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Kidney SPORE

The goals of the Kidney SPORE are to improve the understanding and treatment of kidney cancer that is resistant to standard therapies; to identify biomarkers for the purposes of early detection, monitoring, and predicting treatment outcome; and to use the growing understanding of kidney cancer biology to develop novel immune and targeted therapies that improve the outcome of patients with advanced disease.

Kidney SPORE Principal Investigator: David F. McDermott, MD

Program Accomplishments:
The DF/HCC Kidney Cancer Program is a multifaceted program that encompasses basic science as well as population and clinical science.  Programmatic activities, in large part, complement and interface with the research within the SPORE and demonstrate the rich clinical and research environment that envelopes the SPORE.  Below is a description of research that was performed and/or published within the last 3 years as well as some high profile earlier research. 

a)  Major Scientific Accomplishments- Laboratory
1. VHL Biology
Establishment of HIF transcription factor and its downstream targets as critical components of renal cancer biology.  The VHL gene is mutated and/or methylated in the majority of clear cell RCC. Drs. Kaelin and Iliopoulos were the first to show that the VHL tumor suppressor protein (pVHL) is the substrate recognition module of an E3 ubiquitin ligase that target HIF (hypoxia-inducible factor) for destruction. Moreover, they showed that HIF must undergo, prolyl hydroxylation, an oxygen-dependent posttranslational modification, in order to be recognized and earmarked for destruction by pVHL.  More recent work has shown the critical role of Hif 2 in carcinogenesis associated with VHL loss.  The Kaelin Laboratory showed that disruption of HIF2alpha with shRNA (short hairpin RNA) was like restoration of pVHL function, sufficient to suppress VHL (-/-) renal cancer growth in murine xenograft models (Kondo et al).  This observation was supported by work from the Iliopoulos Lab (Zimmer et al).  Given that HIF has been shown to upregulate the expression of a number of genes including VEGF, TGFalpha, PDGF, Glut 1 and Epo, this work led directly to the study of VEGF binders and VEGF, PDGF and EGF receptor TKIs in patients with RCC and is continuing to influence the development of new therapeutic approaches targeting HIF (as described in new Project 2 and a year 5  Developmental Project led by Dr. Zimmer) or other critical components of the VHL, HIF relationship.
Role of clusterin in VHL Disease. Dr. Kaelin and colleagues also examined proteins that are differentially expressed by isogenic RCC cell line pairs and confirmed that HIF targets IGBP3 and PAI were overproduced by pVHL defective RCCs.   In addition, cells lacking wild-type pVHL, including cells producing type C pVHL mutants linked to familial pheochromocytoma, were defective with respect to expression and secretion of clusterin which did not behave like a HIF target.   Decreased clusterin secretion by pVHL-defective tumors was confirmed in vivo by immunohistochemistry.  This suggested that clusterin was a secreted marker of HIF-independent pVHL function that might be especially important in pheochromcytoma development.   In addition, it is possible that measurement of these secreted biomarkers in bodily fluids might be useful in for early detection or monitoring of pVHL defective renal cell carcinomas.  Clusterin in particular might provide a clue to novel pVHL functions and tumor suppressor mechanisms that might form the basis of the genotype-phenotype correlations observed in VHL disease.   This work was published in the Am J Pathol 2006.

Role of acute VHL Inactivation: Dr. Kaelin and colleagues showed that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo (Young et al Nat Cell Biol 2008). This phenotype was independent of p53 and HIF but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a HIF-independent fashion. These results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.

HIF independent VHL functions- role of Card9 and PARP1 in NFkB activation.  Dr. Kaelin and colleagues also investigated HIF-independent pVHL functions that might affect tumorigenesis and/or response to therapy. For example, kidney cancers are difficult to treat with chemotherapy or radiotherapy and this has been felt to be due, at least partly, to the fact that kidney cancers often display increased NFkB activity. Notably, several recent reports showed that NFkB is also increased upon pVHL inactivation, suggesting that these two observations might be linked.   Kaelin and colleagues were able to show that pVHL serves, in a HIF-independent manner, as an adapter protein that promotes the phosphorylation of the NFkB agonist Card9 by CK2 (Yang et al Mol Cell in press).  Phosphorylation of Card9 by CK2, in turn, prevents it from activating NFkB.  In cells lacking functional pVHL, Card9 remains hypophosphorylated and NFkB activity is increased. They subsequently showed that downregulation of Card9 increases the sensitivity of VHL-/- renal carcinoma growth to cytokines in vitro and suppressed orthotopic tumor formation by VHL-/- renal carcinoma cells in nude mice assays. Therefore deregulation of Card9 might play a role in therapeutic resistance and tumorigenicity of VHL-/- renal carcinoma cells.  More recently, they showed that hypophosphorylated Card9 recruits the enzyme PARP1, which plays a role in the polyadenylation of diverse substrates, and that this recruitment is linked to activation of NFkB.  These results raise the possibility that PARP1 inhibitors might be used to suppress NFkB activity in VHL-/- renal carcinoma cells and therapeutically in RCC.  These observations form the basis of a sample Developmental Project proposed by Dr. Kaelin (see Developmental Research Program), and if initial experiments confirm a role for PARP1 inhibition in VHL -/- renal cancer cells, this work could be the basis of a full SPORE project in future years.

2. Tuberous Sclerosis related studies
Role of Tor pathway regulations, TSC1 and TSC2, in driving VEGF:  Dr. Brugarolas, working in the Kaelin Lab identified a potential role of the mTOR regulators, TSC1 and TSC2, in driving VEGF production via HIF dependent and independent mechanisms (Brugarolas et al). This work contributed to a better understanding of the potential antiangiogenic effects of inhibiting mTOR and laid groundwork for combination therapies involving VEGF and TOR inhibitors.

Relationship of PI3K pathway and PDFFR expression: Dr. Sandra Dabora and colleagues studied the relationship between the RTK/PI3K/Akt/mTOR pathway and PDGFR expression (Zhang et al J Clin Invest 2007). Inactivating mutations of either the TSC1 or the TSC2 tumor-suppressor genes have been shown to cause tuberous sclerosis complex (TSC), a benign tumor syndrome in which there is both hyperactivation of mTOR and inhibition of RTK/PI3K/Akt signaling, partially due to reduced PDGFR expression. They reported that activation of PI3K or Akt, or deletion of PTEN in mouse embryonic fibroblasts (MEFs) also suppresses PDGFR expression. This was a direct effect of mTOR activation, since rapamycin restored PDGFR expression and PDGF-sensitive Akt activation in Tsc1-/- and Tsc2-/- cells. Akt activation in response to EGF in Tsc2-/- cells was also reduced. Furthermore, Akt activation in response to each of EGF, IGF, and PMA was reduced in cells lacking both PDGFRalpha and PDGFRbeta, implying a role for PDGFR in transmission of growth signals downstream of these stimuli. Consistent with the reduction in PI3K/Akt signaling, in a nude mouse model, both Tsc1-/- and Tsc2-/- cells had reduced tumorigenic potential in comparison to control cells, which was enhanced by expression of either active Akt or PDGFR beta. They concluded that PDGFR is a major target of negative feedback regulation in cells with activated mTOR, which limits the growth potential of TSC tumors.  These results have implications for both TSC and the use of TOR inhibitors in renal cancer.
Relationship between genetic alterations and phenotype in Tuberous Sclerosis: Dr. Dabora and colleagues also investigated the relationship between specific genotypic alterations and phenotype in Tuberous Sclerosis. (Kozlowski et al Human Genet 2007) Point mutations and small inragenic deletions account for most TSC1 and TSC2 mutations in tuberous sclerosis. They examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15-50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5', 3' or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Genotype/phenotype analysis showed that all 21 patients with TSC2 deletions extending 3' into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. They concluded that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method.
Combination therapy for Tuberous sclerosis: Dr. Sandra Dabora et al investigated the efficacy of the combination of temsirolimus and IFNg in a mouse model of Tuberous sclerosis complex (TSC) relative to the single agents (Lee et al Genes Chromosomes Cancer 2006).They observed that combination therapy was more effective than single agent therapy in reducing tumor growth and improving survival.  Immunoblot and immunohistochemical analyses showed that tumors treated with temsirolimus plus IFN-gamma had decreased cell proliferation and increased cell death in comparison with untreated tumors or tumors treated with either agent alone. They also observed that temsirolimus resistance could develop with prolonged treatment. Taken together, these results show that targeting multiple cellular pathways is an effective strategy for treating TSC-related tumors, and underscore the importance of investigating combination therapy in future clinical trials for patients with TSC.  These results also have potential implications for the use of temsirolimus in patients with RCC.  This work led directly to a Developmental Project for Dr. Dabora in the initial SPORE.

In more recent studies, (Messina et al. BMC Pharmacol 2008) they examined the effects of treating Tsc2+/- mice and nude mice bearing Tsc2 -/- tumors at different time points with temsirolimus as a single agent or in combination with IFN-gamma. While temsirolimus was more effective in treating Tsc2-/- tumors, they observed no benefit for the addition of IFN gamma or the administration of treatment prior to tumor development in Tsc2 +/- mouse model. This work implied that prevention of the genesis of TSC-related kidney lesions may not be an effective treatment strategy; a finding that will likely influence future preclinical and clinical trials for TSC.

3.  Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) biology
The role of LDH-A in HLRCC:  Drs. Sukhatme, Seth and colleagues have begun to explore the role of LDH-A in HLRCC.  The genetic basis for HLRCC is believed to be a germline inactivating mutation in the gene for the TCA cycle enzyme fumarate hydratase (FH).  Since FH is critical for the conversion of fumarate to malate, inhibition of this process should necessitate that glycolysis followed by fermentation of pyruvate to lactate will be required to provide ATP as well as to regenerate NAD+ required to sustain metabolic pathways.  FH deficiency has been shown to lead to upregulation of HIF1alpha transcript.   RNA for targets of HIF1a, such and LDH-A an enzyme involved in pyruvate lactate interconversion, are increased in tumors of patients with HLRCC.  Drs. Sukhatme and colleagues examined A549 lung cancer cell line (VHL positive) as a model system to generate FH-deficient cell lines and demonstrated that these cell lines have increased lactate accumulation, VEGF production, and VEGF and Glut-1 message levels, relative to controls. These data suggest that glycolysis followed by pyruvate to lactate conversion is enhanced by FH deficiency and that these cells mimic tumors from HLRCC patients.  In addition, they have shown FH deficiency leads to LDH-A activity and have seen enhanced in vivo expression of LDH-A in HLRCC tumor samples.  Furthermore, blocking of LDH-A in a RCC cell line reduces proliferation and induces apoptosis.  This suggests that small-molecule inhibitors that specifically target LDH-A could be used to treat HLRCC and to study the causal role of LDH-A in HLRCC.  This work was presented at the 2007 National SPORE Workshop and resulted in a Kidney Cancer SPORE year 5 Developmental Project. (See DRP Progress Report for and update on this Research). 

4. Wilms Tumor  
Role of WTX in Wilms Tumor:  Drs. Han, Haber and colleagues used the discovery of WTX as a novel tumor suppressor gene whose function is lost in Wilms tumor (SPORE Developmental Project), Science (Rivera et al. Science 2007), to uncover a novel mechanism for Wilms tumor development.  They investigated the possible involvement of WTX in a case of Wilms' tumor containing an apparently balanced reciprocal translocation between chromosomes X and 18 (t(X;18)(q11;p11)) (Han et al Genes Chromosomes Cancer 2007) yielded interesting biologic information.  Fluorescence in situ hybridization (FISH) analysis of paraffin tumor sections revealed a deletion of the WTX locus at Xq11. High-resolution array CGH analysis of tumor DNA revealed a 1.5 Mb chromosome deletion encompassing the WTX gene at Xq11. However, no loss of genetic material was detected on chromosome 18.  Thus unlike most tumors with acquired chromosomal translocations, where a new fusion oncogene or promoter-oncogene fusion is created that drives tumor growth, the t(X;18) in this tumor appears to drive tumorigenesis via deletion of a tumor suppressor. This case demonstrates the importance of array CGH and FISH as adjuncts in tumor cytogenetics.

5. Identification of Novel Targets
Other mutations in kidney tumors: Dr. Signoretti also contributed kidney cancer samples to a large effort involving high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples (Thomas et al Nat Genet 2007). This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, they found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. They identified previously unrecognized oncogene mutations in several tumor types including a B-raf mutation in a kidney tumor (metanephric adenoma) and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention. 

b) Major Scientific Accomplishments-Clinical/Translational
1. Biomarkers

 IMP3 as a potential biomarker for poor prognosis in RCC: Dr. Chin Li Wu working with colleagues at the MGH and University of Massachusetts Medical Center investigated whether tumor tissue expression of IMP3, an oncofetal RNA-binding protein, could be used as a biomarker to predict metastasis and prognosis in RCC. They assessed IMP3 expression by IHC in over 500 RCC specimens including primary tumors from 371 patients with localized disease.  They also examined IMP3 mRNA and protein expression by quantitative RT-PCR wand western blot in selected specimens.  Results were published in Lancet Oncology in July, 2006 (Jiang et al).  They found that IMP3 expression was greatly increased in both metastatic tumors and primary tumor specimens from patients destined to develop metastases. Patients with primary localized tumors that did not express IMP3 had longer metastasis free and overall survivals both as a whole group (p < 0.0001) and stage for stage than those with tumors expressing IMP3.  Multivariate analysis of IMP3 status in primary tumors showed a hazard ratio of 5.84 for metastasis free survival and 4.01 for OS, which were much higher than hazard ratios associated with other independent risk factors.  This suggested that IMP3 is an independent prognostic marker that can be used at initial diagnosis to identify patients at increase risk for relapse and therefore might benefit from adjuvant therapy.  In addition, efforts are underway to determine the biologic basis for IMP3 associated poor prognosis in the hope that either the protein itself or the pathway leading to its expression might serve as a novel therapeutic target.

Dr. Wu and colleagues more recently investigated whether IMP3 could serve as a prognostic biomarker to predict metastasis for patients with localized non clear cell RCC (Jiang Z et al Cancer 2008). The expression of IMP3 in 317 tumors from patients with localized RCC was evaluated by IHC and correlated with clinical outcome.  They found that patients with localized IMP3-positive tumors (n=33; 10%) were over 10 times more likely to metastasize (risk ratio [RR], 11.38; 95% confidence interval [CI], 5.40-23.96; P<.001) and were nearly twice as likely to die (RR, 1.91; 95% CI, 1.13-3.22; P=.016) compared with patients with localized IMP3-negative tumors. The 5-year metastasis-free and overall survival rates were 64% and 58% for patients with IMP3-positive localized papillary and chromophobe RCCs compared with 98% and 85% for patients with IMP3 negative tumors, respectively. In multivariable analysis adjusting for the TNM stage and nuclear grade, patients with IMP3-positive tumors were still over 10 times more likely to progress to distant metastasis (RR, 13.45; 95% CI, 6.00-30.14; P<.001) and were still nearly twice as likely to die (RR, 1.95; 95% CI, 1.15-3.31; P=.013) compared with patients with IMP3-negative tumors. Thus IMP3 appeared to be an independent prognostic biomarker that can be used to identify a subgroup of patients with localized papillary and chromophobe RCC who are at high risk for developing distant metastasis.

Urine specific markers associated with renal inflammation and cancer: Dr. Han and colleagues in the Bonventre Lab have investigated the role of human kidney injury molecule-1 (hKIM-1) a type 1 transmembrane protein that is not detectable in normal kidney tissue but is expressed at high levels in human and rodent kidneys with dedifferentiated proximal tubule epithelial cells after ischemic or toxic injury, in renal cancer.  They hypothesized that renal tumors express hKIM-1 and release this protein into the urine. Urine samples before nephrectomy and nephrectomy tissue samples were collected from 42 patients with renal tumors, 30 normal control subjects, and 10 patients with prostate carcinoma. Tissue was examined for expression of hKIM-1, and cell-free urine supernatants were analyzed for hKIM-1 by ELISA. The normalized urinary hKIM-1 levels were significantly higher in patients with clear cell RCC (0.39 +/- 0.08 ng/mg U(Cr); n = 21), compared with levels in patients with prostate carcinoma (0.12 +/- 0.03 ng/mg U(Cr); P < 0.02; n = 10), or normal control subjects (0.05 +/- 0.01 ng/mg U(Cr); P < 0.005; n = 30). In all patients with a detectable prenephrectomy urinary hKIM-1 level, there was either complete disappearance or marked reduction after nephrectomy. This suggested that the cleaved ectodomain of hKIM-1 can be detected in the urine of patients with RCC and may serve as a new biomarker for early detection of RCC.  The results were published in the J Am Soc of Nephrol (2006).  Dr. Han continued this work through a Career Development Award from the SPORE.  

More recent studies sought to determine what regulates KIM-1 urinary shedding (Zhang Z J Am Soc Nephrol 2007). They found that constitutive cleavage of KIM-1 is mediated by ERK activation, and that cleavage is accelerated by p38 MAP kinase activation. After cleavage, a 14-kD membrane-bound fragment of KIM-1, which contains two highly conserved tyrosine residues, was tyrosine-phosphorylated. Mutagenesis studies demonstrated that the juxtamembrane secondary structure, not the primary amino acid sequence, was critical to the cleavage of KIM-1.  The relevance of this finding to KIM-1 shedding in the setting of RCC or treatment with MAP kinase inhibitors such as sorafenib remains to be determined.

Role of HKim-1 as a biomarker in RCC tissue.  Drs. Han, Joe Bonaventre and colleagues also investigated the role of hKIM-1 as a biomarker in renal cancer tissue.  They evaluated the diagnostic utility of hKIM-1 in a large series of 480 neoplasms including defined subtypes of renal cell tumors, metastatic RCCs, and nonrenal tumors (Lin et al Am J Surg Pathol 2007).  They demonstrated that a membranous/cytoplasmic staining pattern for hKIM-1 was observed in 54 of 73 (74%) clear cell RCCs and 28 of 30 (93%) papillary RCCs on TMA sections. Zero of 54 chromophobe RCCs and 4 of 41 (9.75%) oncocytomas were positive for hKIM-1 when combining TMA and routine sections. Similar staining results were observed in 35 of 45 (78%) metastatic RCCs. Fifteen of 16 cases (93.8%) of clear cell carcinoma of the ovary demonstrated positive reactivity for hKIM-1. These data indicate that hKIM-1 is a relatively sensitive and specific marker for papillary, clear cell, and metastatic RCCs, can be used to distinguish clear cell from chromophobe RCC and may serve as a diagnostic marker for clear cell carcinoma of the ovary.

Biomarkers of sunitinib activity in RCC: Dr. Michaelson and colleagues explored potential biomarkers of sunitinib pharmacological activity in RCC via serial assessment of plasma levels of four soluble proteins VEGF, soluble VEGFR-2 (sVEGFR-2), placenta growth factor (PlGF), and a novel soluble variant of VEGFR-3 (sVEGFR-3). (Primo et al J Translational Med) At the end of cycle 1, VEGF and PlGF levels increased >3-fold (relative to baseline) in 24/54 (44%) and 22/55 (40%) cases, respectively (P < 0.001). sVEGFR-2 levels decreased >or= 30% in 50/55 (91%) cases and >or= 20% in all cases (P < 0.001) during cycle 1, while sVEGFR-3 levels were decreased >or= 30% in 48 of 55 cases (87%), and >or= 20% in all but 2 cases. These levels tended to return to near-baseline after 2 weeks off treatment, indicating that these effects were dependent on drug exposure. Overall, significantly larger changes in VEGF, sVEGFR-2, and sVEGFR-3 levels were observed in patients exhibiting objective tumor response compared with those exhibiting stable disease or disease progression. Thus sunitinib therapy leads to changes in circulating proteins involved in VEGF signaling, including soluble forms of two VEGF receptors. They concluded that this panel of proteins may be of value as biomarkers of the pharmacological and clinical activity of sunitinib in RCC, and of angiogenic processes in cancer and other diseases.

2.  Minimally Invasive Therapy
MRI guided percutaneous cryotherapy for metastatic lesions of soft tissue or bone. Dr. Stuart Silverman and colleagues investigated the safety and feasibility of percutaneous MRI-guided cryotherapy in management of refractory or painful metastatic lesions of soft tissue and bone adjacent to critical structures.  (Tuncali et al Am J Roentgenol. 2007). They found that 22(81%) of 27 tumors were managed without injury to adjacent critical structures. Thirteen (62%) of the 21 tumors for which follow-up information was available either remained the same size as before treatment or regressed. Eight tumors progressed (mean local progression-free interval, 5.6 months; range, 3-18 months). Pain was palliated in 17 of 19 patients; six of the 17 experienced complete relief, and 11 had partial relief. They concluded that MRI-guided percutaneous cryotherapy for metastatic lesions of soft tissue and bone adjacent to critical structures is safe and can provide local tumor control and pain relief in most patients.
External hand compression in percutaneous cryotherapy. Dr. Silverman and colleagues also described the safety and effectiveness of using external hand compression to displace adjacent bowel loops during MRI-guided percutaneous cryoablation of renal tumors.  (Tuncali et al Eur J Radiol 2006). Fourteen patients with 15 renal tumors (mean diameter: 2.4 cm; range: 1.4-4.6 cm) adjacent to bowel were treated with MRI-guided percutaneous cryoablation during which bowel was displaced manually. They reported that the mean distance between tumor margin and closest adjacent bowel increased from 0.8 cm (range: 0-2 cm) before external manual compression to 2.6 cm (range: 1.6-4.1 cm) with manual displacement (p<0.01). Mean minimum distance between iceball edge and closest adjacent bowel during the procedures was 1.6 cm (range: 0.5-3.5 cm). No evidence of bowel injury was encountered. Twelve of 15 tumors had follow-up that showed no tumor recurrence. They concluded that MRI-guided percutaneous cryoablation of renal tumors adjacent to bowel can be done safely and effectively using external hand compression to displace bowel loops.
Incidence and Pattern of residual disease after renal RFA or cryoablation. Drs Gervais, McGovern and colleagues at MD Anderson and Cleveland Clinic examined the incidence and pattern of residual and recurrent disease after radiofrequency ablation or cryoablation of a renal mass in order to determine reasonable minimum recommendations for when to perform surveillance imaging during year 1 after treatment. (Matin et al J Urol, 2006).  They reviewed treatment and follow-up information of 616 patients who underwent RFA or cryoablation for a renal mass at 7 institutions. 63 were found to have residual or recurrent disease after primary RFA(13.4%) or cryoablation (3.9%) for a median of 8.7%.  Most incomplete treatments (70%) were detected within the first 3 months. They concluded that in most cases initial treatment failure was detected within the first 3 months after treatment supporting a recommendation of a minimum of 3 to 4 imaging studies in year 1 after ablative therapy at months 1, 3, 6 (optional) and 12.

3.  IL-2 based therapy
Phase III trial of high dose IL-2 vs low dose IL-2/IFN. Program investigators designed and completed a Cytokine Working Group randomized phase III trial comparing high dose IL-2 to low dose IL-2 and IFN in patients with advance renal cell cancer.   This study that showed that the overall response rate (23 vs 10%), percent CR, and durable CR (8% vs 0) were significantly longer for patients receiving high dose IL-2 as compared to the low dose IL-2 regimen. Even though survival difference did not achieve significance for the population as a whole, high dose IL-2 was associated with significant survival benefit for patients with either liver or bone metastases (p=0.001) or primary tumor in place (p=0.040).  This suggested that in disease presentations typically associated with tumor-induced immune suppression, higher doses of IL-2 were necessary in order to achieve therapeutic benefit.  This study supported the continued use of high dose IL-2 as a standard therapy for advanced renal cancer and considering the toxicity associated with this approach, suggested that efforts to define the population most likely to respond to IL-2 therapy were indicated.  This work was published in Journal of Clinical Oncology (McDermott et al, 2005)

Mechanism of IL-2 induced capillary leak. Investigators in the Sukhatme lab have identified Angiopoeitin 2 (ang 2) as the potential mediator of IL-2 related vascular leak syndrome (VLS) (Gallagher DC, Bhatt RS et al, Clin Ca Res 2007).   They had previously reported that plasma ang 2 levels were elevated in patients with septic shock (Parikh et al. PLoS Medicine, 2006) and hypothesized that ang 2 might similarly contribute to the pathophysiology of HDIL2-related VLS.  They collected serial serum and plasma from patients receiving either HDIL2 alone or in combination with bevacizumab and measured ang 2 and VEGF levels by ELISA.  The effect of ang 2 on vascular endothelial cells was also studied in vitro by testing the effect of serum from patients with high ang 2 levels on cultured endothelial cells.  They concluded that ang 2 is a potential mediator of HDIL2-related VLS as evidenced by the elevation of circulating ang 2 levels in all patients tested and its effect on vascular endothelial cells in vitro.  Furthermore, the lack of correlation between VLS and serum VEGF levels in patients treated with either HDIL2 alone or in combination with bevacizumab suggests that VEGF is not a major contributor to VLS or ang 2 release.  This data suggests that inhibition of ang 2 might be a means of preventing VLS in patients receiving HDIL2. 

Role of IL-2 as salvage therapy following anti-angiogenic therapy. Dr. McDermott and colleagues studied the role of salvage high dose IL-2 therapy in patients whose disease had progressed following anti-angiogenic therapy (Schwartzberg et al, 2007).  Sixteen consecutive patients were analyzed.  All were considered excellent candidates for IL-2 prior to initiating anti-VEGF therapy, but were placed on anti-VEGF agents with the assumption that IL-2 could be given safely at a later date.  Treatment tolerability, as measured by IL-2 doses received, and toxicity, was compared to the HD IL-2 arm of a recent Cytokine Working Group Phase III trial (McDermott, et al, JCO 2005).  Six of 16 patients including 6/10 with prior TKI therapy did not receive week 2 of IL-2 therapy (p=0.034) primarily due to severe cardiac toxicity; 50% (95% CI 18.7% to 82.3%) compared to in 8.5% with first-line IL-2 therapy. No responses were observed.  They concluded that high dose IL-2 therapy following sunitinib or sorafenib treatment was associated with an unacceptably high incidence of severe cardiac toxicity preventing safe administration of a meaningful amount of IL-2 therapy. While further examination of the safety of this sequential approach is necessary, the assumption that IL-2 therapy can be delayed until after TKI failure may not be valid.  This work was selected for Presidential Session presentation at the 22nd meeting of the ISBTC Meeting.

Combination IL-2 and PEG IFN:  Dr. Atkins and colleagues within the Cytokine Working Group reported on the results of a phase I/II trial of PEG interferon-alpha-2b (PEG-IFN) with IL-2 in patients with metastatic RCC. (Clark et al J Immunother 2007) Patients received 1.0, 1.5, 2.0, or 3.0 mcg/kg/wk of PEG-IFN on days 1, 8, 15, and 22; subcutaneous IL-2 was given at a dose of 5 x 10 IU/m2 every 8 hours x 3 on day 1, followed daily at 5 x 10 IU/m2 days 2, 3, 4, and 5 of week 1, then 5 times per week for 3 weeks, followed by 2 weeks off. The MTD of PEG-IFN was 2.0mcg/kg/wk.  Fifty-four patients were enrolled. Frequent grade III/IV cardiac and neurologic toxicities led to an expanded phase I trial.  Eleven serious events in 33 patients in the phase II portion led to early termination. The overall response rate in 53 evaluable patients was 30.2% (95% confidence interval 20.5-39.9), with 2 complete responses and 14 partial responses and at least 1 response at each dose level. The median duration of response was 11 months (range 2 to 65+ mo); median survival was 20 months (range 2 to 71+ mo); median time to progression was 4 months. Despite clinical efficacy the degree of toxicity was felt to be unacceptable for an outpatient treatment regimen.

Clinical Effect of an IL-2 Selective Agonist. Dr. Atkins and colleagues within the CWG performed a Phase I trial to determine the MTD and safety of BAY 50-4798 an analog of interleukin 2 (IL-2) that selectively activates T cells over NK cells raising the potential for dissociating IL-2 toxicity from its antitumor effects (Margolin et al Clin Ca Res 2007). Two of 45 patients achieved confirmed partial responses including one of 21 with RCC treated at the MTD. Pharmacokinetic analysis demonstrated dose-dependent peak concentrations (CMAX) and area under the curve (AUC) with a half-life of approximately 2 hours and no evidence of drug accumulation.  Lymphocyte subset analysis confirmed the preferential expansion of Tcell subsets over NK cells.  Although the antitumor activity of BAY 50-4798 in patients with RCC was low relative to that of high dose IL-2, the possibility exists that BAY 50-4798 might provide advantages over aldesleukin in antigen-specific immunotherapeutic strategies. 

Impact of IL-2 on immunoregulatory subsets. Dr. Atkins and colleagues also investigated the effect of high-dose IL-2 administration on immunoregulatory cell subsets (Van Dervliet et al Clin Ca Res 2007). They 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.  They found that the frequency of both circulating myeloid DC1 and plasmacytoid DC 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. 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. They concluded 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.

4. Clinical investigation of targeted therapeutics in RCC
DF/HCC investigators have been actively involved in the clinical development of all of the new targeted therapies for patients with advanced RCC including studies that led to the FDA approval of sorafenib, sunitinib, and temsirolimus.  Particularly notable accomplishments are cited below

Sunitinib: Drs. George, Michaelson and colleagues within the DF/HCC Kidney Cancer Program participated in the initial limited institution phase II evaluations of sunitinib in patients with renal cancer (Protocol # 02-276 and Protocol #04-039).  These trials showed remarkable response rates of around 40% and median PFS in excess of 9 months in patients with cytokine refractory renal cancer. (Motzer et al JCO 2006, NEJM 2007).   These trials led directly to the phase III trial of sunitinib vs IFN that led to sunitinib’s approval in patients with advanced RCC.

Temsirolimus: Dr. Atkins and colleagues led the initial randomized phase II evaluation of temsirolimus in patients with cytokine refractory renal cancer (Atkins et al JCO 2004).  This trial established the activity of this agent in patients with RCC, apparent preferential effect in patients with clinical poor prognostic features, and the lack of dose dependency above 25ug. All of these findings were critical to the design of the phase III trial that led to the approval of temsirolimus in patients with advanced renal cancer. 

Bevacizumab: DF/HCC investigators were also the lead accruers to a Phase II trial randomizing patients to either the combination of bevacizumab and erlotinib or bevacizumab and placebo (Protocol #04-099) (McDermott (BIDMC-PI). (Bukowski JCO, 2007) This trial was the first randomized trial aimed at isolating the impact of inhibitors of different aspects of HIF inducible proteins in patients with renal cancer.  It established both the surprising single agent activity of bevacizumab as well as the lack of additional contribution from erlotonib in this disease.   
Axitinib: Dr. Michaelson and colleagues within the DF/HCC Kidney Cancer Program participated in a limited institution phase II evaluation of axitinib (AG-013736) a potent and selective inhibitor of VEGFR1-3 in patients with cytokine refractory RCC. (Rixe et al Lancet Oncology 2007) Treatment was well tolerated and responses were seen in 23 (2CR and 21 PR) of 52 (44%) patients.  Median response duration was 23, median time to progression was 15.7 months and median overall survival was 29.9 months.  This data suggests that axitinib is the most active single agent to date in RCC and merits further investigation in this disease.  Along these lines, the DF/HCC Kidney Cancer Program just opened a multi-institutional randomized phase III study of axitinib vs sorafenib in patients exhibiting disease progression on sunitinib with Dr. Michaelson serving as DF/HCC PI.
Sunitinib + gemcitabine: Dr. Michaelson led a phase I trial of the combination of gemcitabine and sunitinib in patients with advanced solid tumors.  Significant antitumor activity was seen in patients with sarcomatoid and undifferentiated RCC and in patients with poor clinical prognostic features.  The MTD was determined to be sunitinib 37.5 mg qd days 1-14 and gemcitabine 1000mg days 1 and 8 of a 21 day schedule.  An abstract detailing these results was submitted to ASCO 2008 and a manuscript is in preparation. This regimen is now being tested within DF/HCC in a phase II study involving patients with poor prognostic features.  In addition, it is being considered for study in ECOG in a randomized phase II comparison with gemcitabine and adriamycin in patients with sarcomatoid histology, and within CALGB in a Phase III trial in comparison with sunitinib alone in patients with poor prognostic features.   Dr. McDermott is slated to be the co-PI of the former cooperative group study and Dr. Michaelson is slated to be the PI of the latter trial.

5. Additional Investigator initiated studies in RCC
DF/HCC KCP investigators are leading additional Phase II investigator-initiated studies involving the AKT inhibitor perifosine (Dr. Cho), the mTor inhibitor RAD 001 (correlating clinical results with PET imaging changes and effects on tissue-based biomarkers) (Dr. Cho) and the combination of AMG386 and sunitinib (Dr. Atkins), temsirolimus and bevacizumab (Dr. Cho) and bevacizumab +/- sunitinib (Dr. Michaelson) These studies are either in progress or will soon open DF/HCC. DF/HCC KCP investigators are responsible for the overall trial design and management in most cases and the correlative science in all. 

6.  Translational Cores
The KCP and SPORE have worked hard to leverage the capabilities of its membership, Cores and the DF/HCC to facilitate research in RCC across the country.  To this end the program has established itself as translational cores for two large intergroup protocols E2805 looking at sorafenib, sunitinib or observation as adjuvant therapy for patients s/p resection of high-risk primary renal cancers and E2804, a randomized phase II trial examining doublet combinations of sorafenib, bevacizumab and temsirolimus and bevacizumab alone in patients with metastatic renal cancer.  The KCP Pathology Core, led by Dr. Signoretti at BWH will be responsible for pathology review, VHL typing from paraffin, and IHC staining for various proteins.   In addition the Monitoring Core led by Dr. Sukhatme from BIDMC will be responsible for circulating endothelial cell assays and measures of angiogenic cytokines using Luminex technology.  These clinical trials are now underway.  These cores are serving similar functions for Cytokine Working Group Trials examining the combination of HD IL-2 and bevacizumab and the high dose IL-2 “Select” trial that aims to prospectively validate the DF/HCC KCP predictive model for selecting patients for IL-2 therapy established in Project 4 of the current SPORE. 

References for DF/HCC Kidney Cancer Program Major Recent Accomplishments1.       Kondo K, Kim WY, Lechpammer M, Kaelin WG, Jr. (2003). Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol 1:E83


2.       Zimmer M, Doucette D, Siddiqui N, Iliopoulos O. Inhibition of hypoxia inducible factor suppresses growth of VHL-/- tumors.  Molecular Cancer Research, 2:89-95, 2004


3.       Nakamura E, Abreu-e-Lima P, Awakura Y, Inoue T, Kamoto T, Ogawa O, Kotani H, Manabe T, Zhang GJ, Kondo K, Nose V, Kaelin WG Jr. Clusterin is a secreted marker for a hypoxia-inducible factor-independent function of the von Hippel-Lindau tumor suppressor protein. Am J Pathol. 2006 Feb;168(2):574-84.


4.       Yang, H., et al., pVHL acts as an Adapter to Promote the Inhibitory Phosphorylation of the NF-κB Agonist Card9 by CK2 Mol. Cell., In Press.


5.       Brugarolas JB, Vazquez F, Reddy A, Sellers WR, Kaelin WG. TSC2 regulates VEGF through mTOR-dependent and -independent pathways. Cancer Cell. 2003 Aug;4(2):147-58.


6.       Zhang H, Bajraszewski N, Wu E, Wang H, Moseman AP, Dabora SL, Griffin JD, Kwiatkowski DJ. PDGFRs are critical for PI3K/Akt activation and negatively regulated by mTOR. J Clin Invest. 2007 Mar;117(3):730-8. Epub 2007


7.       Kozlowski P, Roberts P, Dabora S, Franz D, Bissler J, Northrup H, Au KS, Lazarus R, Domanska-Pakiela D, Kotulska K, Jozwiak S, Kwiatkowski DJ. Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations. Hum Genet. 2007 May;121(3-4):389-400. Epub 2007 Feb


8.       Lee L, Sudentas P, Dabora SL Combination of a rapamycin analog (CCI-779) and interferon-gamma is more effective than single agents in treating a mouse model of tuberous sclerosis complex Genes Chromosomes Cancer. 2006 Oct;45(10):933-44.


9.       Seth P, Tang, Jian, Signoretti S, Sukhatme V: Role of LDH-A in Hereditary Leiomyomatosis and RCC. !5th SP0RE Investigators’ Workshop, Abst 193. 2007. 


10.   Han M, Rivera MN, Batten JM, Haber DA, Cin PD, Iafrate AJ. Wilms' tumor with an apparently balanced translocation t(X;18) resulting in deletion of the WTX gene. Genes Chromosomes Cancer. 2007


11.   Thomas RK, Baker AC, DeBiasi R, Feng W, LaFramboise T, Wang M, Lee JC, Nickoletti R, Hatton C, Goyette M, Girard L, Majmudar K, Ziaugra L, Wong K-K, Gabriel S, Beroukhim R, Peyton M, Barretina J, Dutt A, Emery C, Winckler W, Greulich H, Shah K, Sasaki H, Gazdar A, Minna J, Armstrong S, Mellinghoff I, Mertz K, Rubin MA, Moch H, Loda M, Catalona W, Fletcher J, Signoretti S, Kaye F, Anderson KC, Demetri G, Dummer R, Wagner S, Herlyn M, Sellers WR, Meyerson M and Garraway LA. High-throughput oncogene mutation profiling in human cancer specimens by mass-spectrometric genotyping. Nat Genet. 2007 Feb 11; Erratum in: Nat Genet. 2007 Apr;39(4):567.


12.   Jiang Z, Chu PG, Woda BA, Rock KL, Liu Q, Hsieh CC, Li C, Chen W, Duan HO, McDougal S, Wu CL. Analysis of RNA-binding protein IMP3 to predict metastasis and prognosis of renal-cell carcinoma: a retrospective study. Lancet Oncol. 2006 Jul;7(7):556-64.


13.   Han WK, Alinani A, Wu CL, Michaelson D, Loda M, McGovern FJ, Thadhani R, Bonventre JV. Human kidney injury molecule-1 is a tissue and urinary tumor marker of renal cell carcinoma. J Am Soc Nephrol 2005 Apr;16:1126-34.


14.   Lin F, Zhang PL, Yang XJ, Shi J, Blasick T, Han WK, Wang HL, Shen SS, Teh BT, Bonventre JV. Human kidney injury molecule-1 (hKIM-1): a useful immunohistochemical marker for diagnosing renal cell carcinoma and ovarian clear cell carcinoma. Am J Surg Pathol 2007 Mar;31(3):371-81


15.   15.Tuncali K, Morrison PR, Winalski CS, Carrino JA, Shankar S, Ready JE, vanSonnenberg E, Silverman SG. MRI-guided percutaneous cryotherapy for soft-tissue and bone metastases: initial experience. AJR Am J Roentgenol. 2007 Jul;189(1):232-9.


16.   16.Tuncali K, Morrison PR, Tatli S, Silverman SG.MRI-guided percutaneous cryoablation of renal tumors: use of external manual displacement of adjacent bowel loops. Eur J Radiol. 2006 Aug;59(2):198-202. Epub 2006 May 23.


17.   17. Matin SF, Ahrar K, Cadeddu JA, Gervais DA, McGovern FJ, Zagoria RA, Uzzo RG, Haaga J, Resnick MI, Kaouk J, Gill IS. Residual and recurrent disease following renal energy ablative therapy: a multi-institutional study J Urol. 2006 Nov;176(5):1973-7.


18.   18.McDermott, DF, Regan MM, Clark JI, Flaherty LE, Weiss GR, Logan TF, Kirkwood JM, Gordon MS, Sosman JA, Ersntoff MS, Tretter CPG, Urba WJ, Smith JW, Margolin KA, Mier JW, Gollob JA, Dutcher JP, Atkins MB.  Randomized phase III trial of high-dose Interleukin-2 versus subcutaneous Interleukin-2 and Interferon in patients with metastatic renal cell carcinoma. J Clin Oncol. 2005 Jan 1;23(1):133-41. Erratum in: J Clin Oncol. 2005 Apr 20;23(12):2877.


19.   Gallagher DC, Bhatt RS, Parikh SM, Patel P, Seery V, McDermott DF, Atkins MB, Sukhatme VP. Angiopoietin 2 is a Potential Mediator of High-Dose Interleukin 2 Induced Vascular Leak.  Clin Cancer Res. 2007 April; 13(7):2115-20.


20.   Schwarzberg T, Regan MM, Liu V, Mier JW, Cho D, Koon H, Bhatt RS, Bradley K, Seery V, Lee MY, Atkins MB, and McDermott DF. Retrospective Analysis of Interleukin-2 Therapy in Patients with Metastatic Renal Cell Carcinoma Who had Received Prior Antiangiogenic Therapy ISBTC Annual Meeting.  JIT 2007 Presidential Award Nominee


21.   Margolin K, Atkins MB, Dutcher JP, Ernstoff MS, Smith JW 2nd, Clark JI, Baar J, Sosman J, Weber J, Lathia C, Brunetti J, Cihon F, Schwartz B. Phase I trial of BAY 50-4798, an interleukin-2-specific agonist in advanced melanoma and renal cancer. Clin Cancer Res. 2007 Jun 1;13(11):3312-9


22.   van Dervliet HJ, Koon HB, Yue SC, Uzunparmak B, Seery V, Gavin MA, Rudensky AY, Atkins MB, Balk SP, Exley MA  Effects of the administration of high-dose interleukin-2 on immunoregulatory cell subsets in patients with advanced melanoma and renal cell cancer. Clin Cancer Res. 2007 Apr 1;13(7):2100-8.


23.   Motzer RJ, Michaelson MD, Redman BG, Hudes GR, Wilding G, Figlin RA, Ginsberg MS, Kim ST, Baum CM, DePrimo SE, Li JZ, Bello CL, Theuer CP, George DJ, Rini BI. Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol. 2006 Jan 1;24(1):16-24. Epub 2005 Dec 5.


24.   Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR, Park Y, Liou S-H, Marshall B, Boni JP, Dukart G, Sherman ML. Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol, 2(5):909-918, 2004


25.   Motzer RJ, Hutson TE, Tomczak P, Michaelson MDMGH, Bukowski RM, Rixe O, Oudard S, Negrier S, Szczylik C, Kim ST, Chen I, Bycott PW, Baum CM, Figlin RA. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 2007 Jan 11;356(2):115-24.


26.   Clark JI, Mehrabi J, Sosman JA, Logan TF, Margolin KA, Dutcher JP, Urba WJ, Ernstoff MS, McDermott DF, Lau AM, Atkins MB. Phase I/II Trial of Outpatient PEG-Interferon with Interleukin-2 in Advanced Renal Cell Carcinoma:  A Cytokine Working Group Study. J Immunother. 2007; 30(8):839-846.


27.   Bukowski RM, Kabbinavar FF, Figlin RA, Flaherty K, Srinivas S, Vaishampayan U, Drabkin HA, Dutcher J, Ryba S, Xia Q, Scappaticci FA, McDermott D. Randomized Phase II Study of Erlotinib Combined With Bevacizumab Compared With Bevacizumab Alone in Metastatic Renal Cell Cancer. J Clin Oncol. 2007 Oct 10;25(29):4536-41. Epub 2007 Sep 17. PMID: 17876014


28.   Deprimo SE, Bello CL, Smeraglia J, Baum CM, Spinella D, Rini BI, Michaelson MDMGH, Motzer RJ. Circulating protein biomarkers of pharmacodynamic activity of sunitinib in patients with metastatic renal cell carcinoma: modulation of VEGF and VEGF-related proteins. J Transl Med 2007 5 :32


29.   Young AP, Kaelin WG Jr. Senescence triggered by the loss of the VHL tumor suppressor. Cell Cycle. 2008 Apr 16;7(12)


30.   Young AP, Schlisio S, Minamishima YA, Zhang Q, Li L, Grisanzio C, Signoretti S, Kaelin WG Jr. VHL loss actuates a HIF-independent senescence programme mediated by Rb and p400. Nat Cell Biol. 2008 Mar;10(3):361-9.


31.   Jiang Z, Lohse CM, Chu PG, Wu CL, Woda BA, Rock KL, Kwon ED. Oncofetal protein IMP3: a novel molecular marker that predicts metastasis of papillary and chromophobe renal cell carcinomas. Cancer. 2008 Jun 15;112(12):


32.   Zhang Z, Humphreys BD, Bonventre JVBWH. Shedding of the urinary biomarker kidney injury molecule-1 (KIM-1) is regulated by MAP kinases and juxtamembrane region. J Am Soc Nephrol 2007 Oct 18 (10):2704-14


33.   Messina MP, Rauktys A, Lee L, Dabora SLBWH. Tuberous sclerosis preclinical studies: timing of treatment, combination of a rapamycin analog (CCI-779) and interferon-gamma, and comparison of rapamycin to CCI-779. BMC Pharmacol 2007 7 :14


34.   Rixe O, Bukowski RM, Michaelson MDMGH, Wilding G, Hudes GR, Bolte O, Motzer RJ, Bycott P, Liau KF, Freddo J, Trask PC, Kim S, Rini BI. Axitinib treatment in patients with cytokine-refractory metastatic renal-cell cancer: a phase II study. Lancet Oncol 2007 (11):975-84.