High-dose Recombinant Interleukin-2 Research Articles

Abstract 2812: NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity

Abstract There is a growing interest in combining radiotherapy (RT) with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2Rα (CD25). Here we examined the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway dependency of type I interferon (IFN) response and anti-tumor effect of RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2Rβ and IL2Rγc. In mice implanted with E0771 syngeneic tumors, NL-201 in combination with RT enhanced activation of the STING pathway. This was associated with increased type I IFN production as well as increased activation and number of tumor associated dendritic cells (DCs). In STING (-/-) mice implanted with E0771 tumors, Type I IFN production, tumor-growth inhibition (TGI), and survival advantage was significantly decreased and there were significantly fewer activated CD4+ and CD8+ T-cells. Similarly, CD11c+ knock-out mice implanted with E0771 tumors and treated with the combination exhibited similar TGI, survival, and IFN production compared to vehicle treated mice. Based on these results, we tested the ability of the combination of RT and NL-201 to enhance systemic immunotherapy with an anti-PD-1 checkpoint inhibitor in mice implanted with 4T1 breast tumor cells. Tumor-bearing mice treated with the triple combination exhibited superior TGI and survival, along with increased effector memory T-cell populations as compared to all other treatment groups. These results support our previous findings that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, synergizes with RT, likely via the direct activation of STING. Genetic knockout of STING in tumor-bearing mice abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s immune activation and resulting antitumor effect. These results also describe the important role of STING-mediated activation and tumor penetration of DCs, which were crucial for anti-tumor response. Additionally, when combined with anti-PD-1 antibodies, NL-201+RT combination treatment significantly improved TGI of metastatic breast cancers that were previously resistant to checkpoint blockade. This work provides rationale for the continued exploration of RT in combination with CD25-independent IL-2Rβγ agonism as a means to enhance the systemic immune response to treat immunologically challenging tumors. Citation Format: Kristin Huntoon, DaeYong Lee, Xuefeng Li, Yifan Wang, Wen Jiang, Betty Kim. NL-201, a de novo CD25-independent IL-2Rβγ agonist, synergizes with radiation to generate potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2812.

IMMU-34. NL-201, A DE NOVO ENGINEERED IL2 MIMETIC, SYNERGIZES WITH RADIOTHERAPY TO GENERATE POTENT ANTITUMOR ACTIVITY IN PRECLINICAL GLIOBLASTOMA MODELS

Abstract Combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents has been gaining interest due to improved efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of recombinant IL-2 is limited by toxicity at high doses and the expansion of regulatory T cells (Tregs) at low doses, effects thought to be mediated by preferential binding to IL2Rα (i.e. CD25). In this study we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2/IL-15 agonist with enhanced affinity for the IL-2Rβγ heterodimeric receptor. Single-agent NL-201 was well tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine glioblastoma (GL261 and SB28). In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition. We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen.

Abstract 2067: NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity

Abstract There is a growing interest in combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2RΑ (CD25). In this study, we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2RΒ and IL2RΓ. Single-agent NL-201 was well-tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine breast cancers. In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen-presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition and survival benefit in both localized and metastatic murine breast cancers, including those spread to the central nervous system. Genetic knockout of STING in host cells abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s antitumor responses. Additionally, when combined with PD-1 antibodies, NL-201 significantly improved growth inhibition of metastatic breast cancers that were previously resistant to checkpoint blockade. We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors and brain metastases. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT, likely via the direct activation of STING in DCs. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen in both localized and metastatic settings. Citation Format: Xuefeng Li, Kristin Huntoon, Yifan Wang, Carl Walkey, Betty Y. Kim, Wen Jiang. NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2067.

Tumor-selective activity of XTX202, a protein-engineered IL-2, in mice without peripheral toxicities in nonhuman primates.

2563 Background: High-dose recombinant human interleukin-2 (aldesleukin) elicits anti-tumor immunity and is approved for the treatment of renal cell carcinoma and melanoma based on durable complete remissions. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. Recent second-generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a second generation IL-2 surrogate still experience characteristic dose-limiting toxicities, including vascular leak syndrome. To overcome these toxicities and improve the therapeutic index (TI) of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX202, a highly potent third generation masked IL-2. XTX202 is unmasked in the tumor microenvironment by proteolytic activation resulting in full restoration of binding to IL-2Rβ without binding to IL-2Rα. The current study characterizes the therapeutic index of XTX202 versus aldesleukin and a second generation IL-2 surrogate. Methods: XTX202 bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and peripheral immune activation were evaluated in mice bearing syngeneic tumor models. Safety was evaluated in rodents and Cynomolgus monkeys. XTX200, an unmasked half-life extended IL-2 that does not bind to IL-2Rα, was used as a surrogate second generation IL-2. Results: Masked XTX202 showed limited IL-2R-dependent STAT-5 signaling in vitro. Proteolytic activation of XTX202 resulted in CD8+ T and NK cell activation and over 1000-fold reduction in Treg activation as compared to WT IL-2. XTX202 achieved potent tumor growth inhibition in syngeneic mouse models as a single agent with no evidence of toxicity or peripheral immune activation, thus demonstrating tumor selective activity. XTX202 efficacy in mice at 2 mg/kg dose was equivalent to that achieved with the MTD dose of 0.5 mg/kg of a second generation IL-2 surrogate. XTX202 was well tolerated in NHPs in a 4-week repeat dose study at doses up to 30 mg/kg QW whereas a second generation IL-2 surrogate was not tolerated beyond 0.7 mg/kg QW. Based on these data, XTX202 has a 10 fold improvement in TI vs second generation IL-2. Based on comparative efficacy studies with aldesleukin and literature NHP tolerability data, XTX202 is projected to have a ≥150 fold greater TI than aldesleukin. Conclusions: XTX202, a third generation, tumor-selective IL-2, inhibits tumor growth and is well tolerated in repeat dose studies in NHPs at high doses. GLP toxicity studies with XTX202 are underway and first-in-human studies are expected to initiate this year. XTX202 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of IL-2 while minimizing dose-limiting toxicities.

Human Mesenchymal Stem Cells Overexpressing Interleukin 2 Can Suppress Proliferation of Neuroblastoma Cells in Co-Culture and Activate Mononuclear Cells In Vitro.

High-dose recombinant interleukin 2 (IL2) therapy has been shown to be successful in renal cell carcinoma and metastatic melanoma. However, systemic administration of high doses of IL2 can be toxic, causing capillary leakage syndrome and stimulating pro-tumor immune response. One of the strategies to reduce the systemic toxicity of IL2 is the use of mesenchymal stem cells (MSCs) as a vehicle for the targeted delivery of IL2. Human adipose tissue-derived MSCs were transduced with lentivirus encoding IL2 (hADSCs-IL2) or blue fluorescent protein (BFP) (hADSCs-BFP). The proliferation, immunophenotype, cytokine profile and ultrastructure of hADSCs-IL2 and hADSCs-BFP were determined. The effect of hADSCs on activation of peripheral blood mononuclear cells (PBMCs) and proliferation and viability of SH-SY5Y neuroblastoma cells after co-culture with native hADSCs, hADSCs-BFP or hADSCs-IL2 on plastic and Matrigel was evaluated. Ultrastructure and cytokine production by hADSCs-IL2 showed modest changes in comparison with hADSCs and hADSCs-BFP. Conditioned medium from hADSC-IL2 affected tumor cell proliferation, increasing the proliferation of SH-SY5Y cells and also increasing the number of late-activated T-cells, natural killer (NK) cells, NKT-cells and activated T-killers. Conversely, hADSC-IL2 co-culture led to a decrease in SH-SY5Y proliferation on plastic and Matrigel. These data show that hADSCs-IL2 can reduce SH-SY5Y proliferation and activate PBMCs in vitro. However, IL2-mediated therapeutic effects of hADSCs could be offset by the increased expression of pro-oncogenes, as well as the natural ability of hADSCs to promote the progression of some tumors.

386P - The treatment of disseminated skin melanoma with high-dose recombinant interleukin-2 in combination with chemotherapy

386P - The treatment of disseminated skin melanoma with high-dose recombinant interleukin-2 in combination with chemotherapy

The experience of administration of high-dose recombinant interleukin-2 in combination with chemotherapy in the management of disseminated skin melanoma.

e20008 Background: Interleukin-2 (Biotech, Russia) is a complete structural and functional analogue of human endogenous interleukin-2 produced from nonpathogenic Saccharomyces cerevisiae. Our retro...

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Lymphokine-activated killer cytotoxicity against leukaemic blast cells

While the sensitivity of cell lines and solid tumours to lymphokine-activated killer (LAK) cell cytotoxicity is extensively reported, the ability of these generated cytotoxic cells to lyse leukaemic blast cells is controversial. This study reports the successful generation of LAK cells with specificity for leukaemic blast cells. Control donors, patients in remission and patients with active leukaemia were capable of lysing allogeneic leukaemic blast cells. One patient was also capable of lysing autologous leukaemic blast cells. Successful LAK generation was achieved by the use of high dose recombinant interleukin-2 (rIL-2) while combinations of lymphokines did not improve LAK specificity or efficacy. These findings suggest that LAK immunotherapy may be considered for the treatment of patients with acute leukaemia.

Phase I trial of IL-2 plasmid DNA with electroporation in metastatic melanoma

8578 Background: Significant toxicity limits the systemic delivery of high-dose recombinant Interleukin-2 (IL-2). An alternative method for extended dosing of IL-2 that may reduce toxicity is by intratumoral injection of IL-2 plasmid DNA (pDNA) with electroporation (EP). Methods: A phase I dose-escalation trial is ongoing in subjects with metastatic melanoma to evaluate the safety of intratumoral delivery of IL-2 pDNA (VCL-IM01, Vical Inc., San Diego, CA) followed by EP (MedPulser DNA EPT System, Inovio, San Diego, CA). Eligible subjects have recurrent metastatic melanoma; an injectable lesion = 1 cm2 and < 25 cm2; ECOG 0 or 1; LDH = 1.5 × ULN, and no brain or liver metastases. In the dose-escalation stage of the trial, 3 subjects in each dose cohort received up to 2 cycles of treatment, each consisting of 4 weekly injections followed by a 4-week observation period. Dose levels included 0.5, 1.5, 5.0 mg/tumor, and 15.0 mg (5 mg in each of 3 tumors). A safety assessment was conducted for each cohort prior to enrollment of the next cohort. In the 2nd trial stage, 17 subjects are to be enrolled at the maximum tolerated dose (MTD). The observation period is shortened to 2 weeks between cycles. For all subjects, safety is assessed at every visit. Results: 12 subjects (7 male, 5 female) were enrolled in the dose escalation stage, 3 subjects at each dose. Ages range from 38 to 86 years. No Grade 3 or 4 adverse events (AEs) were reported related to study drug or procedures. All related AEs (12 reported) were Grade 1: 5 related to study drug, 4 to the EP procedure, and 3 to both. Injection site pain was the most common AE. No dose-limiting toxicities occurred; thus the MTD was defined as the 15 mg dose (5 mg/tumor in 3 tumors). To date, 6/17 subjects in Stage 2 of the trial (5 mg/tumor, up to 3 tumors injected) have been enrolled with no Grade 3 or 4 AEs related to study drug or injection/EP procedures. Physicians have observed responses in treated and untreated lesions. Overall response data will be presented. Conclusions: Intratumoral administration of IL-2 pDNA with EP appears safe and well tolerated in 18 patients with metastatic melanoma when given up to a 15 mg dose (5 mg/tumor). Preliminary indications of decreased tumor size suggest local and systemic activity of IL-2 pDNA with EP. No significant financial relationships to disclose.

Effects of the Administration of High-Dose Interleukin-2 on Immunoregulatory Cell Subsets in Patients with Advanced Melanoma and Renal Cell Cancer

High-dose recombinant human interleukin-2 (IL-2) therapy is of clinical benefit in a subset of patients with advanced melanoma and renal cell cancer. Although IL-2 is well known as a T-cell growth factor, its potential in vivo effects on human immunoregulatory cell subsets are largely unexplored. Here, we studied the effects of high-dose IL-2 therapy on circulating dendritic cell subsets (DC), CD1d-reactive invariant natural killer T cells (iNKT), and CD4(+)CD25(+) regulatory-type T cells. The frequency of both circulating myeloid DC1 and plasmacytoid DC decreased during high-dose IL-2 treatment. Of these, only a significant fraction of myeloid DC expressed CD1d. Although the proportion of Th1-type CD4(-) iNKT increased, similarly to DC subsets, the total frequency of iNKT decreased during high-dose IL-2 treatment. In contrast, the frequency of CD4(+)CD25(+) T cells, including CD4(+)Foxp3(+) T cells, which have been reported to suppress antitumor immune responses, increased during high-dose IL-2 therapy. However, there was little, if any, change of expression of GITR, CD30, or CTLA-4 on CD4(+)CD25(+) T cells in response to IL-2. Functionally, patient CD25(+) T cells at their peak level (immediately after the first cycle of high-dose IL-2) were less suppressive than healthy donor CD25(+) T cells and mostly failed to Th2 polarize iNKT. Our data show that there are reciprocal quantitative and qualitative alterations of immunoregulatory cell subsets with opposing functions during treatment with high-dose IL-2, some of which may compromise the establishment of effective antitumor immune responses.

Transient massive peripheral blood blastosis in patient with renal clear-cell carcinoma after treatment with high-dose recombinant interleuklin-2

A 35-year-old woman presented at the Orthopaedic Department with pain in the right hip. Pelvic CT showed a lytic lesion in the sacrum with an associated soft-tissue mass. CT-guided needle biopsy of the bone lesion disclosed clear-cell carcinoma metastasis. Further more, abdominal CT revealed a well-defi ned mass in the left kidney measuring 8×9 cm; thoracic CT showed other abnormalities. A nephrectomy was done for diagnostic and therapeutic purposes. As a result of the diagnosis of stage IV clear-cell renal carcinoma, which had a Fuhrman grade IV, an Eastern Cooperative Oncology Group grade 1, bone dissemination, and a high-risk level according to the University of California Los Angeles integrated staging system, high-dose recombinant interleukin-2 (HD-IL2) was started after recovery from surgery. The patient received 600 000 U/kg of intravenous IL-2 every 8 h for 5 days in two courses, the fi rst course beginning on day 1 and the second beginning on day 15. Throughout HD-IL2 administration, the patient also received commonly used concomitant medication. Baseline blood count before the second course of HD-IL2 was 11·1×10/L of leucocytes (44% neutrophils, 42% lymphocytes, 7% monocytes, 6% eosinophils, and 1% basophils), 114 g/L of haemoglobin, and 391×10/L of platelets. After the fourth dose of the second course of HD-IL2, the patient presented tachycardia (130 bpm), hypotension (70/30 mmHg), and oligoanuria with impairment of renal function (creatinine level 176·8 μmol/L). Metabolic acidosis (pH 7·2; HCO3 14·8 mEq/L) was also recorded. Finally, hypoxaemia (sO2 92%) and radiological bilateral interstitial infi ltrates, shown by chest radiography, revealed pulmonary oedema. The patient was subsequently admitted to the intensivecare unit where supplemental oxygen (3 L/min) and HCO3 were then initiated. Additionally, hypotension was treated with both crystalloids and norepinephrine administration (0·2 μg/kg per min). HD-IL2 was discontinued after the 12th dose of the second course because of its toxic eff ects. Symptoms resolved 2 days after the last dose of HD-IL2 was given; however, blood count showed leucocytosis (22×10/L) with mild anaemia (Haemoglobin 104 g/L) and thrombocytopenia (113×10/L). Diff erential blood count showed 53% undiff erentiated blast cells; 53% erythroblasts were also counted (fi gure). Acute leukaemia was ruled out by means of a bone marrow aspirate done 16 h later. Bone marrow smears showed a hypercellular marrow with a normal myeloid and erythroid ratio, a leftward shift of myeloid series with 2% undiff erentiated blast cells and 11% myeloid blast cells, and predominance (48%) of semimature and, to a lesser extent, mature granulopoietic cells with no dysplastic features. Megakaryocytic hyperplasia was also noted. No metastases were seen. Cytogenetic analysis done on 30 metaphases from unstimulated culture of the bone-marrow sample showed a 46XX karyotype with no structural chromosome anomaly. Blood count returned to baseline levels within the following 2 days. 6 weeks after the last dose of HD-IL2 had been given, an enlargement (35% increase in the larger diameter) of the bone mass was noted. Because the disease was refractory to immunotherapy, the patient has started treatment with sunitinib (SU11248) within a clinical trial. Clear-cell renal carcinoma is the most common adult renal neoplasm. HD-IL2 received approval from the Food and Drug Administration in 1992 for the treatment of patients with stage IV renal-cell carcinoma, on the basis of results of 255 patients who were included in seven phase II trials. In selected patients, the complete response rate was 7% (median follow up of 54 months, range 3–131 months) with actuarial curves predicting that 60% would be free Lancet Oncol 2007; 8: 275–76

Phase I/II study of topical imiquimod and intralesional interleukin-2 in the treatment of accessible metastases in malignant melanoma

Patients with metastatic skin disease in malignant melanoma can be difficult to treat effectively, often requiring repeated treatments with different modalities in an attempt to control their disease. Treatment of nonsurgically resectable melanoma deposits is unsatisfactory, as they are often multiple and recurring. Anecdotal evidence from individual use of imiquimod in superficial metastases and intralesional interleukin (IL)-2 in subcutaneous deposits suggests that the combination may be more effective in bulky subcutaneous disease. To investigate the combination of topical imiquimod and, for selected lesions, intralesional IL-2, to treat a small cohort of patients with accessible melanoma metastases resistant to other treatments. Thirteen patients were recruited: all had evidence of multiple cutaneous and/or subcutaneous metastases. Imiquimod was applied to the metastases on a daily basis for 4 weeks, before the introduction of intralesional IL-2. This was injected up to three times a week, into selected lesions, with 0.1 mL injected per lesion at a concentration of 3.6 MIU mL(-1), a total of 1 mL being given at each session. The treated lesions were assessed individually at intervals of 3 months. Thirteen patients were treated, with 10 being eligible for assessment. In total, 182 lesions were treated: 137 purely cutaneous lesions and 41 subcutaneous lesions. Overall, a clinical response was seen in 92 lesions (50.5%) with 74 (40.7%) of these being a complete response (CR) with 91% of the CRs being in the cutaneous lesions. New lesions did appear during the treatment course; however, patients with cutaneous disease experienced a marked slowing of the appearance of new cutaneous lesions. No cutaneous lesions that responded reappeared on cessation of treatment. The combination of imiquimod and IL-2 is effective in controlling this mixed cutaneous and subcutaneous disease, and is well tolerated. Imiquimod alone is often enough to elicit a response in purely cutaneous lesions. The addition of intralesional IL-2 increases the response rates in subcutaneous lesions, and in otherwise refractory cutaneous lesions.

The Role of Nephron Sparing Surgery for Metastatic (pM1) Renal Cell Carcinoma

Studies have demonstrated increased time to progression when cytoreductive nephrectomy is performed for metastatic renal cell carcinoma. We evaluated the role of nephron sparing surgery in these patients. We selected all patients with pM1 renal cell carcinoma treated with nephron sparing surgery or radical nephrectomy, and all patients with pM0 renal cell carcinoma undergoing nephron sparing surgery for solitary kidney from 1970 to 2002 from the Mayo Clinic Nephrectomy Registry. We identified 16 patients who underwent nephron sparing surgery for pM1 renal cell carcinoma. Solitary kidney was present in 12, 3 had bilateral synchronous disease and 1 had elective nephron sparing surgery. Cancer specific survival rates at 1, 3 and 5 years were 81%, 49% and 49%, respectively. We identified 404 patients who underwent radical nephrectomy for pM1 renal cell carcinoma. Cancer specific survival rates at 1, 3 and 5 years were 51%, 21% and 13%, respectively. The pM1 nephron sparing surgery for solitary kidney cases were more likely to have early (33% vs 10%, p = 0.009) or late (50% vs 19%, p = 0.018) complications compared with pM1 radical nephrectomy cases. There were no significant differences in early (p = 0.475) or late (p = 0.350) complications between pM1 nephron sparing surgery cases and 139 pM0 nephron sparing surgery cases. Cancer specific survival rates in pM1 nephron sparing surgery cases were comparable to pM1 radical nephrectomy cases. Although there were differences in early and late complications between the pM1 nephron sparing surgery and pM1 radical nephrectomy groups, there were no differences when compared with imperative pM0 nephron sparing surgery cases. This study demonstrates that nephron sparing surgery can achieve adequate cytoreductive therapy while preserving renal function, with postoperative complication rates similar to those of pM0 nephron sparing surgery cases.

The MRC Randomised-controlled Trial of Interferon-α, Interleukin-2 and 5-Fluorouracil vs Interferon-α Alone in Patients with Advanced Renal cell Carcinoma (RE04): Rationale and Progress

Renal cell carcinoma is diagnosed in over 6000 people, and accounts for about 2% of malignant disease annually in the UK [1]. For early stage disease, surgery with curative intent is the treatment of choice, although 30–40% of patients diagnosed with disease, apparently limited to the kidney, subsequently will relapse systemically [2]. A further 25% of patients have metastatic disease at presentation. The prognosis for metastatic renal-cell carcinoma is poor, with a median survival of 6–8 months [3]. Response rates to combination chemotherapy [4] and hormonal agents [5] have been around 5–10%. It has been known for some time that the immune system has a role in the natural history of renal-cell carcinoma. For example, spontaneous regressions are rare but well documented [6,7]. Immunotherapy with interferon (IFN) in metastatic disease produces response rates of around 10–20%, with median response durations of 3–16 months [8]. Retrospective data have also shown that patients with a performance status of 0 or 1 are more likely to gain benefit (i.e. increase in median survival) from immunotherapy than are patients with a performance status of 2 or worse [9]. Randomisedcontrolled trials have shown a survival advantage to IFN compared with non-immunotherapy. A randomised trial comparing IFN-a plus vinblastine (VLB) with VLB alone reported a median survival of about 16 months for the 79 patients receiving IFN plus VLB, and 9 months for the 81 patients treated with VLB (PZ 0.0049). Overall response rates were 16.5% for patients treated with IFN plus VLB and 2.5% for patients treated with VLB alone (PZ 0.0025) [10]. The

USING TUMOR MARKERS TO PREDICT THE SURVIVAL OF PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

Approximately 30% of renal cell carcinomas (RCCs) present as metastatic disease. Molecular markers have the potential to characterize accurately the biological behavior of tumors and they may be useful for determining prognosis. A custom tissue array was constructed using clear cell RCC from 150 patients with metastatic RCC who underwent nephrectomy prior to immunotherapy. The tissue array was stained for 8 molecular markers, namely Ki67, p53, gelsolin, carbonic anhydrase (CA)9, CA12, PTEN (phosphatase and tensin homologue deleted on chromosome 10), epithelial cell adhesion molecule and vimentin. Marker status and established clinical predictors of prognosis were considered when developing a prognostic model for disease specific survival. On univariate Cox regression analysis certain markers were statistically significant predictors of survival, namely CA9 (p <0.00001), p53 (p = 0.0072), gelsolin (p = 0.030), Ki67 (p = 0.036) and CA12 (p = 0.043). On multivariate Cox regression analysis that included all markers and clinical variables CA9 (p = 0.00002), PTEN (p <0.0001), vimentin (p = 0.0032), p53 (p = 0.028), T category (p = 0.0025) and performance status (p = 0.0013) were significant independent predictors of disease specific survival and they were used to construct a combined molecular and clinical prognostic model. The bias corrected concordance index (C-index) of this combined prognostic model was C = 0.68, which was significantly higher (p = 0.0033) than that of a multivariate clinical predictor model (C = 0.62) based on the UCLA Integrated Staging System (T category, histological grade and performance status). In patients with clear cell RCC a prognostic model for survival that includes molecular and clinical predictors is significantly more accurate than a standard clinical model using the combination of stage, histological grade and performance status.

Long-term survival in patients with metastatic renal cell carcinoma treated with continuous intravenous infusion of recombinant interleukin-2: the experience of a single institution.

Metastatic renal cell carcinoma is one of the few tumors for which a clear benefit of immunotherapy has been demonstrated. The aim of this study was to evaluate the long-term survival of patients with metastatic renal cell carcinoma, along with response rate and other prognostic and predictive factors. Between July 1989 and May 1995, 56 patients with metastatic renal cell carcinoma were treated in a single institution with high-dose recombinant interleukin-2 in continuous infusion. Survival was measured by the Kaplan and Meier method. Prognostic factors were assessed by univariate and multivariate analyses of survival (Cox proportional hazard ratio model). Of 56 patients, 15 had objective responses (26.8%), 16 stable disease (28.6%), 18 disease progressions (32.1%), and 7 (12.5%) were not valuable for response. Median overall survival was 20 months, and probability of 2- and 5-year survival was 41% and 21%, respectively. At multivariate analysis, the increased risk of death for: performance status > or = 2 vs 0 (HR = 6.20), stable disease (HR = 1.87), disease progression (HR = 10.61) vs partial or complete remission, and for hypotension and oliguria toxicity, G3 + G4 vs G1 + G2 (HR = 2.19). Our study confirms the activity of IL-2 based immunotherapy in renal cell carcinoma. Moreover, ECOG performance status, clinical response, hypotension and oliguria toxicity resulted as independent survival prognostic factors.

How long can we give interleukin-2? Clinical and immunological evaluation of AML patients after 10 or more years of IL2 administration.

We have treated 20 patients, affected by acute myelogenous leukemia in advanced phase of the disease, with intravenous high-dose recombinant interleukin-2 (IL2) as induction treatment, achieving a complete remission (CR) in 11/20 of patients (55%). All CR patients were planned to receive a maintenance program with lower subcutaneous doses of IL2 until relapse. Currently, 5/11 patients are alive in continuous complete remission with a minimum follow-up of 9 years from IL2 induction. In the aim to investigate the treatment's side-effects during or after prolonged IL2 therapy, we decided to submit these patients to a clinical and immunological evaluation. Four patients have been evaluated as one, who independently stopped IL2 after 6 years, refused the check-up. No organ-specific treatment sequelae that may decrease the quality of life or may be life-threatening were found, concerning renal, liver and cardiovascular function. Endocrine abnormalities were detected in three patients, the most serious being a severe hypothyroidism, which prompted cessation of IL2 maintenance after 6 years and required thyroid supplementation treatment. Immunological studies were carried out prior to the last IL2 cycle and showed high levels of CD3-positive T cells expressing the IL2 receptor alpha chain (CD25), both in the peripheral blood and in the bone marrow. Our study shows that low-dose IL2 can be given for a prolonged period of time without serious organ-specific late sequelae and with a good quality of life.

Characterization of novel natural killer (NK)–cell and γδ T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus

AbstractStudies on nasal T/natural killer (NK)–cell lymphoma have been hampered by its tendency to cause necrosis. Thus, the establishment of cell lines of this neoplasm would seem to be valuable. This study attempted to establish cell lines from primary lesions of this tumor, and successfully obtained 2 novel Epstein-Barr virus (EBV)–positive cell lines, SNK-6 and SNT-8, by means of high-dose recombinant interleukin 2. Flow cytometry showed that SNK-6 had an NK-cell phenotype, CD3−CD4−CD8−CD19−CD56+T-cell receptor (TCR) α/β− TCR γ/δ−, whereas SNT-8 was CD3+CD4−CD8−CD19−CD56+TCR α/β− TCR γ/δ+. These were consistent with immunophenotypes of their original tumors, and the cell lines had monoclonal EBV clones identical to ones in their original tumors. Thus, the cell lines developed from cells forming the primary lesions. Genotypic analysis showed that SNK-6 had unrearranged TCR and immunoglobulin heavy-chain genes, supporting the conclusion that SNK-6 was of NK-cell lineage. On the other hand, SNT-8 had rearranged TCR β-, γ-, and δ-chain genes, and together with its phenotype, SNT-8 proved to be a γδ T-cell line. This is the first report of the establishment of cell lines from primary lesions of nasal T/NK cell lymphomas, and the results demonstrated that there are at least 2 lineages, NK- and γδ T-cell, in this neoplasm. Moreover, it has been suggested that nasal T/NK cell lymphomas of these lineages may belong to the same clinicopathologic entity because both types of cases shared common clinical and histopathologic features.

Recurrent metastatic renal cell carcinoma presenting as a bleeding gastric ulcer after a complete response to high-dose interleukin-2 treatment

Immunotherapy with high-dose recombinant interleukin-2 is an effective therapy for selected patients with metastatic renal cell carcinoma (RCC). Objective responses (complete or partial) are observed in about 15% of treated patients. The overall and disease-free survival of patients with a complete response are significantly prolonged. Although RCC is known to spread hematogenously, isolated RCC metastasis to the stomach is a rare event. Recurrent RCC, after a complete response to interleukin-2, presenting clinically as an isolated gastric metastasis, has not been reported to date. In this report, we describe the clinical course of a patient with metastatic RCC who had a complete response to high-dose interleukin-2 and was disease free for 4 years before presenting with massive upper gastrointestinal hemorrhage due to an isolated gastric metastasis. The patient remained disease free for 3 years after resection of the metastasis. Metastatic RCC to the stomach, although rare, should be suspected in any patient with a history of RCC who presents with gastrointestinal symptoms. In the absence of diffuse disease, aggressive therapy, including surgical resection, is appropriate for isolated gastric metastasis, because prolonged survival is possible.