Search

Purpose There is an unmet need for biomarkers for identifying patients likely to benefit from anticancer treatments, selecting dose, and understanding mechanisms of resistance. Plasma vascular endothelial growth factor (VEGF) and soluble VEGF receptor 2 (sVEGFR-2) are known to be modulated by VEGF pathway inhibitors. It is unknown whether chemotherapy or VEGFR inhibitor/chemotherapy combinations induce changes in these or other cytokines and angiogenic factors (CAFs) and whether such changes could be markers of benefit. Methods Thirty-five plasma CAFs were analyzed using multiplexed bead arrays and enzyme-linked immunosorbent assays from 123 patients with non–small-cell lung cancer in a randomized phase II study who received vandetanib, a VEGFR and epidermal growth factor receptor inhibitor, monotherapy carboplatin and paclitaxel (CP), or the combination (VCP). Changes in CAFs at days 8, 22, and 43 from baseline were correlated with progression risk. Results VEGF increased and sVEGFR-2 decreased by day 43 in the vandetanib arm, whereas a distinct pattern was observed in the CP and VCP arms, with significant decreases in interleukin (IL) -12, IL-1 receptor antagonist, and matrix metalloproteinase 9 (MMP-9) and increased macrophage chemoattractant protein 1. In each treatment arm, changes in different markers were associated with progression risk. For example, increases in IL-8 with VCP, MMP-9 with CP, and VEGF with vandetanib monotherapy were associated with increased progression risk, and increase in intercellular adhesion molecule 1 with vandetanib was associated with decreased risk. Conclusion Vandetanib and chemotherapy treatment led to distinct patterns of CAF changes; the combination resembled chemotherapy alone. Changes in specific CAFs correlated with clinical outcome, but markers differed for each treatment arm. CAF profiling may provide insights into the biologic effects of treatment and identify drug-specific markers of activity and clinical benefit.

Background: We investigated cytokines and angiogenic factors (CAFs) in patients with metastatic renal cell carcinoma (mRCC) treated in a randomized phase II clinical trial of sorafenib versus sorafenib+ interferon-a (IFN-a) that yielded no differences in progression-free survival (PFS). We aimed to link the CAF profile to PFS and select candidate predictive and prognostic markers for further study. Methods: The concentrations of 52 plasma CAFs were measured pretreatment (n = 69), day 28, and day 56 using multiplex bead arrays and enzyme-linked immunosorbent assay. We investigated the association between baseline levels of CAFs with PFS and posttreatment changes. Results: Unsupervised CAF clustering analysis revealed two distinct mRCC patient groups with elevated proangiogenic or proinflammatory mediators. A six-marker baseline CAF signature [osteopontin, vascular endothelial growth factor (VEGF), carbonic anhydrase 9, collagen IV, VEGF receptor-2, and tumor necrosis factor-related apoptosis-inducing ligand] correlated with PFS benefit (hazard ratio 0.20 versus 2.25, signature negative versus positive, respectively; P = 0.0002). While changes in angiogenic factors were frequently attenuated by the sorafenib+ IFN combination, most key immunomodulatory mediators increased. Conclusions: Using CAF profiling, we identified two mRCC patient groups, a candidate plasma signature for predicting PFS benefit, and distinct marker changes occurring with each treatment. This platform may provide valuable insights into renal cell carcinoma biology and the molecular consequences of targeted therapies.

CD26 is an antigen with key role in T-cell biology and is expressed on selected subsets of aggressive T-cell malignancies. To elucidate the role of CD26 in tumor behavior, we examine the effect of CD26 depletion by small interfering RNA transfection of T-anaplastic large cell lymphoma Karpas 299. We show that the resultant CD26-depleted clones lose the ability to adhere to fibronectin and collagen I. Because anti–integrin β1 blocking antibodies also prevent binding of Karpas 299 to fibronectin and collagen I, we then evaluate the CD26-integrin β1 association. CD26 depletion does not decrease integrin β1 expression but leads to dephosphorylation of both integrin β1 and p38 mitogen-activated protein kinase (MAPK). Moreover, our data showing that the p38MAPK inhibitor SB203580 dephosphorylates integrin β1 and that binding of the anti-CD26 antibody 202.36 dephosphorylates both p38MAPK and integrin β1 on Karpas 299, leading to loss of cell adhesion to the extracellular matrix, indicate that CD26 mediates cell adhesion through p38MAPK-dependent phosphorylation of integrin β1. Finally, in vivo experiments show that depletion of CD26 is associated with loss of tumorigenicity and greater survival. Our findings hence suggest that CD26 plays an important role in tumor development and may be a novel therapeutic target for selected neoplasms.

CD26 is a Mr 110,000 surface-bound glycoprotein with diverse functional properties, including having a key role in normal T-cell physiology and the development of certain cancers. In this article, we show that surface expression of CD26, especially its intrinsic dipeptidyl peptidase IV (DPPIV) enzyme activity, results in enhanced topoisomerase IIα level in the B-cell line Jiyoye and subsequent in vitro sensitivity to doxorubicin-induced apoptosis. In addition, we show that expression of CD26/DPPIV is associated with increased phosphorylation of p38 and its upstream regulators mitogen-activated protein kinase kinase 3/6 and apoptosis signal-regulating kinase 1 and that p38 signaling pathway plays a role in the regulation of topoisomerase IIα expression. Besides demonstrating that CD26 effect on topoisomerase IIα and doxorubicin sensitivity is applicable to cell lines of both B-cell and T-cell lineages, the potential clinical implication of our work lies with the fact that we now show for the first time that our in vitro results can be extended to a severe combined immunodeficient mouse model. Our findings that CD26 expression can be an in vivo marker of tumor sensitivity to doxorubicin treatment may lead to future treatment strategies targeting CD26/DPPIV for selected human cancers in the clinical setting. Our article thus characterizes the biochemical linkage among CD26, p38, and topoisomerase IIα while providing evidence that CD26-associated topoisomerase IIα expression results in greater in vitro and in vivo tumor sensitivity to the antineoplastic agent doxorubicin.

Lactoferrin has been identified as a factor in human colostrum that accounts for increased incorporation of thymidine into the DNA in an in vitro rat crypt enterocyte bioassay. We have examined lacatoferrin-stimulated thymidine incorporation by comparing the effects of ironfree lactoferrin (apolactoferrin) with those of iron-saturated lactoferrin (diferric lactoferrin) under conditions that inhibit the transfer of iron between these iron-binding proteins in the bioassay system. In addition, we have compared the dose-response relationships of diferric lactoferrin and apolactoferrin. The results demonstrated that lactoferrin, independent of iron-binding states, promoted the incorporation of thymidine into the DNA of rat crypt enterocytes. These observations suggest a previously unreported nutritional role for lactoferrin that is independent of its iron-binding capacity.

e15075 Background: Although recent years have brought great progress into understanding the pathogenesis of RCC and multiple promising biomarkers, no tumor tissue or blood-based test has yet been established to assist in the clinical determination of prognosis. Whether CAFs can be used to classify RCC pts and the set of CAFs that is host vs. tumor-dependent remain unclear. The main objectives of this exploratory analysis were to identify CAFs predictive of recurrence, and to gain insight into coordinated regulation and tumor dependency of individual factors by comparing CAFs pre- and post-nephrectomy (Nx) in RCC pts. Methods: Plasma samples were obtained before and up to 1 year after Nx from 52 pts with sporadic, non-metastatic RCC with dominant clear cell histology. Pts were categorized based on pathologic staging (pT1/pT2 as low-risk [LR] for recurrence, n=32; pT3/pT4 as high-risk [HR], n=20). Multiplex bead suspension arrays and ELISA were used to measure concentrations of 58 CAFs including angiogenesis and invasion mediators (VEGF, VEGF-C, soluble CAIX [sCA9], HGF and TGF-b1, among others), and multiple inflammatory and/or immunomodulatory chemokines and interleukins (IL). t-test and Wilcoxon signed rank or rank-sum tests were respectively used to assess changes in CAFs between pre- and post-Nx or to compare CAFs between subgroups of pts. Results: The concentrations of IL-8 and sCA9, among others, were significantly higher in HR vs. LR RCC pts pre-Nx (P

383 Background: Although recent years have brought great progress into understanding the pathogenesis of RCC and multiple promising biomarkers, no tumor tissue or blood-based test has yet been validated for clinical use. The main objectives of this exploratory analysis were to identify CAFs predictive of recurrence in RCC pts, and to gain insight into tumor dependency of individual factors by comparing CAFs pre- and post-nephrectomy (Nx). Methods: Plasma samples were obtained before and up to 1 year after Nx from pts with sporadic, non-metastatic renal masses suspicious for RCC (n=73). Upon Nx, 52 pts showed dominant clear cell histology and 11 non-clear cell; 10 pts had non-cancerous lesions. RCC pts were categorized by risk of relapse based on pathologic staging (pT1/pT2 as low-risk [LR], n=38; pT3/pT4 as high-risk [HR], n=25). Multiplex bead suspension arrays and ELISA were used to measure concentrations of 58 CAFs including angiogenesis and invasion mediators (VEGF, VEGF-C, soluble CAIX [sCA9], hepatocyte growth factor and transforming growth factor-b1, among others), and multiple inflammatory and/or immunomodulatory chemokines and interleukins (IL). t-test and Wilcoxon signed rank or rank-sum tests were respectively used to assess changes in CAFs between pre- and post-Nx or to compare CAFs between subgroups of pts. Results: Of 58 pre-Nx CAFs available in RCC pts, the concentrations of IL-8, granulocyte-colony stimulating factor, stromal cell-derived factor-1, and osteopontin, among others, were significantly higher in HR vs. LR-RCC (all P Conclusions: We identified pre-Nx CAFs associated with risk for recurrence, and post-Nx changes related to the presence of the tumor in pts with non-metastatic RCC. If validated in a larger study, these CAFs could be used for risk stratification of RCC pts.

362 Background: Cediranib is a highly potent and selective signaling inhibitor of vascular endothelial growth factor (VEGF) receptors−1, −2, −3 and c-Kit. In a phase II trial, patients (pts) with mRCC were randomized 3:1 to treatment with cediranib or placebo to primarily assess the difference in change in tumor size at 12 weeks. Cediranib resulted in a significant reduction in tumor size vs. placebo (Mulders et al. ESMO. 2009). Here we investigated soluble biomarkers in serum and concentration changes during treatment. Methods: Pts received cediranib 45 mg/day PO or placebo for the first 12 weeks on treatment. At that time (or upon progression if earlier), treatment was unblinded and pts on placebo were given the option of receiving cediranib. Serum was collected from 61 pts at baseline (BL; cediranib 46, placebo 15), from 50 pts on day (D) 28, and from 45 pts on D84. Multiplex bead suspension arrays and ELISA were used to measure CAF concentrations including VEGF, placental growth factor (PlGF), sVEGFR-2, PDGFbb, HGF, MMP-9, multiple chemokines and interleukins (IL). The objectives of this exploratory analysis were to assess whether baseline CAFs were associated with changes in tumor size after 12 weeks of treatment and whether CAF levels changed while on treatment. Results: Of 39 CAFs available at BL, lower than median (‘low') concentrations of IL-10, VEGF, PlGF, stem cell factor (SCF), and monokine-induced by interferon-gamma (MIG) were associated with larger decreases in tumor size than high concentrations (independent of treatment arm), whereas the opposite trend was observed for IL-5 and TRAIL. Consistent with inhibition of VEGF signaling, cediranib treatment resulted in changes over time in VEGF (increase), sVEGFR-2 (decrease) and PlGF (increase) concentrations. In addition, SCF, a c-Kit ligand and M-CSF decreased, while VCAM-1 and TRAIL increased during cediranib treatment. Conclusions: Candidate CAFs associated with response to cediranib in mRCC such as IL-10 and VEGF were identified. Patients treated with cediranib showed distinct CAF changes compared with placebo. These results require independent validation in a larger trial. [Table: see text]

5114 Background: SR, an oral inhibitor of Raf kinase, RET-receptor (R), VEGFR-1, 2, and 3, PDGFR-β, FLT-3, and c-KIT tyrosine kinases, has been shown to prolong progression-free survival (PFS) in mRCC after cytokine failure. In a phase II trial, mRCC pts were randomized to first line treatment with SR vs. SR plus interferon (IFN) to assess whether combination therapy improved PFS. Both treatment arms showed similar outcomes (Tannir et al, ASCO. 2008). Here we investigated predictive and prognostic biomarkers in plasma. Methods: Pts received SR 400 mg PO BID or same dose SR plus IFN 0.5 MU SC BID. Plasma was collected from 69 pts at baseline (BL; SR 34, SR+IFN 35), 59 on day (D) 28, and 57 pts on D56. We used multiplex bead suspension arrays to measure concentrations of 54 CAFs, including VEGF, PDGFbb, EGF, HGF, MMP-9, and multiple chemokines and interleukins (IL). Osteopontin (OPN), soluble carbonic anhydrase 9 (sCA9), placental growth factor (PlGF), collagen type IV (ColIV) and sVEGFR-2 were measured by ELISA. The primary objective of this analysis was to establish a CAF signature based on a set of individual markers at BL with a significant and differential impact on the association between treatment arm and PFS. Results: Among 52 CAFs available at BL, higher than median EGF concentrations associated with poor outcome independently of treatment arm, whereas low IL-2 had the opposite effect (p = 0.003 for both). Only OPN showed a significant treatment by factor interaction at BL (p < 0.01), suggesting that OPN has a differential effect on PFS in the two arms. Pts with high OPN benefitted more from single agent SR (7.74 vs. 3.93 mos for the combination; p = 0.007), but no differences were found for those with low OPN. Lower than median on-treatment increases in sCA9 (D28, p = 0.01) and GRO-alpha (D56, p = 0.04) on SR only were also associated with a better outcome. A 6-marker CAF signature at BL containing OPN, sCA9, VEGF, sVEGFR-2, ColIV, and TRAIL demonstrated predictive value on PFS. Conclusions: A CAF signature showed potential value in predicting differential benefit from single agent SR vs. SR+IFN in mRCC. Broad-based screening of circulating CAFs may identify predictive and prognostic biomarkers in the context of clinical trials. [Table: see text]

14687 Background: Recent studies suggest that specific subpopulations of monocytes and myeloid progenitors, particularly those expressing VEGFR-1, play a key role in promoting tumor angiogenesis an...

In a search for dietary factors that might stimulate enterocyte proliferation, we developed an assay for thymidine incorporation into DNA using harvested crypt cells from mature rat small intestine. Human colostrum stimulated a significant increase in thymidine incorporation into rat crypt cell DNA during a 60-min period of incubation. When the protein with biological activity was purified to a single peak by sequential ion exchange and gel filtration chromatography, it was found to have the characteristics of lactoferrin. The protein was identical to lactoferrin standards by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and doublediffusion immunologic precipitation. All available human lactoferrins stimulated thymidine uptake and all reacted with a lactoferrin polyclonal antibody. Human lactoferrin appears to be a potent activator of thymidine incorporation into DNA in incubated rat crypt cells, a nutritional function not previously reported.

Lactoferrin has been identified as the factor in human colostrum that accounts for the increased incorporation of thymidine into DNA in a rat crypt enterocyte bioassay. Commercially available infant formulas used in the refeeding of infants who have severe dietary intolerance associated with mucosal atrophy were tested in this bioassay. In contrast to human milk, no stimulation was observed with these formulas in the assay. All formulas inhibited basal thymidine incorporation. The degree of inhibition of the basal assay progressed from 14% with cow's-milk formula to 30% with soy-based formulas to 45% with hydrolyzed casein formulas. When the formulas were supplemented with lactoferrin to match the level of that in human milk, the result was a 35% relative increase in thymidine incorporation into DNA. The quantitative level of response was primarily influenced by the baseline inhibitions associated with the specific formulas. The most profound effect was observed with the hydrolyzed casein formula, when lactoferrin supplementation reversed the baseline inhibition but did not result in a response greater than that seen in control basal bioassays. The supplementation of infant formulas with lactoferrin improved the thymidine incorporation in rat crypt enterocytes; a response equivalent to that of human milk, however, would require a redesign of other formula components to reduce basal inhibition.