Immunotherapy options in metastatic renal cell cancer: where we are ...

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Immunotherapy options in metastatic renal cell cancer: where we are and where we are going Rodolfo Passalacqua†, Sebastiano Buti, Gianluca Tomasello, Raffaella Longarini, Matteo Brighenti and Matteo Dalla Chiesa

Allogenic transplantation

The treatment of renal cell carcinoma is rapidly changing as a result of recent evidence concerning the efficacy of biological drugs, antiangiogenetic agents and signaltransduction inhibitors. This paper will provide a critical overview of the use of immunotherapy in renal cell carcinoma and review the available data concerning the efficacy of interferons, interleukin-2 and other forms of immunological treatment, particularly allogenic transplantation and vaccines. Moreover, it will focus on the new mechanisms of regulation of the immune system with a better understanding of the interaction between host and tumor, the role of T regulatory cells, heat-shock proteins and vaccines. The mechanism of action and the results obtained in renal cell carcinoma using the new molecular targeted drugs will be examined, along with the possibility of using immunotherapy combined with the new biological agents. Future research will not only need to make every effort to optimize the use of the new molecules and to define their efficacy precisely, but also to consider how to integrate these drugs with the traditional immunotherapy.

Chemoimmunotherapy

Expert Rev. Anticancer Ther. 6(10), 1459–1472 (2006)

New biological therapies

Renal cell carcinoma (RCC) is the tenth most common cancer affecting adults and accounts for approximately 3% of all tumors. It affects more than 32,000 people in the USA every year and approximately 12,000 die due to RCC metastases. Approximately a third of the cases present distant metastases at the time of diagnosis and approximately half of the remaining 60–70% can be expected to develop metastases [1]. The incidence of renal carcinoma increased continuously from 1973 to 1998, and the fact that this was true of both localized and locally advanced or metastatic tumors indicates that the increase was not just a reflection of the identification of more new cases due to improved diagnostic systems [2]. In 1997, international consensus led to the Heidelberg classification of renal cell tumors [3], which identified conventional clear cell, papillary, chromophobe, collecting duct and unclassified RCC as distinct malignant histological subtypes, and oncocytoma and metanephric adenoma as benign tumors. The

CONTENTS Theoretical basis of immunotherapy in renal cell carcinoma Interleukin-2 Interferons Combination immunotherapy Tumor vaccines

Therapies targeting VEGF, PDGF, TGF-α and/or mTOR pathways Expert commentary & five-year view Key issues References Affiliations †

Author for correspondence Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected] KEYWORDS: allogenic transplantation, biological therapy, chemoimmunotherapy, immunotherapy, interferon, interleukin-2, renal cell cancer, tumor vaccine

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10.1586/14737140.6.10.1459

three most common histological subtypes are conventional clear cell RCC (incidence: 65–80%), papillary RCC (7–14%) and chromophobe RCC (6–11%). The collective data from different studies indicate that disease progression and survival are significantly influenced by tumor histology. When controlled for stage and size, chromophobe RCC has a significantly better nonprogression rate; papillary and conventional clear cell RCC have similar clinical outcomes [4,5]. In the case of chromophobe RCC, the median time from nephrectomy to metastasis (32.4 months) and from metastasis to death (33.2 months) is twice as long as in the case of the other subtypes, thus supporting the idea that it has more indolent metastatic potential, whereas the clinical behavior of papillary and conventional clear cell RCC is similar. We have learned recently that the differences in histological pattern, clinical course, therapeutic response and evolution are caused

© 2006 Future Drugs Ltd

ISSN 1473-7140

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Passalacqua, Buti, Tomasello, Longarini, Brighenti & Dalla Chiesa

by different gene alterations. RCC may be inherited or sporadic and four kidney cancer genes have been identified as being associated with four main types of inherited RCC: von Hippel Lindau (VHL) is associated with clear cell RCC; c-Met proto-oncogene with papillary type 1; Birt-Hogg Dubé (BHD) with chromophobe carcinoma; and fumarate hydratase (FH) with papillary type 2 (leiomiomatosis RCC). Various studies are currently underway to develop disease-specific molecular therapies based on an understanding of the RCC gene pathways [6]. The considerable variability in the clinical behavior of RCC reflects its biological complexity. Tumor stage, histological grade and Eastern Cooperative Oncology Group (ECOG) performance status (PS) are the most widely used predictors of survival for patients with newly diagnosed RCC [7], but various predictive models have been constructed with the aim of discriminating different prognostic groups of patients with metastatic (m)RCC. One of these was developed by the Memorial SloanKettering Cancer Center (MSKCC, NY, USA) on the basis of a study of 670 clinical trial patients treated between 1975 and 1996 [8], in which uni- and multivariate analyses identified five prognostic variables as risk factors for short survival: a Karnofsky PS of less than 80%; lactate dehydrogenase (LDH) over 1.5-times the upper normal limit; serum hemoglobin levels below the lower normal limit; corrected serum calcium over 10 mg/dl; and the absence of previous nephrectomy. For patients with a new diagnosis of RCC, nephrectomy (if feasible) should be considered the first-choice treatment not only in early, operable disease (T1–T3), but also in the case of distant metastases that cannot be surgically removed. Two recent randomized studies have independently demonstrated that the overall survival (OS) of patients with metastatic disease is better if they undergo nephrectomy before receiving systemic therapy. However, most of the benefits were seen in patients with PS 0 and with clear cell histology. Extrapolation of these results to less fit patients or nonclear cell histology should be viewed cautiously [9,10]. In the case of patients undergoing radical surgery in whom there are no signs of distant metastases, there is currently no evidence that any benefit can be obtained from immunotherapy or any other kind of adjuvant systemic treatment. The various trials of interferon (IFN), vaccines or other therapies have not yet demonstrated any advantage in terms of OS. Unlike many other types of cancer, mRCC is highly refractory to conventional chemotherapy. In Phase II trials, no single cytotoxic agent has led to a response rate (RR) of more than 10% and an overall response rate (ORR) of just 6% was found in a review of 4093 patients enrolled in 83 chemotherapy trials between 1983 and 1993 [11]. It has been reported more recently that two therapies (thalidomide and gemcitabine plus 5-fluorouracil [5-FU]) have led to low RRs in patients with cytokineresistant RCC, but there is still no consensus concerning the effectiveness of these treatments in terms of clinical responses and there are no randomized trials showing a favorable effect on survival [12,13].

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However, patients with metastases now have new and different treatment options. In the first place, the new biological therapies with molecular or antiangiogenetic targets have proved to be highly effective in controlling the disease in as many as 40–80% of patients, considering both objective responses and stable disease (SD). However, traditional immunotherapy is also still widely used and maintains its major role. The aim of this paper is to provide a critical overview of the current use of immunotherapy in RCC and review the available data concerning the efficacy of IFN, interleukin (IL)-2 and other forms of immunological treatment, such as allogenic transplantation or vaccines. This will be followed by an assessment of its future possibilities in a context of rapidly changing biological knowledge in which molecularly targeted drugs are assuming an increasingly prevalent role. Researchers will need to make every effort in the future to exploit the potential of these new therapies to the full and integrate them with existing immunological therapies with the final objective of eradicating metastatic disease. Theoretical basis of immunotherapy in renal cell carcinoma

The history of the immunotherapy of renal cancer dates back to 1914, when WB Coley reported mixed results from his treatment of renal tumors with a prolonged course of a mixture of bacterial toxins [14] and other investigators subsequently reported various attempts to induce an immune response involving the application of crude tumor preparations (bacillus Calmette–Guerin or Corynebacterium parvum). However, such nonspecific immunotherapy has rarely proved to be effective in RCC and efforts were subsequently directed toward activating a patient’s immune system using biological response modifiers or the adoptive transfer of activated immune cells [15]. Two clinically identifiable events in mRCC can be considered as proving the principle of the role of the immune system: the first is the spontaneous regression of metastases and the second the positive impact of nephrectomy. One randomized trial of IFN-γ-1b in mRCC found a singularly high 6% RR in the placebo control population (similar to that in the IFN-α arm), with remission durations of 2–13 months [16]; this clearly indicates that regressions are possible and can be interpreted as a placebo effect or spontaneous regressions. The results of this study, together with results coming from other series, suggest that spontaneous regressions are not frequent (1–6%) and that the vast majority of these cases will soon relapse with progressive metastatic disease requiring other therapies [18]. Nevertheless, spontaneous regressions of metastatic disease occur more frequently in renal cancer than in any other solid tumor. The role of nephrectomy in mRCC has been clarified recently by the similarly designed SouthWest ONcology Group (SWOG) 8949 and European Organisation for Research and Treatment of Cancer (EORTC) 30947 studies, both of which randomized mRCC patients with the primary in situ to undergo nephrectomy or not before treatment with

Expert Rev. Anticancer Ther. 6(10), (2006)

Immunotherapy in metastatic renal cell cancer

IFN-α. Only 6% of the patients in both arms experienced complete or partial remissions, but OS was significantly longer in the patients undergoing nephrectomy. The difference in median survival was 4.8 months, with a lower risk of death in the first year in the nephrectomy arm (OR: 0.53; 95% confidence interval [CI]: 0.33–0.83; p = 0.006) [9,10]. On the basis of these results, it is appropriate to recommend nephrectomy as an option, if it is possible, before starting systemic immunotherapy in patients with mRCC, mainly in patients with a good PS. The mechanism by which nephrectomy prolongs survival is currently unknown but, given the absence of an increased remission rate in the trials’ IFN-α arms, seems to be unrelated to IFN-α enhancement. Presumably, the bulk of the primary tumor is either immunosuppressive or acts as an immunological sink suppressing cell-mediated immunity until it is removed. Moreover, studies have shown changes in cellmediated immunity after nephrectomy, with lower antitumor responses when the primary is in situ [19]. Interleukin-2

It has been known since the 1970s that mononuclear cells (lymphocytes or monocytes) can secrete a class of nonantibody proteins called cytokines, a new class of hormones with many target cells within and outside the immune system. The cytokines produced by lymphocytes are referred to as lymphokines and are involved in immune function and the regulation of the immune response. They were initially named on the basis of acronyms of their functional properties in vitro (e.g., lymphocyte-activating factor and T-cell growth factor) but, since the introduction of molecular biology techniques to clone their genes, a new nomenclature referring to cytokines as ILs (meaning between leukocytes) has been adopted [20]. IL-2 is a 15 kD protein produced by activated T lymphocytes that was originally discovered as a growth factor for T cells. It acts by binding to a specific receptor on target cells that consists of three (α, β and γ) chains: the β- and γ-chains are essential for signaling, whereas the α chain increases the affinity of the complex. IL-2 induces the proliferation of activated T cells, stimulates the cytotoxicity of natural killer (NK) cells and acts as a cofactor in T cells activating macrophages and B cells. IL-2 has been studied in mRCC since 1984, achieving RRs that range from 15 to 35% [21,22].The fact that nearly half of the responding patients experience a durable complete response (CR) led to its approval for use in mRCC by the US FDA. The long-term disease-free survival data have been confirmed by a follow-up extending to 17 years [23,24]. The early trials escalated the administration of IL-2 to its maximum tolerated dose, which was identified as being an intravenous bolus of 600,000–720,000 IU/kg every 8 h. Highdose IL-2 schedule was considered as more than 65 million units (MU)/m2/day, but was characterized by severe toxicities (such as vascular leak syndrome with fluid retention and pulmonary edema, hypotension and oliguria), a treatmentrelated mortality rate of 2–4% [25], and required treatment in


an intensive-care unit. Unfortunately, no published studies have directly compared high-dose IL-2 with a placebo or non-IL-2 control arm, thus its precise role and impact on overall or progression-free survival (PFS) cannot be defined. A number of different schedules of modified regimens have been developed in order to reduce the toxicity of high-dose IL-2, but no randomized studies of single-agent modified IL-2 regimens versus placebo or a non-IL-2 control arm have been published. However, at the 2005 ASCO annual meeting, the French group presented the results of a four-arm trial (Percy 4) in which 465 mRCC patients with an intermediate risk, a PS of 0–1, more than one metastatic site, no brain metastasis and no major organ failure were randomized to receive medroxyprogesterone acetate (MPA), IFN-α, intermediate-dose IL-2 (18 MU/m2/day subcutaneously) or IFN-α plus IL-2 [27], and found no survival benefit in any of the arms after a median follow-up of 29 months. The RR was higher in the combination arm, but this difference did not lead to a demonstrable benefit for the patients. In our opinion, it is not very clear why the investigators offered MPA as a treatment option for intermediate-risk patients or why they selected this risk group and not a good-risk group of patients for such a trial. The dose-related effects of IL-2 have been investigated directly by two randomized trials. The first compared high-dose IL-2 with a one-tenth dose of IL-2 administered by means of an intravenous bolus or subcutaneously [28]. The RR was significantly better in the high-dose arm (21 vs 13%), as was CR duration, but there was no difference in OS, nor between the intravenous or subcutaneous administration of low-dose IL-2. The toxicity profile of the low-dose IL-2 regimen was clearly better, particularly hypotension (4 vs 43%), although no toxicityrelated death occurred in the high-dose arm. The second trial was conducted by the Cytokine Working Group and the patients were randomized to receive either IL-2 plus IFN or high-dose IL-2 intavenously; the RR was better in the high-dose IL-2 arm, but the difference in median survival was not statistically significant (see next chapter for more details) [29]. In order to reduce the toxicity of high-dose IL-2, alternative regimens have been proposed, including prolonged infusions, lower subcutaneous doses and even chronically administered very low subcutaneous doses. The rationale behind the use of very low doses came from the observation that NK and other subpopulations of T lymphocytes can be effectively stimulated with 0.5–1 MU/m2/day. For example, a Phase II trial of chronic maintenance therapy with a low-dose regimen of IL-2 (1 MU/m2 twice a day) plus IFN-α (3 MU/m2 twice a week) persistently stimulated the immune system and led to a RR that was similar to those obtained with regimens based on higher doses and without any relevant toxic effects [30]. A randomized, multicenter trial by the same group tested the hypothesis that maintenance treatment with this regimen prolonged the time from the first progression to death and OS in 183 immunotherapy-naive mRCC patients. A preliminary analysis showed that a chronic maintenance biotherapy after disease progression is feasible; the RR was 14.6%, but there were no between-group differences

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with regards to the main study end points. Toxicity was mild and no toxicity attributable to the chronic administration of IL-2 was observed [31]. Interferons

The IFNs were first described in 1957 as a family of proteins produced by cells after virus exposure that interfere with viral replication [32] and include IFN-α derived from leukocytes, IFNβ derived from fibroblasts and IFN-γ derived from lymphocytes. The exact mechanism of the antitumoral action of the IFNs is unclear, but they directly inhibit tumor cell proliferation, enhance the lytic activity of NK cells, increase the tumor cell expression of human leukocyte antibody (HLA) class I, and has antiangiogenic effects [33]. Early trials of several different subtypes of recombinant IFN did not suggest any differences in their efficacy, but more recent trials of IFN-β and -γ have indicated that they are both less active than IFN-α [34]. Moreover, a randomized comparison of IFN-γ and placebo showed no differences in RRs (4 vs 6%) or median survival (12 vs 15 months) [35]. IFN-α is the most used form of IFN. Although the best schedule and dose have not yet been established in randomized trials, in most Phase II studies it has been used subcutaneously three times a week at doses of 3–10 MU. As a single agent, IFN-α leads to RRs of between 8 and 26%, but in more recent randomized trials, the RR ranged from 3 to 6%. Median response duration is 5–10 months and median OS approximately 6–13 months, although some rare long-lasting complete remissions have been described. Responses are most frequently seen in lung and, to a lesser extent, lymph nodes. The toxic effects of IFN-α include flu-like symptoms, such as fever, chills, myalgias and fatigue, as well as weight loss, altered taste, depression, anemia, leucopoenia and high liver function test results. The majority of the adverse effects (especially the flu-like symptoms) tend to diminish with time during long-term therapy, but fatigue is worse in the longer-term duration treatment. Only a limited number of randomized studies of IFN-α have been published. One comparing it with MPA revealed a significant improvement in the IFN-α-treated group in terms of median (8.5 vs 6 months) and 1-year survival (43 vs 31%) [36]. Another Phase III trial compared IFN-α plus vinblastine (VBL) with VBL alone and reported median survival rates of 67.6 and 37.8 weeks, respectively (p = 0.0049). Overall responses were observed in 16.5% of the patients treated with the combination and 2.5% of those treated with VBL alone (p = 0.0025) [37]. A recently published Cochrane review of all randomized trials of immunotherapy in mRCC indicated that IFN-α is superior to controls (OR for death at 1 year: 0.56; 95% CI: 0.40–0.77). The pooled overall hazard ratio for death was 0.74 (95% CI: 0.63–0.88) and the weighted average median improvement in survival was 3.8 months. The optimal dose and duration and patient selection for IFN-α treatment remains to be elucidated [38].

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Combination immunotherapy

The combination of IL-2 and IFN-α is particularly interesting for various reasons. Each individual agent is active in RCC and IFN-α upregulates MHC expression, thus leading to synergistic immunological and antitumor effects with IL-2-stimulated T lymphocytes and NK cells. The first nonrandomized studies suggested that the RR among patients with mRCC could be increased by adding IFN-α to high-dose IL-2 [39]. Most studies have used low subcutaneous doses of IL-2 [40]. CRECY was the first randomized study to investigate the role of the combination therapy by randomizing 425 patients to receive high-dose IL-2 in a continuous intravenous infusion, IFN-α subcutaneously three times a week or both agents simultaneously [41]. The combination therapy led to a significantly higher RR (18.6%) than either IL-2 (6.5%) or INF-α alone (7.5%), but without any significant advantage in terms of survival. Further, small-scale randomized studies did not have sufficient statistical power to demonstrate an advantage for combined immunotherapy over IL-2 alone [42,43]. A recently published, randomized Phase III study compared high-dose IL-2 (600,000 U/kg/dose intravenously every 8 h on days 1–5 and 15–19 [maximum 28 doses]) and low-dose IL-2 (5 MIU/m2 subcutaneously every 8 h for three doses on day 1 and then daily 5 days/wk for 4 weeks) in combination with INF-α (5 MIU/m2 subcutaneously three times per week for 4 weeks every 6 weeks) [29] in 192 patients. The RRs were 23.2% for high-dose IL-2 and 9.9% for IL-2/IFN (p = 0.018). There were no statistically significant differences between the two treatment arms in terms of progression-free or median survival. However, the results of this study serve to underline the fact that high-dose IL-2 therapy remains the reference treatment for patients with mRCC. Other studies have confronted the question of lymphokineactivated killer (LAK) cells by seeking to demonstrate that there is an advantage in adding them to standard therapy on the basis of the encouraging early results observed in murine models. However, a randomized comparison of an intravenous highdose IL-2 bolus with or without the addition of LAK cells did not reveal any statistically significant differences in RRs (21% for IL-2 vs 31% for IL-2 plus LAK cells) or survival [44]. Further studies have confirmed these results [45], thus there is no current clinical experience to support the use of LAK cells in patients affected by RCC. Tumor vaccines

One novel approach to the treatment of RCC is a therapeutic vaccine that targets T-cell responses and enhances cell-mediated immunity and tumor rejection. The development of an effective vaccination can be accomplished by fulfilling a series of variables: the identification of antigens, the vector or vehicle of administration, the use of some immune adjuvant and the ability of the vaccine to generate and maintain antigen-specific T-cell responses.



Many tumor-associated antigens have been identified and strategies for delivering such antigens to the immune system can include the use of recombinant viruses, dendritic cells (DCs), or autologous or allogenic whole cell vaccines together with costimulatory molecules able to lower the threshold for T-cell activation. Other autologous vaccines consist of purified complexes of tumor-derived heat-shock proteins (HSPs) linked to tumor antigen peptides. When these HSPs are readministered to a patient after surgery or biopsy of the tumor, the antigenic tumor peptides are expressed on the surface of antigen-presenting cells of the immune system, such as macrophages and DCs. Early randomized studies in adjuvant settings used vaccines that had been prepared using autologous cells from primary tumors that were irradiated or cryopreserved before reinjection. Two prospective controlled trials have been published; one with negative and one with positive results [46,47], but further studies are necessary before any definite conclusions can be drawn. Another approach involves the use of DCs, which initiate a primary immune response by presenting antigen in the context of costimulatory molecules [48] and can be pulsed with tumor protein, DNA, RNA [49] or lysate [50]. The basic theory being tested is that active specific immunotherapy in the form of cytolytic T lymphocytes, can be generated by using, for example, gp96, a chaperone for tightly bound immunogenic peptides. The HSP–peptide complex vaccine (HSPPC-96 Oncophage) uses purified complexes from an individual patient’s tumor, thus making each vaccine unique to each patient’s tumor. HSPs are linked to tumor-antigen peptides [51], which, when readministered to patients, can be expressed on antigenpresenting cells in order to stimulate a potent antitumoral response. Trials using the same technology are currently being carried out in patients with melanoma, or gastric or pancreatic cancer, although the ability to extract HSPs can vary substantially between tumor types owing to the different levels of protease in each tissue. Vaccines may even consist of DCs fused with tumor cells in order to present tumor antigens in a therapy-favorable context [26,52]. A Phase II study, reported at ASCO 2003, assessed the efficacy of HSPPC-96 and the activity of additional IL-2 for patients whose cancer progressed while on HSPPC-96, by administering 25 μg of HSPPC-96 intradermally at weekly intervals in weeks 1–4 and then every 2 weeks until progression. After 10 weeks, the patients were evaluated for progression and then every 8 weeks thereafter. IL-2 added to the vaccine at the time of progression in selected patients, being administered 5 days a week for 4 weeks on and 4 weeks off (11 MU). No adverse events were reported. A total of 61 patients received a minimum of one dose of HSPPC-96, two of whom achieved a partial response (PR), one a CR (with no evidence of disease after 2.5 years) and 18 experienced SD; seven out of 16 progressive patients responded to IL-2 and developed SD. Median progression-free survival was 18 weeks, whereas that in those receiving vaccine and IL-2 was 25 weeks. At 2 years after the initiation of the vaccine, 30% of patients are still alive [53]. Given these encouraging findings,


expanded Phase III clinical trials using HSPPC-96 at 80 clinical sites has completed accrual. The trial has enrolled at least 500 patients who were randomized to receive surgical removal of the primary tumour followed by out-patient treatment with HSPPC-96 or surgery only. The importance of a new class of human regulatory T cells (Treg), characterized as CD4+CD25+ T cells with suppressive function, has emerged recently. These cells typically express a novel transcription factor, Foxp3, that was demonstrated to be exclusively expressed by Tregs and not by activated T cells. Therefore, Foxp3 expression can be used as a surrogate molecular marker for quantifying regulatory T cells in peripheral blood. Recent research has shown that Treg cells act in a regulatory capacity by suppressing the activation and function of other T cells. Their physiological role is to protect the host against the development of autoimmunity by regulating immune responses against antigens expressed by normal tissues. The numbers of Treg cells were elevated in patients with metastatic melanoma and renal carcinoma and remained elevated in patients who failed treatment with high-dose IL-2. However, the Treg cells frequency returned to normal donor levels in those patients who achieved an objective clinical response to IL-2 therapy [54]. Dannull and colleagues have recently shown that the elimination of Treg population by means of the recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (also known as denileukin difitox), followed by vaccination with RNA-transfected DC, significantly improved the stimulation of tumor-specific T-cell responses in mRCC patients in comparison with vaccination alone. These findings may have implications in the design of immune-based strategies that may incorporate the Treg cell depletion strategy to achieve potent antitumor immunity with therapeutic impact [55]. Finally, other authors have proposed a generic DC vaccine strategy for mRCC patients based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). They prepared RNA from a well-characterized, highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for DC loading and used the lipofection of immature DC to achieve efficient RNA transfer [56]. However, although the preclinical and clinical results are promising and the vaccines are generally well tolerated, this strategy requires further optimization. Experimental data suggest that the efficacy of cancer vaccination could be enhanced by treatment with adjuvants or with agents that lead to the preferential depletion of CD4+CD25+ Treg cells. This strategy could potentially be applied to many immune-based approaches of active and passive immunotherapy and serve as a baseline for further clinical investigation in ultimately achieving antitumor immunity with clinical impact. Allogenic transplantation

High-dose chemotherapy followed by allogenic hematopoietic stem cell transplantation has been used for decades to treat lethal hematological malignancies since dose-intensive

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chemotherapy mediates a direct cytotoxic effect on malignant cells, whereas the engrafted immune cells mediate potent graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effects. However, given its associated mortality and morbidity, the treatment is restricted to the young and medically fit, which thus excludes many hematological patients from this therapy. A number of investigators have documented the immunemediated antitumor effects of allogenic transplantation in solid tumors [57]. RCC may be susceptible to a GVT effect as it has been shown that T lymphocytes are an important component of the antitumor immunological response. In addition to the known effects of cytokines, it has also been reported that clonally expanded cytotoxic T lymphocytes are present in primary and mRCC specimens, thus demonstrating HLA-restricted cytotoxicity against RCC cell lines [58]. However, in order to optimize the possibility of generating a GVT effect in vivo, rapid and complete engraftment of the donor immune system is required [59] and, as RCC is quite refractory to chemotherapy, a nonmyeloablative transplantation strategy seemed the best way to test the potential of allogenic immunotherapy against it. In a pioneering study, Childs and colleagues verified the results of nonmyeloablative transplantations in 19 patients with cytokine-refractory mRCC [60]. The patients were administered cyclophosphamide 60 mg/kg for 2 days and fludarabine 25 mg/m2 for 5 days, followed by the infusion of peripheral blood progenitor cells from a 5 or 6 out of 6 HLA-matched sibling; 100% donor chimerism was achieved by tapering posttransplantation cyclosporine and/or giving escalating infusions of donor lymphocytes. There were three lasting CRs and seven PRs, for an ORR of 53%. Clinical response was closely associated with the development of graft-versus-host disease (GVHD) and 100% donor T-cell chimerism, and was most commonly observed in patients with pulmonary metastases of

clear cell histology without any other organ involvement. There was a 4–6 month interval between transplantation and development of a GVT effect. Ten patients experienced acute GVHD: two died of transplantation-related causes and eight from progressive disease. After a median follow-up of 402 days, nine patients were alive 287–831 days after transplantation. An update of this study reported a 48% RR in 52 patients [61]. Rini and colleagues reported a partial RR of 33% in 12 refractory mRCC patients treated with a similar regimen and, in a follow-up study presented at the 2004 ASCO meeting, clarified the real impact of miniallogenic stem cell transplantation [62]. Only 15 (18%) of 84 eligible patients with progressive RCC and at least one sibling were treated (the others had either rapidly progressive disease or no matching sibling), of whom 33% responded (44% achieved full engraftment); the time to response was approximately 180 days and the transplant-related death rate was 33%. Consequently, only five of the 84 patients benefited from this procedure and the cost in term of toxicity was substantial. Other studies have subsequently provided further evidence that RCC may be susceptible to a GVT effect, with RRs ranging widely from 0 to 57% (TABLE 1). In terms of survival, a report from the European Bone and Marrow Transplantation Solid Tumor Working Party analyzed 70 patients with advanced RCC who underwent allogenic transplantations with various reduced-intensity fludarabinebased regimens after failure on immunotherapy and identified two prognostic groups based on PS, and the levels of C-reactive protein and LDH [63]: median survival was only 3.5 months in the case of the patients with a poor prognosis as against 23 months in the case of those with a good prognosis. In conclusion, nonmyeloablative allogenic stem cell transplantation is an interesting strategy that can lead to a disease response when other therapies have failed. However, it is limited by the need for an appropriately matched donor, the fact

Table 1. Clinical trials of nonmyeloablative allogenic transplantation for metastatic RCC. Response rates (%)

Number of patients

Conditioning regimen

GVHD prophylaxis

53

19

Flu/Cy

CSP

Ref. [60]

48

52

Flu/Cy

CSP

[61]

33

12

Flu/Cy

Tacrolimus and MMF

[62]

57

7

Flu/Cy

CSP and MTX

[92]

0

7

Flu/Cy

CSP and MTX

[93]

27

15

Melphalan/Flu

Tacrolimus and MTX

[94]

0

10

2 Gy TBI/Flu

CSP and MMF

[95]

4

25

ATG/BU/Flu

CSP

[96]

11

9

ATG/BU/Flu

CSP

[97]

ATG: Antithymocyte globulin; BU: Busulfan; CSP: Cyclosporin; Cy: Cyclophosphamide; Flu: Fludarabine; GVHD: Graft-versus-host disease; MMF: Mycophenolate mofetil; MTX: Methotrexate; RCC: Renal cell carcinoma; TBI: Total body irradiation.

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that responses are usually delayed until an average of 3–4 months after transplantation, severe toxicity, temporary pancytopenia and acute and chronic GVHD. Only a few patients (5–10%) benefit from this approach and those with rapidly progressive disease cannot be treated long enough to obtain a response. Chemoimmunotherapy

mRCC is refractory to chemotherapeutic agents, with a RR that is generally less than 10% [64,65], but efforts have been made to assess whether there is any advantage in combining chemo- and immunotherapy. Seven randomized clinical trials so far have tested such combinations, with most using 5-FU or VBL and, more recently, the combination of 5-FU and gemcitabine [66,67]. Four studies have compared IFN-α plus VBL with other nonchemoimmunotherapy (TABLE 2). The first prospectively randomized 160 patients with locally advanced or mRCC to receive subcutaneous IFN-α-2a 3 MU three times a week for 1 week followed by 18 MU three times a week for the second and subsequent weeks plus intravenous VBL 0.1 mg/kg once every 3 weeks, or the same dose of VLB alone: chemoimmunotherapy was significantly superior in terms of RR (16.5 vs 2.5%) and median (OS: 16.9 vs 9.4 months) [68]. In the second study, 178 mRCC patients were randomized to receive IFN-α-2a 18 MU three times a week intramuscularly plus intravenous VBL 0.1 mg/kg once every 3 weeks, or the same dose of IFN-α-2a alone: the combination doubled the RR (24 vs 11%) but did not prolong survival (5-year survival was 9% in both treatment arms) [69]. In the third study, 165 mRCC patients were randomized to receive IFN-α-n1 for six induction cycles at daily escalating doses of 3, 5, 20, 20 and 20 MU/m2 during the first 5 days of each 14-day cycle plus intravenous VBL at a dose of 5–10 mg/m2 on day 1 of alternate cycles, or the same schedule of IFN-α-n1 alone. The overall RR was 10%, but there was no significant difference between treatment arms in terms of RR or OS, although a small subset of patients with only lung metastases showed a

high RR (44%) with durable responses and much better OS than the other patients [70]. The fourth prospective study enrolled 76 patients and compared subcutaneous IFN-α 8 MU/day for 3 days per week plus intravenous VBL 0.1 mg/kg once every 3 weeks versus MPA alone: RRs were 20.5 versus 0%, respectively, but there was no statistically significant survival benefit in the IFN-α plus VBL group [71]. Overall, the combination of IFN-α plus VBL led to RR ranging from 10 to 24%, significantly better than those observed with VBL or MPA alone, whereas only one of the two studies comparing it with IFN-α alone showed an absolute difference in RR (13% in favor of the association), and only one of the four found a significant survival advantage in favor of chemoimmunotherapy [68]. On the basis of experimental studies showing some signs of synergism between 5-FU and IFN [72] and clinical data (RR of 2–47% in nonrandomized Phase II studies; an average RR of 24% among 490 patients), Negrier and colleagues enrolled 131 patients and compared the efficacy and toxicity of subcutaneous IFN-α-2a 6 MU three times a week in weeks 1, 3, 5 and 7 plus subcutaneous recombinant (r)IL-2 6 MU/day for 6 days per week in weeks 1, 3, 5, and 7 plus 5-FU administered by means of a continuous intravenous infusion for 5 days at 600 mg/m2/day in weeks 1 and 5, versus the same schedule of IFN-α-2a plus rIL-2 without 5-FU. Although there was a positive trend in favor of the chemoimmunotherapy (RR: 8.2 vs 1.4%), the difference was not statistically significant and the 1-year OS rates were 53 and 52% [73]. Finally, Atzpodien has conducted two studies. The first involved 78 patients and compared IFN-α-2a (5 MU/m2 subcutaneously on day 1 during weeks 1 and 4, and days 1, 3 and 5 during weeks 2 and 3, followed by 10 MU/m2 subcutaneously on days 1, 3 and 5 during weeks 5–8) plus subcutaneous rIL-2 (10 MU/m2 twice daily on days 3–5 during weeks 1 and 4, and 5 MU/m2 on days 1, 3 and 5 during weeks 2 and 3) plus intravenous 5-FU (1000 mg/m2 on day 1 during weeks 5–8) versus tamoxifen (80 mg orally, twice daily for 8 weeks):

Table 2. Clinical trials of chemoimmunotherapy. Number of patients Study design

RR (%)

OS (months)

160

IFN + VBL vs VBL

16.5 vs 2.5 *

16.9 vs 9.4 *

Ref. [68]

178

IFN + VBL vs IFN

24 vs 11

9% at 5 years

[69]

165

IFN + VBL vs IFN

10

Not assessed

[70]

76

IFN + VBL vs MPA

20.5 vs 0

Not assessed

[71]

131

IFN + IL-2 + 5-FU vs IFN + IL-2

8.2 vs 1.4

53% vs 52% at 1 year

[73]

78

IFN + IL-2 + 5-FU vs tamoxifen

39.1 vs 0

24 vs 13

[74]

341

IFN + IL-2 + 5-FU vs IFN + IL-2 + 5-FU + CRA vs IFN + VBL

31 vs 26 vs 20

25 vs 27 vs 16*

[75]

* p < 0.05 5-FU: 5-Fluorouracil; CRA: Cis-retinoic acid; IFN: Interferon; IL: Interleukin; MPA: Medroxiprogesterone acetate; OS: Overall survival; RR: Response rate; VBL: Vinblastine.


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Therapies targeting VEGF, PDGF, TGF-α and/or mTOR pathways Bevacizumab

the RR was 39 and 0%, respectively, and median OS was significantly longer (24 vs 13 months) [74]. The second involved 341 patients who received the same schedule plus 13-cis-retinoic acid (CRA) (20 mg orally, three times daily for 8 weeks), or subcutaneous IFN-α-2a (5 MU/m2 on day 1, 3 and 5 in week 1, followed by 10 MU/m2 on days 1, 3 and 5 in weeks 2–8) plus intravenous VBL (6 mg/m2 on day 1 in weeks 2, 5 and 8). The RRs were 31, 26 and 20%, and the median OSs were 25, 27 and 16 months, respectively [75]. In general, these studies showed considerable variability in clinical response and survival and the results cannot be used to define the role of chemoimmunotherapy in mRCC. The possible reasons for this are the use of different schedules and the absence of a generally accepted standard treatment arm; however, the chemoimmunotherapy regimen demonstrated a good toxicity profile.

Bevacizumab is a monoclonal antibody (MoAb) that binds to and neutralizes all of the active isoforms of VEGF, thus inhibiting the angiogenetic process that is essential for neoplastic growth, progression and metastases. In a Phase II study, 116 RCC patients pretreated with cytokines were randomized to receive bevacizumab 3 or 10 mg/kg every 2 weeks, or placebo [79]. Time to progression was longer in the patients treated with the highest dose than in those treated with placebo (4.8 vs 2.5 months) and their objective RR was 10%. Although no significant survival advantage was described, this study offers the ‘proof of principle’ of the efficacy of antiangiogenic therapy. A Phase III trail of IFN with or without bevacizumab has completed accrual; Phase II studies of low or high doses of IL-2 with bevacizumab are ongoing.

New biological therapies

Sunitinib (SU11248)

Recent progress in our understanding of the biomolecular mechanisms controlling the development and growth of RCC has led to the identification of a number of new biological drugs targeting different pathways (TABLE 3). More than 60% of clear cell renal tumors are associated with a VHL gene loss of function [76] caused by methylations or deletions; this leads to the nonregulation of hypoxia inducible factor (HIF) and the subsequent pathologically increased production of proteins such as vascular enothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-α involved in tumor growth [77,78]. More specifically, VEGF is a tumor-secreted dimeric glycoprotein relevant to the generation and preservation of tumor vasculature. It induces endothelial cell division and migration and protection from apoptosis. VEGF exerts its biological effect through interaction with tyrosine kinase (TK) receptors (VEGFR 1, 2 and 3) selectively expressed on vascular endothelial cells. Upon binding of VEGF to the extracellular domain of the receptor, dimerization and autophosphorylation of the intracellular receptor TK occurs and several downstream protein pathways are activated leading to the biological effects on endothelial cells. PDGF is produced by platelets, endothelial cells, vascular smooth blood vessels and macrophages. The PDGF receptor (PDGFR) is expressed on pericytes that act as structural support for endothelial cells; it may play a role in the later stages of blood vessel formation. TGF-α is a major ligand for the epidermal growth factor receptor (EGFR) and induces cellular proliferation and survival. It can independently increase VEGF expression, thereby providing an additional mitogenic signal from the malignant cell to the supporting blood vessels. Other pathways involved in the action of experimental new drugs include the inhibition of mammalian target of rapamycin (mTOR), a kinase belonging to the PI3K metabolic pathway. mTOR plays a central role in cell growth and survival and it also increases the level of HIF expression [88].

Sunitinib is a small molecule that inhibits the TK of VEGFR–VEGFR-2,VEGFR-3 and PDGFR-α and -β. In a Phase II study, it was given as monotherapy (50 mg/day for 4 weeks, followed by 2 weeks rest) to 63 patients with progressive RCC after cytokine treatment: the partial RR was 40%, mean time to progression (TTP) 8.7 months and mean survival 16.4 months [80]. A Phase III study comparing sunitinib and IFN in untreated patients was presented recently at the 2006 ASCO meeting; 750 patients were randomized 1:1 to receive sunitinib (50 mg orally once daily for 4 weeks, followed by 2 weeks off ) or IFN-α (subcutaneous injection 9 MU given three times weekly, 6-week cycles). Median PFS was 47.3 weeks for sunitinib versus 24.9 weeks for INF-α (harzard ratio: 0.394; p < 0.000001). The RR was 24.8% for sunitinib versus 4.9% for IFN-α ( p < 0.000001) [81]. This is the first trial documenting a superiority of a new biological drug in first-line treatment and sunitinib could be considered as a standard treatment for mRCC.

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Sorafenib (BAY 43–9006)

Sorafenib acts on the TK of VEGFR- 2, VEGFR3 and PDGFR-β, and also inhibits the B-RAF and C-RAF kinase involved in the activation of the RAF–MEK–ERK pathway that stimulates the proliferation of endothelial cells. It showed promising activity and a good tolerability profile in Phase I and II studies [82] and in a double-blind, Phase III study [83] including 769 pretreated patients randomized to receive sorafenib 400 mg twice a day, or placebo. Mean PFS was significantly better with this new drug; 24 versus 12 weeks (p < 0.00001) in patients taking sorafenib or placebo, respectively. A Phase III study comparing sorafenib with IFN-α in untreated mRCC patients is ongoing. Axitinib (AG-013736)

Axitinib is a multitarget TK inhibitor of VEGFR-1, VEGFR-2 and PDGF-β. Recently, some interesting results from a Phase II study have been presented. In this trial, 52 patients progressing on first-line treatment were treated with 5 mg twice a day, and achieved 40% of PRs [84].



Table 3. New active agents for the treatment of metastatic renal cell cancer. Target

Description/ route of administration

Clinical activity in pretreated patients

Common toxicity

Clinical development

BV

VEGF

Monoclonal antibody Phase II random vs Hypertension, i.v. infusion placebo: proteinuria, embolism RR: 10 vs 0% and epistaxis TTP: 4.8 vs 2.5 months p < 0.001

Phase III: IFN-α vs IFN-α + BV (completed accrual) Ongoing trials in association with other cytokines

[79]

SU-11248 Sunitinib

VEGFR PDGFR FLT3-KIT

Selective TK inhibitor Oral

Phase II: RR: 40% PFS: 8.7 months

Fatigue, nausea, diarrhea, stomatitis and lymphopenia

Completed Phase III Sunitinib vs IFN-α in first line treatment. See text for details

[80,81]

Bay 43–9006 Sorafenib

VEGFR PDGFR Raf kinase

Multikinase inhibitor Oral

Phase III random vs placebo: RR: 2 vs 0% PFS: 24 vs 12 weeks p < 0.0001

Rash, diarrhea, hand–foot reaction, hypertension, fatigue and nausea

Phase II random sorafenib vs IFN completed accrual Ongoing trials in association with other cytokines

AG-013736 Axitinib

VEGFR PDGFR

Selective TK inhibitor Oral

Phase II RR: 40% TTP: 8.3 months

Hypertension, fatigue, Phase II in nausea and diarrhea sorafenib-resistant patients (ongoing)

CCI 779 Temsirolimus

mTOR

Ester of rapamycin i.v. infusion

Phase II: RR: 7% PFS: 5.8 months

Rash, mucositis, asthenia, nausea and hyperglycemia

Phase III: CCI779 vs IFNα vs CCI779 + IFN-α in poor risk patients. See text for details

Rad 001 Everolimus

mTOR

mTOR inhibitor Oral

Phase II RR: 28% PFS: >3 months in 86%

Mucositis, skin rash, pneumonitis, hyperglycemia and thrombocytopenia

Phase III: Rad 001 vs placebo in pretreated patients (ongoing)

ABX-EGF Panitumumab

EGFR

Monoclonal antibody Phase II i.v. infusion RR: 5.7% PFS: 3.4 months

Asthenia, abdominal, Continue ABX-EGF bone and back pain, treatment in patients anorexia, cough, previously responding dyspnea and diarrhea

Ref.

[83]

[84]

[89,90]

[91]

[85]

BV: Bevacizumab; EGFR: Epidermal growth factor receptor; FLT: Fms-like tyrosine kinase; IFN: Interferon; i.v.: Intravenous; mTOR: Mammalian target of rapamycin; PFS: Progression-free survival; PDGFR: Platelet-derived growth factor receptor; RR: Response rate; TK: Tyrosine kinase; TTP: Time to progression; VEGF: Vascular endothelial growth factor; VEGFR: VEGF receptor.

Panitumumab (ABX-EGF)

Panitumumab is a fully human MoAb against EGFR that has been evaluated in a Phase II trial. A total of 88 cytokine-resistant mRCC patients were treated with panitumumab at doses of 1.0, 1.5, 2.0 or 2.5 mg/kg weekly by intravenous infusion. Major responses occurred in three patients and two patients had minor responses. In total, 44 patients (50%) also had SD at their first 8-week assessment and the median PFS was 100 days (95% CI: 58–140 days) [85]. Cetuximab

Cetuximab is a MoAb against EGFR but, at the present time, it has not proved to be efficacious and no further studies of it as a single agent in RCC are planned [86]. Gefitinib & erlotinib

Gefitinib and erlotinib are two small molecules that block the TK of EGFR but have shown poor results as single agents [86]. There are some ongoing studies of combinations of these drugs


with other target agents. Moreover, a SWOG Phase II study of erlotinib in patients with locally advanced or metastatic papillary RCC has completed accrual. Temsirolimus (CCI 779)

Temsirolimus is a derivate of rapamycin that acts by inhibiting mTOR. In a randomized Phase II study, 100 patients with RCC refractory to standard treatment were randomized to receive weekly intravenous doses of 25, 75 or 250 mg [89]. Overall, the objective RR was 7% (1 CR and 7 PRs), without any significant differences between the three arms. The TTP was 5.8 months and the median survival was 15 months. The results of a Phase III randomized, three-arm study of temsirolimus versus IFN-α vs the combination of temsirolimus plus IFN-α, in poor-risk mRCC patients, was recently presented at the 2006 ASCO annual meeting. A statistically significant longer survival was found in favor of patients treated with temsirolimus alone: 10.9 versus 7.3 versus 8.4 months in the temsirolimus versus IFN versus the combination, respectively

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(hazard ratio: 0.73; range: 0.57–0.92; p = 0.015) [90]. The results of this study suggest a potential new standard therapy for this poor-risk group of patients. Everolimus (RAD 001)

This is an oral serine-threonine kinase inhibitor of mTOR that has led to promising results in a recent Phase II study of 25 patients. The partial RR was 28% with a TTP of over 3 months for 86% of patients [91]. A Phase III, placebocontrolled trial is planned in patient with cytokine- and anti-VEGF-resistant mRCC. Expert commentary & five-year view

In no other human tumor is the treatment as rapidly evolving as in mRCC. The clinical results of traditional immunotherapy using IFNs, IL-2 and their combinations, as well as other forms of immunotherapy, cannot be expected to improve any further, thus new therapeutic agents and strategies are required, including the new biological drugs and therapies targeting the VEGF, PDGF, TGF-α or mTOR pathways. Future research will not only need to make every effort to optimize the use of these new molecules, define their efficacy precisely and compare them with traditional immunotherapy, but also consider how to integrate immunotherapy with the new biological drugs. It is in fact possible to hypothesize that immunotherapy will retain its weight, since its mechanism of action is different from that of the new kinase-targeting biological therapies and it has an excellent safety profile, particularly in the case of low and intermediate IL-2 and IFN doses. It is likely that many target therapies, especially those based

on MoAbs, will act synergistically with IL-2 and IFN and that this will lead to higher clinical RRs and, above all, a longer response duration. Another exciting area of interest comes from the recent research concerning the regulation of the host immune response and particularly the role of Treg cells and vaccines using HSPs. The mechanism of IL-2-mediated tumor regression is largely unclear and the balance between activation and expansion of tumor-reactive effectors T cells relative to Treg cells by IL-2 may determine the clinical outcome for a patient [54]. Depletion of Tregs specific, using recombinant IL-2 diphtheria toxin conjugate, may enhance the ability of IL-2 to elicit antitumor immune responses with clinical impact in cancer patients. In the past, we have learned that RCC is a heterogeneous disease. Now, we can understand two main aspects of this disease: the genetic and molecular pathways responsible for the clinical heterogeneity and the mechanisms of host–tumor interaction and the way to positively modify such an interaction. In the next few years we will need to better define the activity of the new biological targeted drugs and their clinical impact. At the same time, an optimal stimulation of the immune system should be defined, that takes into account the identification of Treg cells and an optimal use of vaccination. The challenge facing researchers in the next few years will be the identification of combination regimens of biological and immunotherapy drugs, specifically targeted considering the genetic and molecular alterations of each individual tumor and patient’s immunological status and capable of attacking neoplastic cells via different mechanisms of action. Immunotherapy will have a particular place in this future and will continue to play a key role in the treatment of RCC.

Key issues • For patients with a new diagnosis of renal cell carcinoma, nephrectomy (if feasible) should be considered as the first choice treatment in both operable (T1-T3) and metastatic disease. • There is no evidence of any survival benefit from immunotherapy or any other kind of adjuvant systemic treatment. • At present, effective immunotherapy options are represented by high-dose interleukin (IL)-2 (which induces more and durable complete responses), interferon (IFN)-α, or low/intermediate doses of IL-2 with or without IFN-α. • Vaccines based on heat-shock protein–peptide complex (HSPPC)-96 have shown promising results recently. • The efficacy of cancer vaccination could be enhanced by treatment with agents that lead to preferential depletion of CD4+CD25+ T regulatory cells. • Nonmyeloablative allogenic stem cell transplantation is an interesting strategy when other therapies have failed; limits are represented by severe toxicity, costs and benefit limited to a small percentage of patients treated. • Based on recent results of randomized trials, different molecular targets(e.g., new biological agents such as sunitinib, sorafenib and temsirolimus) are more effective than IFN in renal cell carcinoma. • The challenge facing researchers in the next few years will be the identification of combination regimens of biological and immunotherapy drugs, specifically targeted considering the genetic and molecular alterations of each individual tumor and patient’s immunological status.

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Gianluca Tomasello, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected]

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Raffaella Longarini, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected]

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Matteo Brighenti, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected]

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Matteo Dalla Chiesa, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected]

Affiliations •

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Rodolfo Passalacqua, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected] Sebastiano Buti, MD Istituti Ospitalieri, Department of Internal Medicine, Medical Oncology Division, Viale Concordia 1, 26100, Cremona, Italy Tel.: +39 037 240 5242 Fax: +39 037 240 5235 [email protected]
