyte mean count observed at the end of the chemotherapeutic treatment was significantly higher with respect to the values seen before the onset of treatment. On the contrary, lymphocyte mean number decreased on chemotherapy in patients with SD or PD, even though the decline was statistically significant with respect to the pretreatment values in the only patients who had a PD in response to chemotherapy. This study would suggest that chemotherapy itself may paradoxically act, at least in part, as a cancer immunotherapy by inducing lymphocytosis, as well as previously demonstrated for the only immunotherapy with IL-2, probably by modulating the cytokine network and correcting the altered endogenous production of cytokines, responsible for cancer-related immunodeficiency.     

Combined Use of Anticancer Drugs and an Inhibitor of Multiple Drug Resistance-Associated Protein-1 Increases Sensitivity and Decreases Survival of Glioblastoma Multiforme Cells In Vitro

Glioblastoma multiforme (GBM) is a brain tumour characterised by a remarkably high chemoresistance and infiltrating capability. To date, chemotherapy with temozolomide has contributed only poorly to improved survival rates in patients. One of the most important mechanisms of chemoresistance comes about through the activity of certain proteins from the ATP-binding cassette superfamily that extrudes antitumour drugs, or their metabolites, from cells. We identify an increased expression of the multiple drug resistance-associated protein 1 (Mrp1) in glioblastoma multiforme biopsies and in T98G and G44 cell lines. The activity of this transporter was also confirmed by measuring the extrusion of the fluorescent substrate CFDA. The sensitivity of GBM cells was low upon exposure to temozolomide, vincristine and etoposide, with decreases in cell viability of below 20% seen at therapeutic concentrations of these drugs. However, combined exposure to vincristine or etoposide with an inhibitor of Mrp1 efficiently decreased cell viability by up to 80%. We conclude that chemosensitization of cells with inhibitors of Mrp1 activity might be an efficient tool for the treatment of human GBM.     

Restoration of expression of signal-transduction molecules in lymphocytes from patients with metastatic renal cell cancer after combination immunotherapy

A decrease in lymphocyte signal-transduction molecules, described in cancer patients and patients with chronic infectious diseases, has been proposed as a possible mechanism leading to an impaired immune response in cancer patients. Here we report the effects of combination immunotherapy on the levels of T cell receptor zeta chain and p56lck tyrosine kinase in a retrospective study of cryopreserved lymphocytes from 26 metastatic renal cell carcinoma patients treated with high-dose interleukin-2 (IL-2), interferon alpha (IFNalpha) and ex vivo IL-2-activated lymphocytes. Of the 26 patients, 12 were responders (5 complete and 7 partial) and 14 were non-responders (6 stable and 8 with progressive disease). Prior to treatment, 21 of 26 patients (81%) and 13 of 21 patients (62%) respectively expressed zeta chain and p56lck at less than 50% of the levels observed in healthy controls. During therapy, this low zeta chain and p56lck expression increased to at least 50% of normal in 13 of the 21 patients (62%) and in 6 of the 13 patients (46%) respectively; in the remaining patients expression levels remained at 50% of normal or more, or declined. Although, in this limited study, pretreatment levels of and p56lck did not show significant correlation with antitumor response, 4 of 5 patients that achieved a complete response (80%) corrected both zeta chain and p56lck levels to at least 50% of normal, while restoration of both signal-transduction molecules to such levels was only observed in 3 of 7 partial responders (43%), 1 of 5 patients with stable disease (20%) and 2 of 7 patients with progressive disease (29%). Thus, these results suggest that analysis of changes in signal-transduction molecules may a be useful tool for immunological monitoring of patients throughout immunotherapy, and could provide important information for designing new clinical trials that restore impaired signal transduction while activating T cell responses.     

Phase II study of interleukin-2 and 13-cis-retinoic acid as maintenance therapy in metastatic colorectal cancer

Purpose We have previously shown that low-dose interleukin-2 (IL-2) and 13-cis-retinoic acid (13-cis-RA) improved lymphocyte and natural killer (NK) cell count of patients with advanced tumors showing a clinical benefit from chemotherapy. The primary endpoint of this study was to ask whether IL-2 and 13-cis-RA improved (≥30%) lymphocyte and NK cell count in patients with metastatic colorectal cancer (MCRC) that had a clinical benefit from induction chemotherapy. Secondary endpoint was the evaluation of toxicity, progression-free survival (PFS), and overall survival (OS). Patients and methods Forty patients with MCRC, showing a clinical benefit from chemotherapy, were treated with subcutaneous low-dose IL-2 (1.8 × 10⁶ IU) and oral 13-cis-RA (0.5 mg/kg) in order to maintain responses and improve survival through the increase of lymphocyte and NK cells. The biological parameters and the clinical outcome of these patients were compared with those of a control group of patients (80) with a similar disease status, including clinical benefit from chemotherapy. Results The most common adverse events were mild cutaneous skin rash and fever. After 4 months and 2 years of biotherapy, a statistically significant improvement was observed in lymphocyte and number of NK cells with respect to baseline values and to controls. After a median follow-up of 36 months, median PFS was 27.8 months, while median OS was 52.9 months. Conclusion These data show that maintenance immunotherapy with low-dose IL-2 and oral 13-cis-RA in patients with MCRC showing a clinical benefit from chemotherapy is feasible, has a low toxicity profile, improves lymphocyte and NK cell count. An improvement in the expected PFS and OS was also observed. A randomized trial is warranted.