IL-33 facilitates rapid expulsion of the parasitic nematode Strongyloides ratti from the intestine via ILC2- and IL-9-driven mast cell activation.

Parasitic helminths are sensed by the immune system via tissue-derived alarmins that promote the initiation of the appropriate type 2 immune responses. Here we establish the nuclear alarmin cytokine IL-33 as a non-redundant trigger of specifically IL-9-driven and mast cell-mediated immunity to the intestinal parasite Strongyloides ratti. Blockade of endogenous IL-33 using a helminth-derived IL-33 inhibitor elevated intestinal parasite burdens in the context of reduced mast cell activation while stabilization of endogenous IL-33 or application of recombinant IL-33 reciprocally reduced intestinal parasite burdens and increased mast cell activation. Using gene-deficient mice, we show that application of IL-33 triggered rapid mast cell-mediated expulsion of parasites directly in the intestine, independent of the adaptive immune system, basophils, eosinophils or Gr-1+ cells but dependent on functional IL-9 receptor and innate lymphoid cells (ILC). Thereby we connect the described axis of IL-33-mediated ILC2 expansion to the rapid initiation of IL-9-mediated and mast cell-driven intestinal anti-helminth immunity.

Limited role of mast cells during infection with the parasitic nematode Litomosoides sigmodontis.

Mast cells are innate effector cells that due to their localization in the tissue form the first line of defense against parasites. We have previously shown that specifically mucosal mast cells were essential for the termination of the intestinal Strongyloides ratti infection. Here, we analyze the impact of mast cells on the immune response and defense against the tissue-dwelling filarial nematode Litomosoides sigmodontis using mast cell-deficient Cpa3cre mice. Despite an increase and an activation of mast cells at the site of infection in wildtype BALB/c mice the outcome of L. sigmodontis infection was not changed in mast cell-deficient BALB/c Cpa3cre mice. In Cpa3cre mice neither vascular permeability induced by blood-sucking mites nor the migration of L3 was altered compared to Cpa3 wildtype littermates. Worm burden in the thoracic cavity was alike in the presence and absence of mast cells during the entire course of infection. Although microfilaremiae in the peripheral blood increased in mast cell-deficient mice at some time points, the infection was cleared with comparable kinetics in the presence and absence of mast cells. Moreover, mast cell deficiency had no impact on the cytokine and antibody response to L. sigmodontis. In summary, our findings suggest that mast cells are not mandatory for the initiation of an appropriate immune response and host defense during L. sigmodontis infection in mice.

Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite Strongyloides ratti.

Infections with helminth parasites are controlled by a concerted action of innate and adaptive effector cells in the frame of a type 2 immune response. Basophils are innate effector cells that may also contribute to the initiation and amplification of adaptive immune responses. Here, we use constitutively basophil-deficient Mcpt8-Cre mice to analyze the impact of basophils during initiation and execution of the protective type 2 responses to both, a primary infection and a challenge infection of immune mice with the helminth parasite Strongyloides ratti. Basophil numbers expanded during parasite infection in blood and mesenteric lymph nodes. Basophil deficiency significantly elevated intestinal parasite numbers and fecal release of eggs and larvae during a primary infection. However, basophils were neither required for the initiation of a S. ratti-specific cellular and humoral type 2 immune response nor for the efficient protection against a challenge infection. Production of Th2 cytokines, IgG1 and IgE as well as mast cell activation were not reduced in basophil-deficient Mcpt8-Cre mice compared to basophil-competent Mcpt8-WT littermates. In addition, a challenge infection of immune basophil-deficient and WT mice resulted in a comparable reduction of tissue migrating larvae, parasites in the intestine and fecal release of eggs and L1 compared to mice infected for the first time. We have shown previously that S. ratti infection induced expansion of Foxp3+ regulatory T cells that interfered with efficient parasite expulsion. Here we show that depletion of regulatory T cells reduced intestinal parasite burden also in absence of basophils. Thus basophils were not targeted specifically by S. ratti-mediated immune evasive mechanisms. Our collective data rather suggests that basophils are non-redundant innate effector cells during murine Strongyloides infections that contribute to the early control of intestinal parasite burden.

Interleukin-9 promotes early mast cell-mediated expulsion of Strongyloides ratti but is dispensable for generation of protective memory.

IL-9 is a cytokine with pleiotropic function that mediates allergic inflammation and immunity to intestinal helminth parasites. Accumulating evidence suggests that IL-9 acts via both, initiation and regulation of adaptive immune responses and direct activation of intestinal effector pathways. Here we use IL-9 receptor deficient mice on BALB/c and C57BL/6 genetic background to dissect effector and regulatory functions of IL-9 during infection with the parasitic nematode Strongyloides ratti. IL-9 receptor-deficient mice displayed increased intestinal parasite burden and prolonged infection irrespective of the genetic background of the mice. Increased parasite burden was correlated to a reciprocally reduced early degranulation of mucosal mast cells, reduced intestinal IL-13 expression and caused by IL-9 receptor deficiency on hematopoietic cells. We observed additional significant changes in the adaptive immune response to S. ratti infection in the absence of the IL-9 receptor that depended on the mouse strain. However, the generation of protective memory to a second infection was intact in IL-9 receptor-deficient mice, irrespective of the genetic background. In summary, our results support a central role for IL-9 as an early mast cell activating effector cytokine during intestinal helminth infection while non-redundant functions in the initiation and amplification of adaptive immune responses were not apparent.

Basophils Are Dispensable for the Control of a Filarial Infection.

Basophils are innate effector cells that contribute to allergic reactions and provide protection against parasites. Using basophil-deficient Mcpt8-cre mice, we have previously shown that these granulocytes contributed to the immune mediated early control of the gastrointestinal helminth Strongyloides ratti in mice. In this study, we analyze the impact of basophils on the immune response and defense against the tissue-dwelling filarial helminth parasite Litomosoides sigmodontis Although basophils and IgE increased at the site of infection, the absence of basophils did not change the outcome of L. sigmodontis infection. Worm burden in the thoracic cavity and microfilaremiae in the peripheral blood were alike in L. sigmodontis-infected Mcpt8-cre mice compared with Mcpt8 wild type littermates during the entire course of infection. Analysis of the cytokine and Ab response to L. sigmodontis revealed no consistent alterations in the absence of basophils. Furthermore, basophil-deficient and -competent mice were protected to the same extent during a secondary infection with L. sigmodontis In summary, our findings suggest that basophils are dispensable for the initiation of the appropriate immune response and host defense against L sigmodontis infection in mice.

Filariae-Retrovirus Co-infection in Mice is Associated with Suppressed Virus-Specific IgG Immune Response and Higher Viral Loads.

Worldwide more than 2 billion people are infected with helminths, predominantly in developing countries. Co-infections with viruses such as human immunodeficiency virus (HIV) are common due to the geographical overlap of these pathogens. Helminth and viral infections induce antagonistic cytokine responses in their hosts. Helminths shift the immune system to a type 2-dominated immune response, while viral infections skew the cytokine response towards a type 1 immune response. Moreover, chronic helminth infections are often associated with a generalized suppression of the immune system leading to prolonged parasite survival, and also to a reduced defence against unrelated pathogens. To test whether helminths affect the outcome of a viral infection we set up a filarial/retrovirus co-infection model in C57BL/6 mice. Although Friend virus (FV) infection altered the L. sigmodontis-specific immunoglobulin response towards a type I associated IgG2 isotype in co-infected mice, control of L. sigmodontis infection was not affected by a FV-superinfection. However, reciprocal control of FV infection was clearly impaired by concurrent L. sigmodontis infection. Spleen weight as an indicator of pathology and viral loads in spleen, lymph nodes (LN) and bone marrow (BM) were increased in L. sigmodontis/FV-co-infected mice compared to only FV-infected mice. Numbers of FV-specific CD8+ T cells as well as cytokine production by CD4+ and CD8+ cells were alike in co-infected and FV-infected mice. Increased viral loads in co-infected mice were associated with reduced titres of neutralising FV-specific IgG2b and IgG2c antibodies. In summary our findings suggest that helminth infection interfered with the control of retroviral infection by dampening the virus-specific neutralising antibody response.

Foxp3⁺ regulatory T cells delay expulsion of intestinal nematodes by suppression of IL-9-driven mast cell activation in BALB/c but not in C57BL/6 mice.

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.

Age has no significant impact on overall survival and on treatment tolerability in relapsed stage IV cutaneous malignant melanoma patients receiving Cisplatin, Gemcitabine, and Treosulfan.

OBJECTIVES: We compared the efficacy and tolerability of cisplatin, gemcitabine, and treosulfan (CGT) therapy in younger patients (age, <60 years) and in elderly patients (age, ≥60 years) with pretreated relapsed American Joint Committee on Cancer stage IV cutaneous malignant melanoma. PATIENTS AND METHODS: A total of 91 patients at the age of 18 to 80 years, in relapse after first-, second-, or third-line therapy received 40 mg/m intravenous (i.v.) cisplatin, 1000 mg/m i.v. gemcitabine, and 2500 mg/m i.v. treosulfan on days 1 and 8. CGT-therapy was repeated every 5 weeks until progression of disease occurred. RESULTS: Younger (n = 49) and elderly (n = 42) patients showed a significant difference in disease stabilization in 25% versus 7% (P ≤ 0.05), as opposed to 69% versus 91% patients exhibiting disease progression. In contrast, the overall median survival probability was not significantly different (P = 0.8153). Neither treatment-related toxicity nor toxicity-associated dose reduction showed substantial differences. CONCLUSIONS: Our results demonstrated that CGT therapy could be safely administered to a patient up to age 80 years.

Bleomycin, vinorelbine and trofosfamide in relapsed stage IV cutaneous malignant melanoma patients.

PURPOSE: To evaluate the efficacy of bleomycin, vinorelbine, and trofosfamide (BVT) in 28 patients with pretreated relapsed AJCC stage IV cutaneous malignant melanoma. METHODS: Patients in relapse after first- or second-line therapy received 8 mg/m(2) intravenous (i.v.) bleomycin, 25 mg/m(2) i.v. vinorelbine, on days 1 and 6, each, and oral (p.o.) trofosfamide 60 mg/m(2)/day, days 1-7. BVT therapy was repeated every 5 weeks until progression of disease occurred. A maximum of six BVT cycles (mean, 2.2 cycles) was administered per patient. RESULTS: Three patients (11%) reached a partial response; 5 (18%) patients showed stable disease, and 20 (71%) patients progressed upon BVT therapy. Median overall survival of all 28 patients was 6 months (6-month survival rate, 52%). Patients with partial remission or stable disease (n = 8) exhibited a median overall survival of 10 months (6-month survival rate, 75%), while patients with disease progression (n = 20) showed a median overall survival of 3 months (6-month survival rate, 43%). Most side effects were limited to WHO grade I/II mild anemia, leucocytopenia, fatigue, nausea/vomiting, pain, and anorexia. WHO grade III/IV side effects occurred in 7% (anorexia) and 4% (fatigue) of patients. CONCLUSION: Treatment with BVT was efficient in 29% of pretreated relapsed stage IV cutaneous melanoma patients, with overall good tolerability and safety.

Increased expression of the tumor suppressor PLZF is a continuous predictor of long-term survival in malignant melanoma patients.

Promyelocytic leukemia zinc finger (PLZF) is a transcriptional repressor and tumor suppressor inhibiting melanoma cell growth in vitro and in vivo in animal models. In this study, we analyzed the impact of in vivo primary tumor gene expression of PLZF on the long-term survival of malignant melanoma patients. PLZF expression was assessed by using DNA microarray and real-time polymerase chain reaction analysis of 41 primary malignant melanomas from patients with a defined histology and a close to 20-year clinical follow-up, of 29 melanoma metastases, and of 6 different melanoma cell lines. Kaplan-Meier survival analyses, log-rank statistics and Cox regression analysis were employed to identify the impact of PLZF expression on long-term survival. We detected PLZF expression in 92% of primary melanoma tumors in vivo but not in melanoma cell lines in vitro. By univariate analysis, we identified: (1) PLZF mRNA expression < or = 10,000 mRNA copies/mug total tumor RNA, (2) Breslow tumor thickness >4 mm, and (3) American Joint Committee on Cancer stages IIC, IIIB, IIIC, and IV as statistically significant pretreatment risk factors. We defined a continuous prognostic index (i.e., risk score) for primary melanoma patients based on the regression coefficient of PLZF mRNA expression. Applying a cutpoint to the prognostic index at - 1.65, patients were assigned to one of two risk groups: low-risk patients (n = 28) with a median overall survival of 79 months (5-year survival of 61%) and high-risk patients (n = 13) with a median overall survival of 32 months (5-year survival of 23%) (p < 0.05). This is the first time that PLZF mRNA expression has been linked to a prognostic model for primary malignant melanoma patients to derive prognostic groups for clinical purposes (e.g., improved melanoma immunotherapies).

Peripheral blood neutrophils as independent immunologic predictor of response and long-term survival upon immunotherapy in metastatic renal-cell carcinoma.

The aim of this study was to evaluate the prognostic impact of pretreatment neutrophils, previously rendered statistically independent, on the response and on long-term survival of metastatic renal carcinoma patients treated with outpatient subcutaneous (s.c.) interleukin-2 (IL-2) and s.c. interferon (IFN)-alpha. We assessed a total of 495 patients receiving s.c. IL-2/s.c. IFN-alpha-based therapy. While 417 patients with neutrophil counts <6500 cells/microL at baseline achieved 30% objective responses and a median survival of 22 months, 78 patients with pretreatment neutrophil counts >or= 6500 cells/microL had 18% objective responses and a median survival of 9 months (p=0.0000). In conclusion, pretreatment peripheral blood neutrophils <6500/microL constitute an immunologic predictor of tumor response and long-term survival in metastatic renal-cell carcinoma patients treated with s.c. IL-2 and s.c. IFN-alpha-based regimens.

Cisplatin, gemcitabine and treosulfan is effective in chemotherapy-pretreated relapsed stage IV uveal melanoma patients.

PURPOSE: The efficacy of cisplatin, gemcitabine, and treosulfan (CGT) was evaluated in patients with chemotherapy pretreated relapsed AJCC stage IV uveal malignant melanoma. METHODS: Patients received i.v./intrahepatic cisplatin, i.v. gemcitabine, and i.v. treosulfan (CGT) on day 1 and 8 as first-line (n = 1), second-line (n = 9), third-line (n = 1) or fourth-line (n = 1) therapy. Cisplatin, gemcitabine, and treosulfan (CGT)-therapy was repeated every 5 weeks until progression of disease occurred. A maximum of six CGT-cycles (mean, 2 cycles) was administered per patient. RESULTS: No objective response was observed, six patients (50%) had stable disease and six (50%) patients progressed upon first reevaluation. Overall survival of all the 12 patients was 6 months. Patients with stable disease reached a median overall survival of 12 months, while patients with disease progression upon first reevaluation had a median overall survival of 4 months, only. Grade III/IV related hematotological side effects were experienced in six (leukopenia) and four (thrombocytopenia) patients. CONCLUSIONS: Treatment with CGT may lead to disease stabilization and prolonged survival in a substantial proportion of progressive stage IV uveal melanoma patients, even following heavy chemotherapy treatment.

Fractional polynomials in a new metastatic renal carcinoma continuous prognostic index involving histology, laboratory, and clinical predictors.

BACKGROUND: We analyzed the effect of histology and various clinical and laboratory predictors in a new continuous prognostic index for metastatic renal-cell carcinoma based on fractional polynomials. PATIENTS AND METHODS: We evaluated 322 metastatic renal-cell carcinoma patients treated with subcutaneous recombinant cytokine-based home therapies in consecutive trials. We evaluated papillary histology, along with age, disease localizations, C-reactive protein, and neutrophil count in a new prognostic index, which was based on the multivariable fractional polynomial (MFP) algorithm. RESULTS: The MFP model allowed us to divide patients into three risk groups, using seven selected significant prognostic factors: histology, neutrophils, C-reactive protein, bone metastases, liver metastases, lymph node metastases, and age. Using the multivariable fractional polynomial model, median overall survival for high-, intermediate-, and low-risk patients was 10 months (n=80), 23 months (n=162), and 41 months (n=80) (scheme A; p

GM-CSF plus antigenic peptide vaccination in locally advanced melanoma patients.

Twenty-four (24) pretreated patients with relapsed high-risk resected The American Joint Committee on Cancer (AJCC) stage IIA-IV melanoma received adjuvant peptide vaccinations derived from the melanosomal antigens MelanA/MART1, MAGE-1, gp100, and tyrosinase, according to patient tumor-associated human leukocyte antigen (HLA) restricted antigen expression, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Pretreatment was comprised of surgery (n=23 primary tumor; n=23 metastases), local radiotherapy (n=2), immunotherapy (n=23), chemotherapy (n=10), and chemoimmunotherapy (n=1), respectively. All patients received peptide vaccines in an adjuvant setting. Seven (7) patients were relapse free for 3+ up to 25+ months. Of the patients exhibiting progressive disease (n=17), 13 patients developed metastases during vaccination (9 local, 4 distant), and 4 patients developed metastases (2 local, 2 distant) after finishing vaccine therapy. Two (2)-year local and distant metastases-free survival, 2-year distant metastases-free survival, and 2-year overall survival were calculated as 8.6%, 68%, and 85%, respectively. Vaccine treatment was well tolerated, with no severe side-effects. Twenty (20) of 24 patients developed local delayed-type hypersensitivity (DTH) reactions to synthetic peptide vaccination. Transient fever (n=2) and pain in muscle/bone (n=2) occurred rarely. In conclusion, antigenic peptide vaccination, combined with GM-CSF, is safe and may yield clinical benefits in relapsed high-risk resected melanoma patients.

Long-term maintenance therapy in interferon-alpha2a/interleukin-2-pretreated advanced renal-cell carcinoma patients.

PURPOSE: In this paper, we report on the long-term therapeutic efficacy of maintenance treatment in 12 advanced renal carcinoma patients. PATIENTS AND METHODS: Following prior systemic treatment with 8-week cycles of subcutaneous interferon- alpha2a (s.c. IFN-alpha2a) and subcutaneous interleukin-2 (s.c. IL-2)-based immunotherapy (5-day standard Atzpodien regimen), patients received prolonged maintenance treatment consisting of intermittent s.c. IFN-alpha2a and s.c. IL-2, combined with long-term daily peroral 13-cis-retinoic acid (p.o. 13cRA). RESULTS: Patients received a mean of 10 months (range, 0-27 months) of maintenance treatment. Median progression-free survival was calculated at 6 months (range, 0-61 months), and median overall suvival was 22 months (range, 2-65 months) from the start of maintenance therapy. Of 12 patients, 5 were still alive (> or = 60 months) and 2 patients remained progression-free (61 months) at last follow-up. Maintenance treatment was well or moderately tolerated. CONCLUSIONS: Maintenance treatment with s.c. IL-2/s.c. INF-alpha2a/p.o. 13-cRA may support long-term efficacy of prior s.c. IL-2/s.c. INF-alpha2a-based therapy in advanced renal carcinoma patients.

Metastatic renal carcinoma long-term survivors treated with s.c. interferon-alpha and s.c. interleukin-2.

AIM: The aim of this retrospective analysis was to identify common features of long-term survivors among 218 advanced renal cell carcinoma patients sequentially entered on subcutaneous-recombinant-cytokine- based therapies between 1988 and 1993. PATIENTS AND METHODS: All patients were treated with subcutaneous (s.c.) interferon-alpha2a (IFN-alpha2a) and s.c. interleukin-2 (IL-2) alone (n = 98 pts) or in combination with intravenous (i.v.) 5-fluorouracil (5-FU) (Atzpodien regimen; n = 120 pts); those patients who survived more than 10 years were classified as long-term survivors. RESULTS: Thirteen patients (6.3%) were identified as long-term survivors with a median follow-up of 141 months (range, 122-174 months). According to a validated model of known clinical predictors, the long-term survivor group consisted of 6 low-risk, 5 intermediate-risk, and 2 high-risk patients, respectively. Within their clinical course, 9 longterm survivors achieved a complete response with a median duration of 141 months (range, 91-161 months), 1 patient yielded a partial remission, and 3 patients achieved stable disease. Maximum response was observed between 2 and 40 months after treatment initiation (median, 4 months), while treatment time to maximum response ranged from 2 to 14 months (median, 4 months). There was no correlation between treatment time and maximum response. Overall, long-term survivors underwent treatment for 4 and up to 80 months (median, 8 months). CONCLUSION: Our data suggest that long-term survival of metastatic renal carcinoma patients beyond 10 years is independent of known clinical risk factors and treatment time. However, long-term survival of cytokine-treated, advanced renal cell carcinoma (RCC) patients remains a rare event and, thus, emphasizes the need for further investigations toward more effective therapies.

Dose-dependent treatment benefit in high-risk melanoma patients receiving adjuvant high-dose interferon alfa-2b.

UNLABELLED: This retrospective analysis of 150 consecutive high-risk melanoma patients treated with high-dose interferon alfa-2b at a single institution demonstrates similar relapse-free and overall survival data, as previously published from Eastern Cooperative Oncology Group (ECOG) and Intergroup trials. The data suggest at least a transient dose dependency of the treatment effect on relapse-free and overall survival with high-dose interferon in high-risk melanoma patients. BACKGROUND: Adjuvant high-dose interferon seems to be the best adjuvant treatment option for patients with high-risk melanoma (AJCC-stage IIC, III) after definitive surgery. METHODS: One-hundred fifty consecutive patients were treated at our institution during the period from September 1997 to March 2003 were retrospectively studied. RESULTS: After a median follow-up of 35 months, 63% of patients had developed a melanoma relapse, and 37% were relapse- free. Fifty-five percent of patients are still alive, and 45% had died-all but 3 patients from melanoma. Patients with stage IIC disease demonstrated a similar unfavorable course of disease as patients with stage IIIC disease (2-year relapse-free survival 18% and 26%). We identified two groups of patients with different cumulative interferon dose-levels (> or =90% and <90% of the projected dose, according to the protocol), who demonstrated at least transient differences, both in terms of relapse-free and overall survival; the predictive impact was statistically independent upon the Cox regression analysis. CONCLUSIONS: Our clinical data are consistent with the published ECOG and Intergroup data dealing with highdose interferon in high-risk melanoma patients. The data suggest a dose-dependency on the treatment effect and, therefore, support further prospective trials comparing different dose-distribution patterns in high-dose interferon.

Predictive impact of retinoid X receptor-alpha-expression in renal-cell carcinoma.

PURPOSE: Retinoid receptors are nuclear transcription factors that mediate the effects of retinoids on gene expression. In our study, we analyzed the expression of retinoid X receptor-alpha (RXR-alpha) and its prognostic value in renal-cell carcinoma (RCC) patients exhibiting stage IV disease upon first diagnosis or thereafter. MATERIALS AND METHODS: Detection of RXR-alpha was performed on tumor specimens from 63 patients with primary RCC, using immunohistochemical techniques. For our evaluation of the immunostaining results, we developed a new cell-counting system based on the subcellular distribution of immunoreactivity. The impact of the subcellular distribution of RXR-alpha on the prognosis of patients with RCC was analyzed statistically among other clinicopathologic factors. The primary end point was survival. RESULTS: In 34 RCC samples (54.0%), RXR-alpha was detected predominantly in the nucleus, while 25 RCC specimens (39.7%) displayed an aberrant subcellular distribution pattern, with a predominantly cytoplasmic staining with nuclear sparing in 15 specimens (23.8%), and a combined nuclear and cytoplasmic staining in 10 specimens (15.9%). Very faint to undetectable immunoreactivity was noted in 4 cases (6.3%) of RCC. Univariate Kaplan-Meier analysis showed that patients with a predominantly nuclear localization of RXR-alpha had a significantly prolonged survival after primary tumor diagnosis, when compared to patients with a predominantly aberrant subcellular distribution profile (p < 0.01). Furthermore, multivariate analysis revealed that an aberrant subcellular distribution of RXR-alpha in RCC was an independent predictor of poor survival (p < 0.01). CONCLUSIONS: Our study indicated that the subcellular intratumoral distribution pattern of RXR-alpha could independently predict the survival of RCC patients. However, the exact mechanisms underlying the aberrant compartmentalization and the functions of RXR-alpha in RCC remain to be determined.

Individualized synthetic peptide vaccines with GM-CSF in locally advanced melanoma patients.

We report on 10 patients with resected American Joint Committee on Cancer (AJCC)stage IIA-IIIC melanoma receiving individualized adjuvant peptide vaccinations derived from the melanosomal antigens MelanA/MART1, gp100 and tyrosinase, according to patient tumor associated HLA restricted antigen expression, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Except for 1 patient, all patients had received systemic pretreatment with immunotherapy (n = 8), chemoimmunotherapy (n = 1), chemotherapy (n = 1), or cefalectin therapy (n = 1). Upon prior therapy, 7 of 10 patients had progressed with subcutaneous/cutaneous (n = 2), lymph node (n = 3), or subcutaneous/cutaneous and lymph node (n = 2)metastases, which were subsequently resected prior to vaccination. After a mean of 6.5 vaccination cycles, progression-free survival was 6 months (median, range 2-10). Five patients were relapse-free for 1+ up to 21+ months, 3 patients developed a solitary cutaneous metastasis, and 2 patients developed multiple metastases during vaccination. Overall, vaccine treatment was well tolerated, with no severe side-effects. Eight of 10 patients developed local delayed type hypersensitivity (DTH)reactions to synthetic peptides after the first or second injection. In 2 patients, transient fever, nausea, diarrhea, and muscle pain of National Cancer Institute (NCI)Grade I occurred. In summary, individualized synthetic peptide vaccination, combined with GM-CSF, was feasible and warrants further clinical investigation.

Thirteen-year, long-term efficacy of interferon 2alpha and interleukin 2-based home therapy in patients with advanced renal cell carcinoma.

BACKGROUND: The goal of the current report was to demonstrate the long-term efficacy of outpatient subcutaneous (sc) interferon alpha (IFN-alpha) and sc interleukin 2 (IL-2)-based combination regimens in patients with metastatic renal cell carcinoma. METHODS: In three consecutive clinical trials, 443 patients received combined sc IFN-alpha and sc IL-2 (n = 97 patients); combined sc IFN-alpha, sc IL-2, and intravenous (iv) 5-fluorouracil (5-FU) (n = 260 patients); or combined sc IFN-alpha, sc IL-2, and iv 5-FU with oral 13cis-retinoic acid (n = 86 patients). RESULTS: The median overall survival was 21+ months. The 2-year, 5-year, and 13-year survival rates were calculated at 45.26%, 15.96%, and 8.96%, respectively. The median time to disease progression was 6 months. The 2-year, 5-year, and 13-year progression free survival rates were 17.84%, 9.54%, and 9.20%, respectively. CONCLUSIONS: The current data suggest that combined outpatient sc IFN-alpha and sc IL-2, according to the Atzpodien regimen, achieves long-term survival benefits in a subset of patients with metastatic renal cell carcinoma, both with and without 13-cis-retinoic acid and/or 5-fluorouracil.