Pieter H.M. De Mulder

Vaccination of patients with metastatic renal cell carcinoma with autologous dendritic cells pulsed with autologous tumor antigens in combination with interleukin-2: a phase 1 study.

Dendritic cells (DC) have been recognized as the most potent antigen presenting cells (APC) of the immune system. We performed a phase 1 study in twelve patients with metastatic renal cell carcinoma (RCC) using autologous immature DC loaded with autologous tumorlysate (TuLy) as a vaccine based on our earlier in vitro observations that such DC can activate tumor-specific cytotoxic T-lymphocytes. The treatment was combined with low-dose interleukin (IL)-2, as this has shown benefit in DC-based therapies. Patients received three intradermal vaccinations at two weekly intervals, and, after each vaccination, IL-2 was administered for 5 consecutive days. In six patients, keyhole-limpet hemocyanin (KLH) was added to the DC culture for immunologic monitoring purposes. In general, DC phenotype was CD14low, CD86high, CD40high, CD80low, and CD83low. We noticed that the number of CD14+ cultured DC increased during treatment. Nevertheless, ovalbumin uptake remained high, underlining that these cells were still functional immature DC. The vaccine was able to elicit cellular anti-KLH responses, emphasizing the ability of the injected DC to mount an immunologic response. However, proliferative responses against TuLy were not detected, and humoral responses against TuLy or KLH were absent. Objective clinical responses were not observed, but extended stable disease was noted. The absence of cellular, humoral, or clinical antitumor responses suggests that the vaccination strategy with immature DC has little benefit for patients with advanced RCC. Nevertheless, this study shows the feasibility of a completely autologous DC and tissue culture methodology for the generation of TuLy pulsed DC.

Management of intermediate-prognosis germ-cell cancer: results of a phase I/II study of Taxol-BEP.

The standard chemotherapy regimen in metastatic germ‐cell cancer is bleomycin, etoposide and cisplatin (BEP). Chemotherapy studies testing cisplatin dosage and the substitution of ifosfamide for bleomycin have not shown this to be superior to BEP. Paclitaxel (Taxol) has demonstrated promising activity as a second‐line treatment in patients with relapsing or cisplatin‐refractory germ‐cell cancer. Hence, the potential of incorporating paclitaxel in first‐line chemotherapy should be investigated. We assessed the feasibility of the addition of paclitaxel to BEP (T‐BEP) in a phase I/II study in patients with intermediate‐ or poor‐prognosis germ‐cell cancer or with carcinoma of unknown primary (CUP). Paclitaxel was investigated at dose levels of 75, 125, 175 and 200 mg/m2 given as a 3 hr infusion on day 1, before the start of BEP. BEP comprised etoposide at a dose of either 120 mg/m2 on days 1, 3 and 5 or 100 mg/m2 on days 1–5. To deliver the highest possible dose of paclitaxel into BEP, all patients received filgrastim (G‐CSF). Thirty patients were entered, 14 of whom had intermediate‐ (n =7) or poor‐ (n = 7) prognosis germ‐cell cancer. Paclitaxel up to 200 mg/m2 and BEP at 360 mg/m2 was well tolerated. There was minimal neurosensory and no neuromotor toxicity with the use of 4 T‐BEP cycles. More pronounced myelotoxicity and diarrhea at the higher dose level of etoposide resulted in a recommended dose level for multicenter phase II/III testing of paclitaxel 175 mg/m2 and BEP 500 mg/m2. Of the 13 evaluable patients with intermediate‐ or poor‐prognosis germ‐cell cancer, all achieved complete response. With a median follow‐up of 18 months, none of these patients has relapsed. We conclude that T‐BEP is a well‐tolerated induction regimen that should be further tested for its therapeutic potential. A randomized phase II/III study of T‐BEP vs. BEP has been started as an EORTC trial in patients with intermediate‐prognosis disease. Int. J. Cancer 83:831–833, 1999.

Intratumoral Recombinant Human Interleukin-12 Administration in Head and Neck Squamous Cell Carcinoma Patients Modifies Locoregional Lymph Node Architecture and Induces Natural Killer Cell Infiltration in the Primary Tumor

The objective of this study was to evaluate the histologic and immunohistopathologic effects of intratumorally given recombinant human interleukin-12 on the immune cells in the primary tumors and regional lymph nodes. Ten previously untreated patients with head and neck squamous cell carcinoma (HNSCC) were injected in the primary tumor twice to thrice, once weekly, at two dose levels of 100 or 300 ng/kg, before surgery. These patients were compared with 20 non-IL-12-treated control HNSCC patients. In the primary tumor, the number of CD56+ natural killer (NK) cells was increased in IL-12-treated patients compared with control patients. In some IL-12-treated patients, an impressive peritumoral invasion of CD20+ B cells was noticed. No differences were seen in the CD8+ or CD4+ T lymphocytes. Interestingly, major differences were apparent in the architecture of the enlarged lymph nodes of IL-12-treated patients; in particular, the distribution of B cells differed and fewer primary and secondary follicles with smaller germinal centers were observed. In addition, a decrease of dendritic cell lysosyme-associated membrane glycoprotein–positive cells in the paracortex was noted, resulting in a reduction of paracortical hyperplasia. In the lymph nodes, especially the CD56+ NK cells but also the CD8+ and CD4+ T lymphocytes, produced a high amount of IFN-γ. Patients, irrespectively of IL-12 treatment, with a high number of CD56+ cells in the primary tumor had a better overall survival than those with a low number. In conclusion, after i.t. IL-12 treatment in HNSCC patients, the largest effect was seen on the NK cells, with a higher number in the primary tumor and a high IFN-γ mRNA expression in the lymph nodes. Significant effects were noted on B cells, with altered lymph node architecture in every IL-12-treated patient and excessive peritumoral infiltration in some patients.

Intratumoral administration of recombinant human interleukin 12 in head and neck squamous cell carcinoma patients elicits a T-helper 1 profile in the locoregional lymph nodes.

The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary tumor, and peripheral blood. Ten previously untreated patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients. Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-γ and IL-10 increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections. A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed, due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells in the lymph nodes showed a 128-fold (mean) increase of IFN-γ mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.

Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation.

The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin‐12 (rhIL‐12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL‐12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B‐blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL‐12 treated patients, increased IFN‐γ mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL‐12 treatment. In conclusion, these data indicate that after i.t. IL‐12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients.

A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis.

CD8+ T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.

Phase I/II study of recombinant interferon α and γ in advanced progressive renal-cell carcinoma

SummaryIn vitro studies have documented the synergistic antiviral and antiproliferative activity of recombinant interferon α (rIFNα) and rIFNγ. Furthermore, rIFNγ is a strong immunomodulator with optimal effects at a relative low dose (0.1 mg/m2). On the basis of these observations, we began a phase I/II study with the combination of rIFNγ at 100 µg/m2 (2 × 106 IU/m2) and rIFNα2c 6 µg/m2 (2 × 106 IU/m2), injected twice a week subcutaneously. In cases of stable or progressive disease we increased the dose of rIFNα2c every 2 weeks by 6 µg/m2 until the maximum tolerated dose was reached. A total of 32 patients with proven progressive renal-cell carcinoma were included. Of the 31 eligible patients, 21 were male and 10 female, their average age was 57.2 years (range 35–72), 28 had had nephrectomy, their median Karnofsky performance status was 90% (70%–100%), and their tumors were localized predominantly to visceral tissue. In 2, response was complete and in 6 it was partial, for a response rate of 25%. The disease had stabilized in 5 patients and progressed in 16. The median duration of partial response was 14 months (8–16 months); of 2 cases of complete response, 1 persists (23+ months), and the other suffered a relapse after 22 months. The median time to response was 24 weeks (18–24 weeks). The maximum tolerated dose of rIFNα was 30 µg/m2 (range of 6–36 µg/m2). Side-effects included those known to be associated with interferon treatment. One patient developed septicemia during a period with grade 4 leukopenia. Our study permits no conclusion regarding the additional value of rIFNγ.

Targeted Approaches for Treating Advanced Clear Cell Renal Carcinoma

The mainstay of any curative treatment in renal cell carcinoma (RCC) is surgery. In the case of metastatic disease at presentation, a radical nephrectomy is recommended to good performance status patients prior to the start of cytokine treatment. Interferon (IFN)-a offers in a small but significant percentage of patients advantage in overall survival. Interleukin (IL)-2-based therapy gives similar survival rates. To date, hormonal therapy and chemotherapy do not have a proven impact on survival. Recent insights demonstrate that the majority of clear cell RCC harbor abnormalities of the von Hippel-Lindau (VHL) gene. This gene plays a key role in the stimulation of angiogenesis by vascular endothelial growth factor (VEGF) in this highly vascularized tumor. This opens interesting new treatment strategies including blockade of VEGF with the monoclonal antibody bevacizumab (Avastin?) and inhibition of VEGF receptor tyrosine kinases with small oral molecules such as sunitinib (SU11248, Sutent?) or PTK787. Likewise, inhibition of the Raf kinase pathway with oral sorafenib (Bay 43-9006, Nexavar?) or inhibition of the mTOR pathway with intravenous CCI-779 are under investigation. Preliminary clinical results with all these compounds are promising, and the results of ongoing first-line phase III studies will become available in the next years.

Prevention of Acute Cisplatin-Induced Nausea and Vomiting

All cytotoxic drugs can induce nausea and vomiting depending on the type of agent, dose, route and schedule of administration [1]. This variability underlines the differences in mechanisms responsible for the difference in onset and duration of this side-effect. Cisplatin is one of the most widely used cytotoxic agents and has significantly contributed to the curative potential of chemotherapy. However, the spectrum of side-effects is substantial and its capacity to induce nausea and vomiting is unprecedented. High doses of cisplatin produce acute nausea and vomiting in almost all patients unless antiemetic drugs are used. Therefore much effort has been made to control this distressing side-effect and the present status will be updated in this chapter.