F-18 labelled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients.

PURPOSE: The prostate-specific membrane antigen (PSMA) targeted positron-emitting-tomography (PET) tracer 68Ga-PSMA-11 shows great promise in the detection of prostate cancer. However, 68Ga has several shortcomings as a radiolabel including short half-life and non-ideal energies, and this has motivated consideration of 18F-labelled analogs. 18F-PSMA-1007 was selected among several 18F-PSMA-ligand candidate compounds because it demonstrated high labelling yields, outstanding tumor uptake and fast, non-urinary background clearance. Here, we describe the properties of 18F-PSMA-1007 in human volunteers and patients. METHODS: Radiation dosimetry of 18F-PSMA-1007 was determined in three healthy volunteers who underwent whole-body PET-scans and concomitant blood and urine sampling. Following this, ten patients with high-risk prostate cancer underwent 18F-PSMA-1007 PET/CT (1 h and 3 h p.i.) and normal organ biodistribution and tumor uptakes were examined. Eight patients underwent prostatectomy with extended pelvic lymphadenectomy. Uptake in intra-prostatic lesions and lymph node metastases were correlated with final histopathology, including PSMA immunostaining. RESULTS: With an effective dose of approximately 4.4-5.5 mSv per 200-250 MBq examination, 18F-PSMA-1007 behaves similar to other PSMA-PET agents as well as to other 18F-labelled PET-tracers. In comparison to other PSMA-targeting PET-tracers, 18F-PSMA-1007 has reduced urinary clearance enabling excellent assessment of the prostate. Similar to 18F-DCFPyL and with slightly slower clearance kinetics than PSMA-11, favorable tumor-to-background ratios are observed 2-3 h after injection. In eight patients, diagnostic findings were successfully validated by histopathology. 18F-PSMA-1007 PET/CT detected 18 of 19 lymph node metastases in the pelvis, including nodes as small as 1 mm in diameter. CONCLUSION: 18F-PSMA-1007 performs at least comparably to 68Ga-PSMA-11, but its longer half-life combined with its superior energy characteristics and non-urinary excretion overcomes some practical limitations of 68Ga-labelled PSMA-targeted tracers.

[The impact of the androgen receptor splice variant AR-V7 on the prognosis and treatment of advanced prostate cancer]. / Bedeutung der Androgenrezeptor-Spleißvariante AR-V7 für Prognose und Therapie des fortgeschrittenen Prostatakarzinoms.

A recently discovered mechanism enabling prostate cancer cells to escape the effects of endocrine therapies consists in the synthesis of C-terminally truncated, constitutively active androgen receptor (AR) splice variants (AR-V). Devoid of a functional C-terminal hormone/ligand binding domain, various AR-Vs are insensitive to therapies targeting the androgen/AR signalling axis. Preliminary studies suggest that AR-V7, the most common AR-V, is a promising predictive tumour marker and a relevant selection marker for the treatment of advanced prostate cancer. This review critically outlines recent advances in AR-V7 diagnostics and presents an overview of current AR-V7 targeted therapies.

RNA polymerase II transcription is required for human papillomavirus type 16 E7- and hydroxyurea-induced centriole overduplication.

Aberrant centrosome numbers are detected in virtually all human cancers where they can contribute to chromosomal instability by promoting mitotic spindle abnormalities. Despite their widespread occurrence, the molecular mechanisms that underlie centrosome amplification are only beginning to emerge. Here, we present evidence for a novel regulatory circuit involved in centrosome overduplication that centers on RNA polymerase II (pol II). We found that human papillomavirus type 16 E7 (HPV-16 E7)- and hydroxyurea (HU)-induced centriole overduplication are abrogated by alpha-amanitin, a potent and specific RNA pol II inhibitor. In contrast, normal centriole duplication proceeded undisturbed in alpha-amanitin-treated cells. Centriole overduplication was significantly reduced by siRNA-mediated knock down of CREB-binding protein (CBP), a transcriptional co-activator. We identified cyclin A2 as a key transcriptional target of RNA pol II during HU-induced centriole overduplication. Collectively, our results show that ongoing RNA pol II transcription is required for centriole overduplication whereas it may be dispensable for normal centriole duplication. Given that many chemotherapeutic agents function through inhibition of transcription, our results may help to develop strategies to target centrosome-mediated chromosomal instability for cancer therapy and prevention.

Centriole overduplication through the concurrent formation of multiple daughter centrioles at single maternal templates.

Abnormal centrosome numbers are detected in virtually all cancers. The molecular mechanisms that underlie centrosome amplification, however, are poorly characterized. Based on the model that each maternal centriole serves as a template for the formation of one and only one daughter centriole per cell division cycle, the prevailing view is that centriole overduplication arises from successive rounds of centriole reproduction. Here, we provide evidence that a single maternal centriole can concurrently generate multiple daughter centrioles. This mechanism was initially identified in cells treated with the peptide vinyl sulfone proteasome inhibitor Z-L(3)VS. We subsequently found that the formation of more than one daughter at maternal centrioles requires cyclin E/cyclin-dependent kinase 2 as well as Polo-like kinase 4 and that overexpression of these proteins mimics this phenotype in the absence of a proteasome inhibitor. Moreover, we show that the human papillomavirus type 16 E7 oncoprotein stimulates aberrant daughter centriole numbers in part through the formation of more than one daughter centriole at single maternal templates. These results help to explain how oncogenic stimuli can rapidly induce abnormal centriole numbers within a single cell-division cycle and provide insights into the regulation of centriole duplication.

Cyclin-dependent kinase 2 is dispensable for normal centrosome duplication but required for oncogene-induced centrosome overduplication.

Cyclin-dependent kinase 2 (CDK2) has been proposed to function as a master regulator of centrosome duplication. Using mouse embryonic fibroblasts (MEFs) in which Cdk2 has been genetically deleted, we show here that CDK2 is not required for normal centrosome duplication, maturation and bipolar mitotic spindle formation. In contrast, Cdk2 deficiency completely abrogates aberrant centrosome duplication induced by a viral oncogene. Mechanistically, centrosome overduplication in MEFs wild-type for Cdk2 involves the formation of supernumerary immature centrosomes. These results indicate that normal and abnormal centrosome duplication have significantly different requirements for CDK2 activity and point to a role of CDK2 in licensing centrosomes for aberrant duplication. Furthermore, our findings suggest that CDK2 may be a suitable therapeutic target to inhibit centrosome-mediated chromosomal instability in tumor cells.

[Level IV inferior vena cava tumor thrombus : A rare diagnosis in patients with renal cell carcinoma]. / Level-IV-Cavathrombus : Eine seltene Diagnose bei Patienten mit Nierenzellkarzinom.

Renal cell carcinoma in combination with a supradiaphragmatic tumor thrombus is a rare tumor entity. Radical surgery including nephrectomy and thrombectomy is still considered standard treatment. The extent of the tumor thrombus should be preoperatively evaluated by MRI and TEE. An interdisciplinary team is important for surgery planning and realization. Despite the known risks of an operation, a longer overall survival is achieved.

Ecotropic viral integration site 1, a novel oncogene in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men in the western world. Mutations in tumor suppressor genes and in oncogenes are important for PCa progression, whereas the role of stem cell proteins in prostate carcinogenesis is insufficiently examined. This study investigates the role of the transcriptional regulator Ecotropic Viral Integration site 1 (EVI1), known as an essential modulator of hematopoietic and leukemic stem cell biology, in prostate carcinogenesis. We show that in healthy prostatic tissue, EVI1 expression is confined to the prostate stem cell compartment located at the basal layer, as identified by the stem cell marker CD44. Instead, in a PCa progression cohort comprising 219 samples from patients with primary PCa, lymph node and distant metastases, EVI1 protein was heterogeneously distributed within samples and high expression is associated with tumor progression (P<0.001), suggesting EVI1 induction as a driver event. Functionally, short hairpin RNA-mediated knockdown of EVI1 inhibited proliferation, cell cycle progression, migratory capacity and anchorage-independent growth of human PCa cells, while enhancing their apoptosis sensitivity. Interestingly, modulation of EVI1 expression also strongly regulated stem cell properties (including expression of the stem cell marker SOX2) and in vivo tumor initiation capacity. Further emphasizing a functional correlation between EVI1 induction and tumor progression, upregulation of EVI1 expression was noted in experimentally derived docetaxel-resistant PCa cells. Importantly, knockdown of EVI1 in these cells restored sensitivity to docetaxel, in part by downregulating anti-apoptotic BCL2. Together, these data indicate EVI1 as a novel molecular regulator of PCa progression and therapy resistance that may control prostate carcinogenesis at the stem cell level.

Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma.

BACKGROUND: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. PURPOSE: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. METHODS: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. RESULTS: Tyrosinase mRNA was detected by RT-PCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P < or = .05). CONCLUSIONS: The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

Human papillomavirus type 16 E7 oncoprotein-induced abnormal centrosome synthesis is an early event in the evolving malignant phenotype.

Genomic instability is a hallmark of malignant growth that frequently involves mitotic defects associated with centrosome abnormalities. However, the question of whether abnormal centrosomes cause genomic instability or develop secondary to other changes has not been conclusively resolved. Here we show that human papillomavirus (HPV)-16 E7 can induce abnormal centrosome synthesis before the development of extensive nuclear abnormalities. In contrast, expression of HPV-16 E6 is associated with marked nuclear atypia and concomitant accumulation of centrosomes. Our results demonstrate that HPV-16 E7-induced centrosome abnormalities represent an early event during neoplastic progression potentially driving genomic destabilization.

Biological activities and molecular targets of the human papillomavirus E7 oncoprotein.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPV-associated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular differentiation and proliferation in normal epithelium, thus allowing the virus to replicate in differentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely reflect this important function.

Centrosome abnormalities, genomic instability and carcinogenic progression.

Centrosome abnormalities are a frequent finding in various malignant tumors. Since centrosomes form the poles of the mitotic spindle, these abnormalities have been implicated in chromosome missegregation and the generation of aneuploid cells which is commonly found in many human neoplasms. It is a matter of debate, however, whether centrosome alterations can drive cells into aneuploidy or simply reflect loss of genomic integrity by other mechanisms. Since these two models have fundamentally different implications for the diagnostic and prognostic value of centrosome abnormalities, we will discuss the relevance of abnormal centrosomes in the context of different oncogenic events as exemplified by high-risk human papillomavirus-associated carcinogenesis.

[Metastasized prostate cancer. Position paper on the use of chemotherapy]. / Metastasiertes Prostatakarzinom. Positionspapier zum Einsatz der Chemotherapie.

BACKGROUND: Antihormonal and cytotoxic therapy options are available for the therapy of metastasized prostate cancer (mPC). Because no comparative studies are available, especially for castration-resistant prostate cancer (mCRCP), it remains unclear which patients will profit best from which therapy. OBJECTIVES: Previous data on the sequence of the various therapy options show that correct selection of the first line therapy for mCRPC can have an influence on the prognosis of the patient. In this position paper the various therapy options are critically illustrated and the clinical and pathohistological criteria for selection of the first line therapy of mCRPC are discussed. RESULTS: Molecular markers are an important aid for future patient selection and individualized therapy for optimal use of the available forms of therapy.

[Structure of biobanks for urological research]. / Struktur von Biobanken für die urologische Forschung.

Biomedical research plays an important role in the development of novel diagnostic procedures, drugs and treatment strategies with regard to cancerous and chronic inflammatory diseases. Biobanks are essential tools in this process. The complex structures and benefits of biobanks are presented in this article.

[Intratumoral heterogeneity in renal cell carcinoma. Molecular basis and translational implications]. / Intratumorale Heterogenität des Nierenzellkarzinoms. Molekulare Grundlagen und translationale Implikationen.

Advanced clear cell renal cell carcinoma is characterized by extensive intratumoral genomic heterogeneity and branched as well as convergent evolutionary traits with genomically different subclones evolving in parallel in the same tumor. Distinct driver mutations can be found in spatially separated subclones, which may hinder the development of novel targeted therapies. However, truncal mutations of the VHL tumor suppressor gene and chromosome 3p loss were ubiquitously detected and will hence continue to be a focus of future drug development. Nevertheless, genomic instability, enhanced tumor genome plasticity and intratumoral heterogeneity are likely to represent major challenges towards biomarker development and personalized patient care.

Chemoimmunotherapy of advanced malignant melanoma: sequential administration of subcutaneous interleukin-2 and interferon-alpha after intravenous dacarbazine and carboplatin or intravenous dacarbazine, cisplatin, carmustine and tamoxifen.

Both chemotherapy and interleukin-2 and/or interferon-alpha produce objective responses in a proportion of advanced malignant melanoma patients. While duration of response to chemotherapy is short, i.e. usually below 4 months, immunotherapy has resulted in a small number of long-lasting remissions in patients with metastatic melanoma. In two consecutive phase II trials in a total of 67 patients, we assessed the potential synergism between both modalities, i.e. chemo- and immunotherapy. Treatment consisted of intravenous (i.v.) carboplatin (CBDCA, 400 mg/m2) and dacarbazine (DTIC, 750 mg/m2) given twice (i.v. bolus over 30 min) at 3-week intervals, or 4 cycles of DTIC (220 mg/m2 i.v. 3 days), cisplatin (DDP, 35 mg/m2 i.v. 3 days), carmustine (BCNU, 150 mg/m2 i.v. cycles 1 and 3) and tamoxifen (TAM, 20 mg oral/daily) at 3-week intervals. Chemotherapy was followed by immunotherapy with combined subcutaneous (s.c.) interleukin-2 (rIL-2) and SC interferon-alpha 2 (rIFN-alpha). Among 40 patients who received a full cycle of chemotherapy with CBDCA/DTIC and sequential immunotherapy, there were 3 (7.5%) complete remissions (CRs) with a median duration of 19 months (range 13-26+). Partial remissions (PRs) were noted in 11 (27.5%) patients with a median response duration of 8 (range 5-14) months. Among 27 patients who received DTIC/DDP/BCNU/TAM and rIL-2/rIFN-alpha, there were 3 (11%) complete remissions and 12 (44.5%) partial remissions. Duration of complete and partial remissions ranged from 9+ to 13+ (median, 11+), and 5 to 15+ (median, 7+) months, respectively. Chemotherapy produced mostly moderate toxicity. Thrombocytopenia was common with the nadir after a median time of 18 days following start of CBDCA/DTIC and DTIC/DDP/BCNU, respectively. 10 patients required transfusion of thrombocytes. Nausea and vomiting due to chemotherapy were well tolerated using concomitant ondansetrone (8 mg i.v.). Immunotherapy was self-administered at home with mild to moderate side effects; malaise, fever, chills, nausea/vomiting, diarrhoea, anorexia and arthralgias were most frequent, but were spontaneously reversible after ending rIL-2/IFN-alpha. A mean 87 and 88% of the projected doses of rIL-2 and rIFN-alpha were administered on either protocol. There were no life-threatening complications and no treatment-related deaths. The sequential combination of chemotherapy and rIL-2 plus rIFN-alpha had at least additive therapeutic activity against metastatic malignant melanoma. The schedules produced long-lasting remissions and were tolerated well overall. These trials substantiate a potential role for low to intermediate dose immunotherapy in maintaining and consolidating therapeutic effects of chemotherapy in metastatic melanoma.

Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia.

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

Increased serum levels of basic fibroblast growth factor (bFGF) are associated with progressive lung metastases in advanced renal cell carcinoma patients.

Basic fibroblast growth factor (bFGF) has been detected in body fluids of patients with various malignancies including renal cancer. Cytoplasmatic expression of bFGF in primary renal cell carcinoma cells has been reported recently to correlate with an impaired patient survival. In the present study, we analysed the statistical association of spontaneous serum bFGF levels in 23 patients with advanced renal cell carcinoma and progressive metastasis in different organ sites. Increased bFGF serum levels (>90% percentile for healthy donors i.e., > 14 pg/ml) were found in eight patients (35%) with a mean of 24.1 pg/ml. All patients in this subgroup presented with progressive pulmonary metastases at the time of sample collection (p < or = 0.007). In a total of fifteen patients exhibiting progressive pulmonary metastasis, bFGF serum levels were found to be significantly higher when compared to patients lacking progressive lung lesions (p < or = 0.0006). Of fifteen patients with bFGF levels lower than 14 pg/ml, six showed bone metastases at the time of sample collection (p < or = 0.04). Our results suggest that increased serum bFGF levels may be associated with a higher frequency of progressive pulmonary metastases. Interactions between soluble angiogenic factors and components of the extracellular matrix or basement membranes in remote sites of metastasis will be subject to further experiments.

Altered expression of beta 1 integrins in renal carcinoma cell lines exposed to vinblastine.

BACKGROUND: Cellular expression of P-glycoprotein (P-gp) which mediates a well characterized mechanism of multidrug resistance (MDR) has been reported previously to be associated with an enhanced tumor dissemination. Since adhesion receptors of the beta 1 integrin family play a substantial role in tumor spread, we studied expression of VLA-1 to -6 in a total of four renal carcinoma cell (RCC) lines prior to and after induction of MDR via exposure to vinblastine. MATERIAL AND METHODS: Surface expression of P-gp and VLA-1 to -6 was determined immunocytochemically in untreated pre-established renal carcinoma cell lines (Caki-1, Caki-2, A498) and a cell line derived from a RCC patient who had received a vinblastine-containing therapy regimen prior to the resection of a local relapse of the tumor (EH). Resistant sublines were cultivated in the presence of 1 ng/ml and 10 ng/ml of vinblastine sulfate, respectively. RESULTS: In all cell lines examined, an increased number of P-gp expressing cells was observed upon exposure to vinblastine. Significant changes of beta 1 integrin expression were observed in three of four RCC cell lines. A de novo expression of VLA-1, VLA-2, and VLA-4 as detected by immunocytochemistry occurred in resistant Caki-1 cells. A498 cells showed an increasing number of VLA-2 positive cells in drug resistant sublines. In contrast, a decrease of VLA-2 and VLA-5 expression was found in EH cells, the only cell line exhibiting P-gp expression prior to vinblastine exposure. Caki-2 cells showed no significant changes of surface integrin expression upon treatment with vinblastine. CONCLUSIONS: Our results demonstrate that induction of drug resistance can be associated with substantial changes of the integrin phenotype in renal carcinoma cell lines. In our experiments, among all VLAs studied, VLA-2 was most frequently altered in expression by RCC cell lines. The significance of these observations for aberrant metastatic properties of multidrug resistant tumor cells will be the subject of further studies.

In vivo tumor necrosis factor-alpha as indicator of biologic and clinical response to low-dose SC recombinant interleukin 2.

The effect of low-dose human recombinant interleukin-2 (rIL-2) on the induction of secondary tumor necrosis factor-alpha (TNF-alpha) in vivo was studied in 16 patients with metastatic renal cell carcinoma. In all patients s.c. rIL-2 resulted in a significant increase in TNF-alpha serum levels within 4 to 8 hours, as determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha serum concentrations remained elevated up to 24 hours following single s.c. administration of rIL-2. Total secondary TNF-alpha release, as assessed by the area under the curve (AUC), appeared to be independent of dose distribution of rIL-2 (10 million IU rIL-2 q12 hours versus 20 million IU rIL-2 q24 hours). rIL-2 induced TNF-alpha release was significantly higher in patients who had received prior rIL-2 immunotherapy, while steroids resulted in a significant suppression of TNF-alpha release. Secondary TNF-alpha release was statistically associated with progression-free survival of renal cell carcinoma patients and may be a prognostic factor in patients receiving rIL-2.