Three articles in this issue of Clinical Cancer Research show how multiple vaccinations can lead to immunosuppression. Moreover, two studies in patients show that granulocyte macrophage colony-stimulating factor (GM-CSF) as an adjuvant immunostimulant to different kind of vaccines can lead to adverse outcome in terms of relapse-free and overall survival. Modulation of regulatory T-cell activity may be required to overcome this outcome and may be crucial for the successful development of therapeutic vaccines. (Clin Cancer Res 2009;15(22):6745–7)

There is high morbidity associated with local recurrence of rectal cancer. However, the adjuvant therapies given to prevent such recurrences also have significant side effects and associated risks. The ability to select patients with the highest risk of recurrence and greatest therapeutic response will improve rectal cancer care. (Clin Cancer Res 2009;15(22):6748–50)

Covalent and reversible post-translational modifications of proteins are a common theme in signaling. Ubiquitin conjugation was originally described to target proteins to proteasomal degradation by ubiquitin polymerization involving lysine (K) 48 residues. Differently linked polymers of polyubiquitin have been found that modify proteins without targeting to proteasomal degradation. Instead this pathway creates docking sites for signaling scaffolds that are key to control the nuclear factor-B (NF-B) pathway. Indeed TRAF-2, TRAF-6, and TRAF-3 are E3 ubiquitin ligases that form K63-linked ubiquitin polymers. Therefore signaling via TNF family receptors, IL1R, IL-18R, T-cell receptor (TCR), and Toll-like receptors (TLR) use this type of post-translational modification. Specific enzymes exist (DUBs) that deactivate this system, degrading K63 polyubiquitin chains. Interestingly, mice deficient in these deubiquitinases develop autoimmunity and inflammation. In carcinogenesis, the K63 polyubiquitin pathway is possibly critical for inflammation-driven tumor promotion. The pathway is also critically involved in costimulation of tumor immunity/immunotherapy as well as in the biology of malignant cells themselves. The elements of this new signaling paradigm offer the opportunity for therapeutic exploitation and drug discovery. (Clin Cancer Res 2009;15(22):6751–7)

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with an annual incidence of approximately 400,000 worldwide. Although the principal risk factors for head and neck cancer remain tobacco and alcohol use, human papillomavirus (HPV) has recently been found to be etiologically associated with 20 to 25% of HNSCC, mostly in the oropharynx. HPV causes human cancers by expressing two viral oncoproteins, E6 and E7. These oncoproteins degrade and destabilize two major tumor suppressor proteins, p53 and pRb, through ubiquitination. Additional studies have shown that E6 and E7 can directly bind to multiple host proteins other than p53 and pRb (e.g., Bak and p21^Cip1), further contributing to genetic instability. However, expression of E6 and E7 alone is not sufficient for cellular transformation, and the additional genetic alterations necessary for malignant progression in the setting of virus-induced genomic instability are unknown. In addition to the etiological differences, HPV-positive cancers are clinically distinct when compared with HPV-negative cancers with regard to treatment response and survival outcome, with tumor HPV-positivity being a favorable prognostic biomarker. Further understanding of carcinogenesis and clinical behavior of HPV-positive cancers will improve disease prevention, patient care, and surveillance strategies for HNSCC patients. (Clin Cancer Res 2009;15(22):6758–62)

Class 3 semaphorins (SEMA3) were first identified as glycoproteins that negatively mediate neuronal guidance by binding to neuropilin and repelling neurons away from the source of SEMA3. However, studies have shown that SEMA3s are also secreted by other cell types, including tumor cells, where they play an inhibitory role in tumor growth and angiogenesis (specifically SEMA3B and SEMA3F). SEMA3s primarily inhibit the cell motility and migration of tumor and endothelial cells by inducing collapse of the actin cytoskeleton via neuropilins and plexins. Besides binding to SEMA3s, neuropilin also binds the protumorigenic and proangiogenic ligand vascular endothelial growth factor (VEGF). Although some studies attribute the antitumorigenic and antiangiogenic properties of SEMA3s to competition between SEMA3s and VEGF for binding to neuropilin receptors, several others have shown that SEMA3s display growth-inhibitory activity independent of competition with VEGF. A better understanding of these molecular interactions and the role and signaling of SEMA3s in tumor biology will help determine whether SEMA3s represent potential therapeutic agents. Herein, we briefly review (a) the role of SEMA3s in mediating tumor growth, (b) the SEMA3 receptors neuropilins and plexins, and (c) the potential competition between SEMA3s and VEGF family members for neuropilin binding. (Clin Cancer Res 2009;15(22):6763–70)

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with Epstein-Barr virus and has poor prognosis because of local invasion and/or multiple dissemination. Recently, the role of chemokines/chemokine receptors in tumor proliferation and invasion has been shown. In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal NK/T-cell lymphoma.

Experimental Design: A chemokine protein array was used to examine specific chemokines produced by SNK-6 and SNT-8 (Epstein-Barr virus–positive nasal NK/T-cell lymphoma lines). The expression of interferon –inducible protein 10 (IP-10) and the IP-10 receptor CXCR3 was investigated by ELISA and flow cytometry. Cell growth and invasion were assessed by the MTT and Matrigel invasion assays, respectively. Immunohistologic staining and ELISA were used to examine IP-10 expression in biopsies and sera from patients, respectively.

Results: IP-10 was specifically produced by SNK-6 and SNT-8. Moreover, CXCR3 was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in the lymphoma cells. Serum IP-10 levels in the patients were much higher than those of healthy controls and the levels were decreased during the complete remission phase after treatments.

Conclusions: These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism. (Clin Cancer Res 2009;15(22):6771–9)

Purpose: Colon cancer is a major cause of cancer deaths. Dietary factors contribute substantially to the risk of this malignancy. Western-style diets promote development of azoxymethane-induced colon cancer. Although we showed that epidermal growth factor receptors (EGFR) controlled azoxymethane tumorigenesis in standard fat conditions, the role of EGFR in tumor promotion by high dietary fat has not been examined.

Experimental Design: A/J x C57BL6/J mice with wild-type Egfr (Egfr^wt) or loss-of-function waved-2 Egfr (Egfr^wa2) received azoxymethane followed by standard (5% fat) or western-style (20% fat) diet. As F₁ mice were resistant to azoxymethane, we treated mice with azoxymethane followed by one cycle of inflammation-inducing dextran sulfate sodium to induce tumorigenesis. Mice were sacrificed 12 weeks after dextran sulfate sodium. Tumors were graded for histology and assessed for EGFR ligands and proto-oncogenes by immunostaining, Western blotting, and real-time PCR.

Results: Egfr^wt mice gained significantly more weight and had exaggerated insulin resistance compared with Egfr^wa2 mice on high-fat diet. Dietary fat promoted tumor incidence (71.2% versus 36.7%; P < 0.05) and cancer incidence (43.9% versus 16.7%; P < 0.05) only in Egfr^wt mice. The lipid-rich diet also significantly increased tumor and cancer multiplicity only in Egfr^wt mice. In tumors, dietary fat and Egfr^wt upregulated transforming growth factor-, amphiregulin, CTNNB1, MYC, and CCND1, whereas PTGS2 was only increased in Egfr^wt mice and further upregulated by dietary fat. Notably, dietary fat increased transforming growth factor- in normal colon.

Conclusions: EGFR is required for dietary fat-induced weight gain and tumor promotion. EGFR-dependent increases in receptor ligands and PTGS2 likely drive diet-related tumor promotion. (Clin Cancer Res 2009;15(22):6780–9)

Purpose: Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown.

Experimental Design: All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry, and analyzed by array-based comparative genomic hybridization. Previously untreated tumors were subjected to global gene expression arrays.

Results: We analyzed 45 thymic tumors [thymoma, n = 38 (type A, n = 8; type B2, n = 22; type B3, n = 8); thymic carcinoma, n = 7]. One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, whereas the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A to B2 thymomas versus type B3 and thymic carcinomas. Moreover, array-based comparative genomic hybridization could readily distinguish squamous cell carcinomas of the thymus versus the lung, which can often present a diagnostic challenge.

Conclusions: Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathologic, and therapeutic implications for the treatment of thymic malignancies. (Clin Cancer Res 2009;15(22):6790–9)

Purpose: Recent studies suggest that children <24 months with stage I favorable histology Wilms tumors <550 g [very low risk Wilms tumors (VLRWT)] have an excellent prognosis when treated with nephrectomy only, without adjuvant chemotherapy. The identification of risk categories within VLRWT may enable refinement of their definition and optimization of their therapy.

Experimental Design: To define biologically distinct subsets, global gene expression analysis was done on 39 VLRWT that passed all quality-control parameters and the clusters identified were validated in an independent set of 11 VLRWT. Validation of select differentially expressed genes was done with immunohistochemistry on a tissue microarray from 20 of 39 tumors. Loss of heterozygosity (LOH) for 11p15, 1p, and 16q was analyzed in 52 tumors using PCR.

Results: Two distinctive clusters were identified. One cluster included 9 tumors with epithelial differentiated tubular histology, paucity of nephrogenic rests, lack of LOH for 1p, 16q, and 11p, absence of relapse, and a unique gene expression profile consistent with arrest following mesenchymal-to-epithelial transition. The second cluster included 13 tumors with mixed histology, intralobar nephrogenic rests, and decreased expression of WT1. Three of 6 relapses occurred in this cluster. Of 43 informative tumors, 11p LOH was present in 5 of 5 relapses and 11 of 38 nonrelapses.

Conclusions: Two subsets comprising a total of 56% of VLRWT are identified that have pathogenetic and molecular differences and apparent differences in risk for relapse. If these predictors can be prospectively validated, this would enable the refinement of clinical stratification and less arbitrary definition of VLRWT. (Clin Cancer Res 2009;15(22):6800–9)

Purpose: Glycogen synthase kinase-3β (GSK3β) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3β in gastrointestinal, pancreatic, and liver cancers.

Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3β expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3β activity by in vitro kinase assay. The effects of small-molecule GSK3β inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3β inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells.

Results: Cancer cell lines and most cancer tissues showed increased GSK3β expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3β resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3β inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity.

Conclusion: The results indicate that deregulated GSK3β sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. (Clin Cancer Res 2009;15(22):6810–9)

Purpose: As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition, invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non–small cell lung cancer (NSCLC).

Experimental Design: Immunohistochemistry was done to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis was used to correlate these findings with survival. NSCLC cell lines gene-modified to stably overexpress Snail were evaluated in vivo in two severe combined immunodeficiency murine tumor models. Differential gene expression between Snail-overexpressing and control cell lines was evaluated using gene expression microarray analysis.

Results: Snail is upregulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (P < 0.026). In a heterotopic model, mice bearing Snail-overexpressing tumors developed increased primary tumor burden (P = 0.008). In an orthotopic model, mice bearing Snail-overexpressing tumors also showed a trend toward increased metastases. In addition, Snail overexpression led to increased angiogenesis in primary tumors as measured by MECA-32 (P < 0.05) positivity and CXCL8 (P = 0.002) and CXCL5 (P = 0.0003) concentrations in tumor homogenates. Demonstrating the importance of these proangiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression.

Conclusion: Snail upregulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands. (Clin Cancer Res 2009;15(22):6820–9)

Purpose: Despite the strong association between malignant disease and thromboembolic disorders, the molecular and cellular basis of this relationship remains uncertain. We evaluated the hypothesis that tumor-derived tissue factor–bearing microparticles in plasma contribute to cancer-associated thrombosis.

Experimental Design: We developed impedance-based flow cytometry to detect, quantitate, and size microparticles in platelet-poor plasma. We evaluated the number of tissue factor–bearing microparticles in a cohort of cancer patients of different histologies (N = 96) and conducted a case-control study of 30 cancer patients diagnosed with an acute venous thromboembolic event (VTE) compared with 60 cancer patients of similar age, stage, sex, and diagnosis without known VTE, as well as 22 patients with an idiopathic VTE.

Results: Tissue factor–bearing microparticles were detected in patients with advanced malignancy, including two thirds of patients with pancreatic carcinoma. Elevated levels of tissue factor–bearing microparticles were associated VTE in cancer patients (adjusted odds ratio, 3.72; 95% confidence interval, 1.18-11.76; P = 0.01). In cancer patients without VTE, a retrospective analysis revealed a 1-year cumulative incidence of VTE of 34.8% in patients with tissue factor–bearing microparticles versus 0% in those without detectable tissue factor–bearing microparticles (Gray test P = 0.002).The median number of tissue factor–bearing microparticles in the cancer VTE cohort (7.1 x 10⁴ microparticles/µL) was significantly greater than both the idiopathic VTE and cancer–no VTE groups (P = 0.002 and P = 0.03, respectively). Pancreatectomy in three patients eliminated or nearly eliminated these microparticles which coexpressed the epithelial tumor antigen, MUC-1.

Conclusion: We conclude that tumor-derived tissue factor–bearing microparticles are associated with VTE in cancer patients and may be central to the pathogenesis of cancer-associated thrombosis. (Clin Cancer Res 2009;15(22):6830–40)

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a "second-generation" class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy.

Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors.

Results: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC₅₀, 0.16 nmol/L).

Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising "second-generation" HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):6841–51)

Purpose: Signal transducer and activator of transcription-3 (STAT3) is downstream of growth factor and cytokine receptors, and regulates key oncogenic pathways in non–small cell lung cancer (NSCLC). Activation of STAT3 by cellular Src (c-Src) promotes tumor progression. We hypothesized that c-Src inhibition could activate STAT3 by inducing a homeostatic feedback loop, contributing to c-Src inhibitor resistance.

Experimental Design: The effects of c-Src inhibition on total and phosphorylated STAT3 were measured in NSCLC cell lines and in murine xenograft models by Western blotting. c-Src and STAT3 activity as indicated by phosphorylation was determined in 46 human tumors and paired normal lung by reverse phase protein array. Modulation of dasatinib (c-Src inhibitor) cytotoxicity by STAT3 knockdown was measured by MTT, cell cycle, and apoptosis assays.

Results: Depletion of c-Src by small interfering RNA or sustained inhibition by dasatinib increased pSTAT3, which could be blocked by inhibition of JAK. Similarly, in vivo pSTAT3 levels initially decreased but were strongly induced after sustained dasatinib treatment. In human tumors, phosphorylation of the autoinhibitory site of c-Src (Y527) correlated with STAT3 phosphorylation (r = 0.64; P = 2.5 x 10^–6). STAT3 knockdown enhanced the cytotoxicity of dasatinib.

Conclusions: c-Src inhibition leads to JAK-dependent STAT3 activation in vitro and in vivo. STAT3 knockdown enhances the cytotoxicity of dasatinib, suggesting a compensatory pathway that allows NSCLC survival. Data from human tumors showed a reciprocal regulation of c-Src and STAT3 activation, suggesting that this compensatory pathway functions in human NSCLC. These results provide a rationale for combining c-Src and STAT3 inhibition to improve clinical responses. (Clin Cancer Res 2009;15(22):6852–61)

Purpose: Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT^AY502-3ins exon 9 mutation being the most sensitive.

Experimental Design: As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT^AY502-3ins mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib.

Results: Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT^AY502-3ins mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib.

Conclusions: Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT^AY502-3ins showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains. (Clin Cancer Res 2009;15(22):6862–70)

Purpose: The vascular targeting antibody bavituximab is being combined with chemotherapy in clinical trials in cancer patients. Bavituximab targets the membrane phospholipid, phosphatidylserine, complexed with β2-glycoprotein I. Phosphatidylserine is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors. Phosphatidylserine exposure on tumor vessels is increased by chemotherapy and irradiation. Here, we determined whether treatment with the murine equivalent of bavituximab, 2aG4, combined with irradiation can suppress tumor growth in a rat model of glioblastoma.

Experimental Design: F98 glioma cells were injected into the brains of syngeneic rats where they grow initially as a solid tumor and then infiltrate throughout the brain. Rats with established tumors were treated with 10 Gy whole brain irradiation and 2aG4.

Results: Combination treatment doubled the median survival time of the rats, and 13% of animals were rendered disease free. Neither treatment given individually was as effective. We identified two mechanisms. First, irradiation induced phosphatidylserine exposure on tumor blood vessels and enhanced antibody-mediated destruction of tumor vasculature by monocytes/macrophages. Second, the antibody treatment induced immunity to F98 tumor cells, which are normally weakly immunogenic. Surviving rats were immune to rechallenge with F98 tumor cells. In vitro, 2aG4 enhanced the ability of dendritic cells (DCs) to generate F98-specific cytotoxic T cells. Phosphatidylserine exposure, which is induced on tumor cells by irradiation, likely suppresses tumor antigen presentation, and 2aG4 blocks this tolerogenic effect.

Conclusion: Bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma. (Clin Cancer Res 2009;15(22):6871–80)

Purpose: A single vaccination of intact or reconstituted-lymphopenic mice (RLM) with a granulocyte macrophage colony-stimulating factor–secreting B16BL6-D5 melanoma cell line induces protective antitumor immunity and T cells that mediate the regression of established melanoma in adoptive immunotherapy studies. We wanted to study if multiple vaccinations during immune reconstitution of the lymphopenic host would maintain a potent antitumor immune response.

Experimental Design: RLM were vaccinated multiple times over a 40-day period. Spleens were isolated from these mice, activated in vitro, and adoptively transferred into mice bearing 3-day experimental pulmonary metastases.

Results: Multiple vaccinations, rather than boosting the immune response, significantly reduced therapeutic efficacy of adoptive immunotherapy and were associated with an increased frequency and absolute number of CD3+CD4+Foxp3+ T regulatory (T_reg) cells. Anti-CD4 administration reduced the absolute number of T_reg cells 9-fold. Effector T-cells generated from anti-CD4–treated mice were significantly (P < 0.0001) more therapeutic in adoptive transfer studies than T cells from multiply vaccinated animals with a full complement of CD4+ cells.

Conclusion: These results suggest that CD4+ T_reg cells limit the efficacy of multiple vaccinations and that timed partial depletion of CD4+ T cells may reduce suppression and "tip-the-balance" in favor of therapeutic antitumor immunity. The recent failure of large phase III cancer vaccine clinical trials, wherein patients received multiple vaccines, underscores the potential clinical relevance of these findings. (Clin Cancer Res 2009;15(22):6881–90)

Purpose: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome–negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy.

Experimental Design: A novel inhibitor of JAK1/2 was characterized using kinase assays. Cellular effects of this compound were measured in cell lines bearing the JAK2V617F or JAK1V658F mutation, and its antiproliferative activity against primary polycythemiavera patient cells was determined using clonogenic assays. Antineoplastic activity in vivo was determined using a JAK2V617F-driven xenograft model, and effects of the compound on survival, organomegaly, body weight, and disease-associated inflammatory markers were measured.

Results: INCB16562 potently inhibited proliferation of cell lines and primary cells from PV patients carrying the JAK2V617F or JAK1V658F mutation by blocking JAK-STAT signaling and inducing apoptosis. In vivo, INCB16562 reduced malignant cell burden, reversed splenomegaly and normalized splenic architecture, improved body weight gains, and extended survival in a model of JAK2V617F-driven hematologic malignancy. Moreover, these mice suffered from markedly elevated levels of inflammatory cytokines, similar to advanced myeloproliferative disease patients, which was reversed upon treatment.

Conclusions: These data showed that administration of the dual JAK1/2 inhibitor INCB16562 reduces malignant cell burden, normalizes spleen size and architecture, suppresses inflammatory cytokines, improves weight gain, and extends survival in a rodent model of JAK2V617F-driven hematologic malignancy. Thus, selective inhibitors of JAK1 and JAK2 represent a novel therapy for the patients with myeloproliferative diseases and other neoplasms associated with JAK dysregulation. (Clin Cancer Res 2009;15(22):6891–900)

Purpose: Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors.

Experimental Design: We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models.

Results: HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a ⁵¹Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1–treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors.

Conclusions: These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy. (Clin Cancer Res 2009;15(22):6901–11)

Purpose: MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs, as well as with ionizing radiation. In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low–dose rate ionizing radiation (LDR-IR), which is similar to the clinical use of cancer brachytherapy.

Experimental Design: An isogenic pair of MMR⁺ (MLH1⁺) and MMR^– (MLH1^–) human colorectal cancer HCT116 cells was exposed to prolonged LDR-IR (1.3-17 cGy/h x 24-96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins, and cell death marker proteins were examined with Western blotting.

Results: MLH1⁺ HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers, a reduced HPRT gene mutation rate, and more pronounced cell cycle alterations (increased late-S population and a G₂/M arrest) following LDR-IR compared with MLH1^– HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1⁺ cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination) levels.

Conclusions: MLH1 status significantly affects cellular responses to prolonged LDR-IR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of homologous recombination (through inhibition of Rad51). (Clin Cancer Res 2009;15(22):6912–20)

Purpose: Resistance to anti-HER2 monoclonal antibody trastuzumab is a relevant issue in breast cancer patients. Among the mechanisms implicated in trastuzumab resistance, increasing evidence supports a role of tumor microenvironment. We previously found that a novel toll-like receptor 9 agonist, referred to as immune modulatory oligonucleotide (IMO) and currently under clinical investigation, acts through epidermal growth factor receptor (EGFR) and shows direct antiangiogenic effects by cooperating with anti-EGFR or anti-VEGF drugs, thus interfering with cancer cells and microenvironment.

Experimental Design: In this study, we used KPL-4 and JIMT-1 trastuzumab-resistant breast cancer cells to evaluate the combination IMO plus trastuzumab as a therapeutic option for trastuzumab-resistant breast cancers.

Results: IMO inhibits KPL-4 and JIMT-1 xenografts growth and potentiates trastuzumab antitumor effect, with complete suppression of tumor growth, potent enhancement of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity, and strong inhibition of EGFR/HER2-related signaling. In KPL-4 xenografts, IMO alone interferes with HER signal transduction, whereas trastuzumab is ineffective. IMO induces an HER-dependent signal inhibition also in vitro by modulating a functional interaction between toll-like receptor 9 and HER receptors occurring at membrane level. Finally, IMO plus trastuzumab produces a cooperative antiangiogenic effect related to suppression of endothelial HER-related signaling.

Conclusions: We showed a cooperative effect of IMO plus trastuzumab in trastuzumab-resistant breast cancers due to IMO direct antitumor and antiangiogenic activity and antibody-dependent cell-mediated cytotoxicity enhancement. Moreover, we provided first evidence of a toll-like receptor 9/HER interaction at membrane level as novel mechanism of action. Altogether, we propose IMO plus trastuzumab as an effective strategy in trastuzumab-resistant breast cancers. (Clin Cancer Res 2009;15(22):6921–30)

Purpose: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A_–317 and C_–1610 alleles. Haplotype *1 was also associated higher DHFR expression.

Experimental Design: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript.

Results: Six polymorphisms were identified, of which five were single nucleotide polymorphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into five subtypes (*1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G₃₀₈A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels.

Conclusions: The study provides a new insight into DHFR regulatory variations predisposing to an event in ALL patients. (Clin Cancer Res 2009;15(22):6931–8)

Purpose: Gene expression studies in melanoma have been few because tumors are small and cryopreservation is rarely possible. The purpose of this study was to evaluate the Illumina DASL Array Human Cancer Panel for gene expression studies in formalin-fixed melanoma primary tumors and to identify prognostic biomarkers.

Experimental Design: Primary tumors from two studies were sampled using a tissue microarray needle. Study 1: 254 tumors from a melanoma cohort recruited from 2000 to 2006. Study 2: 218 tumors from a case-control study of patients undergoing sentinel node biopsy.

Results: RNA was obtained from 76% of blocks; 1.4% of samples failed analysis (transcripts from <250 of the 502 genes on the DASL chip detected). Increasing age of the block and increased melanin in the tumor were associated with reduced number of genes detected. The gene whose expression was most differentially expressed in association with relapse-free survival in study 1 was osteopontin (SPP1; P = 2.11 x 10^–6) and supportive evidence for this was obtained in study 2 used as a validation set (P = 0.006; unadjusted data). Osteopontin level in study 1 remained a significant predictor of relapse-free survival when data were adjusted for age, sex, tumor site, and histologic predictors of relapse. Genes whose expression correlated most strongly with osteopontin were PBX1, BIRC5 (survivin), and HLF.

Conclusion: Expression data were obtained from 74% of primary melanomas and provided confirmatory evidence that osteopontin expression is a prognostic biomarker. These results suggest that predictive biomarker studies may be possible using stored blocks from mature clinical trials. (Clin Cancer Res 2009;15(22):6939–46)

Purpose: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients.

Experimental Design: We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemoimmunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts, representing a total of 301 CLL patients.

Results: A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training data set. The progression signature was validated in two independent data sets, showing a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and nonresponding CLL patients.

Conclusions: Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients. (Clin Cancer Res 2009;15(22):6947–55)

Purpose: Identifying rectal cancer patients at risk for local recurrence would allow for refinement in the selection of patients who would benefit from preoperative radiotherapy. PIK3CA, KRAS, and BRAF mutations are commonly found in colon cancers, but their prevalence has not been clearly assessed in rectal cancer. In this study, we aim to determine the mutation frequencies of PIK3CA, KRAS, and BRAF and to investigate whether a mutation may be used as a prognostic parameter in rectal cancer patients.

Experimental Design: We evaluated DNA mutations in PIK3CA, KRAS, and BRAF in 240 stage I to III rectal tumors obtained from nonirradiated patients from the Dutch Total Mesorectal Excision trial.

Results: PIK3CA, KRAS, and BRAF mutations were identified in 19 (7.9%), 81 (33.9%), and 5 (2.1%) rectal cancers. Patients with PIK3CA mutations developed more local recurrences (5-year risks, 27.8% versus 9.4%; P = 0.006) and tended to develop these recurrences more rapidly after surgery (median local recurrence-free interval since surgery: 7.9 versus 19.6 months; P = 0.07) than patients without PIK3CA mutations. In multivariate analysis, PIK3CA mutations remained as an independent predictor for the development of local recurrences (hazard ratio, 3.4; 95% confidence interval, 1.2-9.2; P = 0.017), next to tumor-node-metastasis stage.

Conclusion: PIK3CA mutations can be used as a biomarker in identifying rectal cancer patients with an increased risk for local recurrences. Currently, our findings suggest that prospective evaluation of PIK3CA mutation status could reduce overtreatment by preoperative radiotherapy for the low-risk patients who might otherwise only experience the side effects. (Clin Cancer Res 2009;15(22):6956–62)

Purpose: Chromosome 9 is frequently deleted in high-risk gastrointestinal stromal tumors (GISTs), whereas its specific tumor suppressor genes (TSGs) are less understood. We did an integrative study of MTAP gene at 9p21 to analyze its implication in GISTs.

Experimental Design: To search TSGs on chromosome 9, we used ultrahigh-resolution array comparative genomic hybridization to profile DNA copy number alterations of 22 GISTs, with special attention to MTAP gene. MTAP immunoexpression was assessable for 306 independent GISTs on tissue microarrays, with 146 cases analyzed for MTAP homozygous deletion, 181 for mutations of KIT and PDGFRA receptor tyrosine kinase genes, and 7 for MTAP hypermethylation.

Results: Array comparative genomic hybridization identified 11 candidate TSGs on 9p and six on 9q. MTAP and/or CDKN2A/CDKN2B at 9p21.3 were deleted in one intermediate-risk (11%) and seven high-risk (70%) GISTs with two cases homozygously codeleted at both loci. MTAP homozygous deletion, present in 25 of 146 cases, was highly associated with larger size and higher mitotic rate, Ki-67 index, and risk level (all P < 0.01) but not with receptor tyrosine kinase genotypes. Whereas MTAP homozygous deletion correlated with MTAP protein loss (P < 0.001), 7 of 30 GISTs without MTAP expression did not show homozygous deletion, including three MTAP-hypermethylated cases. MTAP homozygous deletion was univariately predictive of decreased disease-free survival (P < 0.0001) and remained multivariately independent (P = 0.0369, hazard ratio = 2.166), together with high-risk category (P < 0.0001), Ki-67 index >5% (P = 0.0106), and nongastric location (P = 0.0416).

Conclusions: MTAP homozygous deletion, the predominant mechanism to deplete protein expression, is present in 17% of GISTs. It correlates with important prognosticators and independently predicts worse outcomes, highlighting the role in disease progression. (Clin Cancer Res 2009;15(22):6963–72)

Purpose: The average weight-losing pancreatic cancer patient undergoing palliative therapy is frequently overweight rather than underweight, and this can confound conventional measures used for risk stratification. The aim of this study was to evaluate if weight and body composition, specifically sarcopenia, assessed from diagnostic computed tomography (CT) scans, is of prognostic value in patients with pancreatic cancer. The nature and extent of tissue loss over subsequent months was also evaluated.

Experimental Design: A total of 111 patients entering a palliative therapy program, who had CT images and had undergone nutritional screening, were studied. In patients for whom follow-up scans were available (n = 44), longitudinal changes in body composition were studied at a mean of 230 ± 62 and 95 ± 60 days prior to demise.

Results: Sixty-two patients (55.9%) were sarcopenic, 44 (39.6%) were overweight/obese, and 18 (16.2%) were both. Age ≥59 years (hazard ratio, 1.71; 95% confidence interval, 1.10-2.66; P = 0.018), and overweight/obese sarcopenia (hazard ratio, 2.07; 95% confidence interval, 1.23-3.50; P = 0.006) were identified as independent predictors of survival on multivariate analysis. Longitudinal analysis revealed that total fat-free mass index decreased from 15.5 ± 2.5 kg/m² to 14.5 ± 2.0 kg/m² (P = 0.002), and total fat mass index decreased from 7.5 ± 2.0kg/m² to 6.0 ± 1.5kg/m² (P < 0.0001) over 135 days.

Conclusions: Sarcopenia in overweight/obese patients with advanced pancreatic cancer is an occult condition but can be identified using CT scans. This condition is an independent adverse prognostic indicator that should be considered for stratification of patients' entering clinical trials, systemic therapy, or support care programs. (Clin Cancer Res 2009;15(22):6973–9)

Purpose: To investigate the diagnostic performance of circulating tumor cells (CTC) in discrimination between primary lung cancer and nonmalignant diseases as well as in prediction of distant metastasis.

Patients and Methods: We prospectively evaluated CTCs in 7.5-mL samples of peripheral blood sampled from patients with a suspicion or a diagnosis of primary lung cancer. A semiautomated system was used to capture CTCs with an antibody against epithelial cell adhesion molecule.

Results: Of 150 eligible patients, 25 were finally diagnosed as having nonmalignant disease, and 125 were diagnosed as having primary lung cancer with (n = 31) or without (n = 94) distant metastasis. CTCs were detected in 30.6% of lung cancer patients and in 12.0% of nonmalignant patients. CTC count was significantly higher in lung cancer patients than in nonmalignant patients, but a receiver operating characteristic (ROC) curve analysis showed an insufficient capability of the CTC test in discrimination between lung cancer and nonmalignant diseases with an area under ROC curve of 0.598 (95% confidence interval, 0.488-0.708; P = 0.122). Among lung cancer patients, CTC count significantly increased along with tumor progression, especially with development of distant metastasis. The area under ROC curve for CTC count in prediction of distant metastasis was 0.783 (95% confidence interval, 0.679-0.886; P < 0.001). When patients with one or more CTCs were judged as having metastatic disease, sensitivity and specificity of the CTC test were 71.0% and 83.0%, respectively.

Conclusions: CTC is a useful surrogate marker of distant metastasis in primary lung cancer. (Clin Cancer Res 2009;15(22):6980–6)

Purpose: To determine the prognostic significance of a multimarker assay incorporating expression levels of three molecular markers in primary cutaneous melanoma.

Experimental Design: We assessed expression levels of NCOA3, SPP1, and RGS1 using immunohistochemical analysis in a tissue microarray cohort of 395 patients. For each marker, we identified optimal cut-points for expression intensity to predict disease-specific survival (DSS) and, as a secondary endpoint, sentinel lymph node (SLN) status. The cumulative overexpression of all three markers was embodied in a multimarker index, and its prognostic effect on DSS and SLN status was assessed using Cox regression, Kaplan-Meier analysis, and logistic regression. The prognostic effect of this multimarker assay on DSS was assessed in an independent cohort of 141 patients, in which marker expression levels were scored using immunohistochemical analysis of stained tissue sections.

Results: Increasing multimarker index scores were significantly predictive of reduced DSS and increased SLN metastasis in the 395-patient cohort. Multivariate logistic regression analysis revealed multimarker expression scores as an independent predictor of SLN status (P = 0.001). Multivariate Cox regression analysis showed the independent effect of the multimarker index on DSS (P < 0.001). The multimarker index was the most significant factor predicting DSS when compared with other clinical and histologic factors, including SLN status (P = 0.002). Multimarker expression scores were also the most significantly predictive of DSS in the independent cohort (P = 0.01).

Conclusions: These results describe a multimarker assay with independent prognostic effect on the prediction of survival associated with melanoma in two distinct cohorts. (Clin Cancer Res 2009;15(22):6987–92)

Purpose: NKG2D (natural killer group 2, member D) binds to cellular ligands of the MIC and ULBP/RAET family. These ligands have restricted expression in normal tissue, but are frequently expressed on primary tumors. The role of NKG2D ligands is thought to be important in carcinogenesis but its prognostic effect has not been investigated in such a large cohort.

Experimental Design: In our study, 462 primary colorectal tumors were screened for the expression of all MIC/ULBP/RAET proteins and NK cell infiltration. Tumor microarray technology was used for the purpose of this investigation.

Results: NKG2D ligands were expressed by the majority of colorectal tumors; however, the level of expression varied considerably. High expression of MIC (68 versus 56 months) or RAET1G (74 versus 62 months) showed improved patient survival. Tumors expressing high levels of MIC and RAET1G showed improved survival of 77 months over tumors that expressed high levels of one ligand or low levels of both. High-level expression of all ligands was frequent in tumor-node-metastasis stage I tumors, but became progressively less frequent in stages II, III, and IV tumors. Expression of MIC was correlated with NK cellular infiltration.

Conclusion: The observations presented are consistent with an immunoediting mechanism that selects tumor cells that have lost or reduced their expression of NKG2D ligands. The combination of MIC and tumor-node-metastasis stage was found to be the strongest predictor of survival, splitting patients into eight groups and suggesting prognostic value in clinical assessment. Of particular interest were stage I patients with low expression of MIC who had a similar survival to stage III patients, and may be candidates for adjuvant therapy. (Clin Cancer Res 2009;15(22):6993–7002)

Purpose: The level of estrogen receptor (ER), progesterone receptor (PR), and HER2 aids in the determination of prognosis and treatment of breast cancer. Immunohistochemistry is currently the predominant method for assessment, but differences in methods and interpretation can substantially affect the accuracy, resulting in misclassification. Here, we investigated the association of microarray-based mRNA expression levels compared with immunohistochemistry.

Experimental Design: Microarray mRNA quantification of ER, PR, and HER2 was done by the developed TargetPrint test and compared with immunohistochemical assessment for breast tumors from 636 patients. Immunohistochemistry was done in a central laboratory and in an independent reference laboratory according to American Society of Clinical Oncology/College of American Pathologists guidelines for 100 cases. For HER2 immunohistochemistry 2+ cases, additional chromogenic in situ hybridization (CISH) was used to determine the final status.

Results: ER concordance between microarray and central immunohistochemistry was 93% [95% confidence interval (95% CI), 91-95%]. Only 4% of immunohistochemistry-positive samples were classified negative using microarray, whereas 18% of immunohistochemistry-negative samples showed a positive microarray ER status. Concordance for PR was 83% (95% CI, 80-86%) and 96% of all samples showed an identical classification of HER2 status by microarray and immunohistochemistry/CISH (95% CI, 94-98%). Nine percent of immunohistochemistry HER2-positive samples showed a negative microarray classification. Detailed review of 11 cases with discordant classifications by American Society of Clinical Oncology/College of American Pathologists and central immunohistochemistry indicated that microarray assessment was likely to add additional information in 5 cases.

Conclusion: Microarray-based readout of ER, PR, and HER2 shows a high concordance with immunohistochemistry/CISH and provides an additional, objective, and quantitative assessment of tumor receptor status in breast cancer. (Clin Cancer Res 2009;15(22):7003–11)

Purpose: The aim of the current study was to externally validate the value of survivin as a prognostic marker for bladder cancer in a large multi-institutional cohort of patients treated with radical cystectomy.

Methods: The study comprised 726 patients treated with radical cystectomy and bilateral pelvic lymphadenectomy. Survivin staining and scoring were done with automated systems coupled with advanced color detection software. Specimens showing at least 10% reactivity were considered altered. Predictive accuracy was quantified using the concordance index and 200-bootstrap resamples were used to reduce overfit bias.

Results: Survivin was an independent predictor of disease recurrence and cancer-specific survival in multivariable analyses that controlled for the effects of standard clinicopathologic features (hazard ratios, ~1.6; P values ≤ 0.002). In all patients (n = 726), addition of survivin to a model including standard clinicopathologic variables did not improve its predictive accuracy (P = 0.67 for disease recurrence and P = 0.27 for cancer-specific survival). In the subgroup of patients with pT_1-3N₀M₀ disease (n = 398), addition of survivin improved the accuracy of standard clinicopathologic features for prediction of disease recurrence and cancer-specific survival (1.3%, P < 0.001 and 1.2%, P < 0.001, respectively).

Conclusions: Survivin expression improves our accuracy for prediction of cancer recurrence and survival in pT_1-3N₀M₀ patients by a small but statistically significant margin. Our findings support the need for further evaluation of survivin and its signaling pathways as well as survivin-targeted therapies in bladder cancer. (Clin Cancer Res 2009;15(22):7012–9)

Purpose: Cancer progression is mediated by processes that are also important in wound repair. As a result, cancers have been conceptualized as overhealing wounds or "wounds that do not heal," and gene expression signatures reflective of wound repair have shown value as predictors of breast cancer survival. Despite the widespread acknowledgment of commonalities between host responses to wounds and host responses to cancer, the gene expression responses of normal tissue adjacent to cancers have not been well characterized.

Experimental Design: Using RNA extracted from histologically normal breast tissue from 107 patients, including 60 reduction mammoplasty patients and 47 cancer patients, we measured whole genome expression profiles and identified a gene expression signature that is induced in response to breast cancer.

Results: This signature represents an in vivo "wound response" signature that is differentially expressed in the normal tissue of breast cancer patients compared with those without disease and is highly accurate (at least 92% sensitivity and 98% specificity) in distinguishing diseased and nondiseased. The in vivo wound response signature is highly prognostic of breast cancer survival, and there is a strong association between the groups identified by this signature and those identified using serum-treated fibroblasts and other microenvironment-derived or microenvironment-related signatures.

Conclusions: The prevalence of the wound response signature in histologically normal tissue adjacent to breast cancer suggests that microenvironment response is an important variable in breast cancer progression and may be an important target for clinical interventions. (Clin Cancer Res 2009;15(22):7020–8)

Purpose: The availability of a variety of immune response modifiers creates an opportunity for improved efficacy of immunotherapy, but it also leads to uncertainty in how to combine agents and how to assess those combinations. We sought to assess the effect of the addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) to vaccination with a melanoma vaccine.

Experimental Design: Ninety-seven patients with resected melanoma (stage II-IV) were enrolled, stratified by stage, and randomized to receive a cellular melanoma vaccine with or without GM-CSF. The primary endpoint was delayed-type hypersensitivity (DTH) response to melanoma cells. Antibody responses, peripheral leukocyte counts, and survival were also examined.

Results: The GM-CSF arm showed enhanced antibody responses with an increase in IgM titer against the TA90 antigen and increased TA90 immune complexes. This arm also had diminished antimelanoma cell delayed-type hypersensitivity response. Peripheral blood leukocyte profiles showed increases in eosinophils and basophils with decreased monocytes in the GM-CSF arm. These immune changes were accompanied by an increase in early melanoma deaths and a trend toward worse survival with GM-CSF.

Conclusion: These data suggest that GM-CSF is not helpful as an immune adjuvant in this dose and schedule and raise concern that it may be harmful. Based on the discordant findings of an immune endpoint and clinical outcome, the use of such surrogate endpoints in selecting treatments for further evaluation must be done with a great deal of caution. (Clin Cancer Res 2009;15(22):7029–35)

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites.

Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class I–restricted melanoma peptides to stimulate CD8⁺ T cells plus a HLA-DR–restricted tetanus helper peptide to stimulate CD4⁺ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 µg GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded.

Results: CD8⁺ T-cell response rates to the 12 MHC class I–restricted melanoma peptides (by day 50) with or without GM-CSF were 34% and 73%, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4⁺ T-cell responses to tetanus helper peptide were higher without GM-CSF (95% versus 77%; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95% confidence interval) were 76% (67-83%) and 52% (43-61%), respectively, with too few events to assess differences by study group.

Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8⁺ and CD4⁺ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):7036–44)

Purpose: To evaluate the effects of sunitinib, a multitargeted tyrosine kinase inhibitor, on the QT interval in patients with cancer.

Experimental Design: Patients received sunitinib loading doses (150-200 mg) on days 3 and 9 and maintenance doses (50 mg/d) on days 4 to 8. Moxifloxacin (day 1), placebo (day 2), and granisetron [with placebo (day 2) or sunitinib (days 3 and 9)] were also administered. Treatment effects were evaluated by time-matched, serial electrocardiograms, and manually overread.

Results: Twenty-four of 48 patients were QT/PK evaluable. Moxifloxacin produced a time-matched, maximum mean placebo-adjusted corrected QT interval (QT_cF) of 5.6 ms [90% confidence interval (CI), 1.9-9.3]. Sunitinib QT_cF changes correlated with exposure, but not T_max. Maximum mean time-matched, placebo-adjusted QT_cF was 9.6 ms (90% CI, 4.1-15.1) at steady state/therapeutic concentrations (day 3) and 15.4 ms (90% CI, 8.4-22.4) at supratherapeutic concentrations (day 9). No patient had a QT_cF >500 ms. Concomitant granisetron produced no significant QT_cF prolongation. Sunitinib-related adverse events were as previously described.

Conclusions: Sunitinib has a dose-dependent effect on QT interval. The increased risk of ventricular arrhythmias must be weighed against the therapeutic benefit sunitinib provides to patients with advanced cancer. (Clin Cancer Res 2009;15(22):7045–52)

Purpose: In the Anastrozole, Tamoxifen Alone or in Combination trial, the combination arm was inferior to anastrozole alone in terms of disease-free survival possibly due to an adverse pharmacokinetic interaction or a predominant estrogenic effect of tamoxifen under estrogen deprivation. We assessed whether the addition of a lower dose of tamoxifen influenced anastrozole bioavailability and favorably modulated biomarkers of bone fracture, breast cancer, cardiovascular disease, and endometrial cancer risk. The influence of CYP2D6 genotype on tamoxifen effects was also determined.

Experimental Design: Seventy-five postmenopausal women with breast intraepithelial neoplasia were randomly allocated to either 1 mg/d anastrozole or 10 mg/wk tamoxifen or their combination for 12 months. Study endpoints were plasma drug concentrations and changes of C-telopeptide, osteocalcin, estradiol/sex hormone binding globulin (SHBG) ratio, estrone sulfate, insulin-like growth factor-I (IGF-I)/insulin-like growth factor binding protein-3 (IGFBP-3), C-reactive protein, antithrombin-III, endometrial Ki-67 expression, and thickness.

Results: Anastrozole concentrations were not affected by the combination with low-dose tamoxifen, whereas endoxifen levels were lower in poor CYP2D6 metabolizers. C-telopeptide increased by 20% with anastrozole and decreased by 16% with tamoxifen and by 7% with their combination (P < 0.001); osteocalcin showed similar changes. Compared with anastrozole, the combination arm showed lower IGF-I/IGFBP-3 levels (–17% versus –9%; P = 0.004) and lower estradiol/SHBG and estrone sulfate reductions (–15% versus –29% and –30% versus 38%, respectively). However, IGF-I/IGFBP-3 and estradiol/SHBG did not decrease in poor CYP2D6 metabolizers. Endometrial thickness was not greater in the combination than in the anastrozole arm.

Conclusions: The addition of a weekly tamoxifen administration did not impair anastrozole bioavailability and modulated favorably its safety profile, providing the rationale for further studies. (Clin Cancer Res 2009;15(22):7053–60)

Purpose: We evaluated the safety, maximum tolerated dose, pharmacokinetics, and biological effects of the combination of the Raf-1, RET, KIT, platelet-derived growth factor receptor, and vascular endothelial growth factor receptor 2 kinase inhibitor sorafenib and the farnesyltransferase inhibitor tipifarnib.

Experimental Design: A standard 3 + 3 phase I dose-escalation design was used with a 28-day cycle (sorafenib daily and tipifarnib for 21 days, by mouth).

Results: Fifty patients were treated; 43 reached restaging evaluation after cycle 2. The most common side effects were grade 1 to 2 rash, hyperglycemia, and diarrhea. Dose-limiting toxicity was rash, and the recommended phase II dose is sorafenib 400 mg p.o. qam/200 mg p.o. qpm and tipifarnib p.o. 100 mg bd. Despite the low doses of tipifarnib, one quarter of patients had ≥50% reduction in farnesyltransferase levels. Interestingly, six of eight patients with medullary thyroid cancer had durable stable disease (n = 3) or partial remissions (n = 3), lasting 12 to 26+ months. Five of the six responders had available tissue, and RET gene mutations were identified in them. Prolonged (≥6 months) stable disease was also seen in nine patients as follows: papillary thyroid cancer (n = 4; 18+ to 27+ months), adrenocortical cancer (n = 2; 7 and 11 months), and one each of melanoma (platelet-derived growth factor receptor mutation positive; 14 months), renal (6 months), and pancreatic cancer (6 months).

Conclusions: Our study shows that the combination of tipifarnib and sorafenib is well tolerated. Activity was seen, especially in patients with medullary thyroid cancer, a tumor characterized by RET mutations. (Clin Cancer Res 2009;15(22):7061–8)

Purpose: Anti–vascular endothelial growth factor therapy enhances the activity of radiotherapy in experimental models, and bevacizumab has therapeutic activity in patients with metastatic colorectal cancer.

Experimental Design: Twenty-two patients with locally advanced inoperable colorectal carcinomas (LA/I-CRC) were treated with conformal hypofractionated (3.4 Gy/fraction x 15) split-course accelerated radiotherapy (biological equivalent dose, 67.2 Gy) supported with amifostine, capecitabine (600 mg/m² daily, 5 days/week), and bevacizumab (5 mg/kg every 2 weeks, five cycles). Biopsies from nine patients, performed before and 1 week after bevacizumab administration, were analyzed for changes in mRNA expression with Illumina gene arrays.

Results: No serious grade 3 chemotherapy-related side effects were recorded. There was low acute toxicity, with moist perineal desquamation noted in 2 of 22 patients, diarrhea grade 2 to 3 in 5 of 22 patients, and severe proctalgia in 2 of 22 patients. One patient died from Fournier's gangrene before treatment completion. Within a median follow-up of 18 months, two patients with preradiotheraphy direct involvement of adjacent organs expressed recto-vaginal/perineal fistula. Out of 19 evaluable cases, 13 (68.5%) showed complete response and 4 showed (21.1%) partial response. Fourteen patients are alive with no evidence of loco-regional relapse. In the gene array analysis, 30 known genes associated with transcription factors, DNA repair, and proliferation were downregulated by bevacizumab. DUSP1 gene was the most consistently downregulated transcript.

Conclusions: The combination of radiotherapy with bevacizumab is feasible and results in a high rate of durable complete responses in patients with LA/I-CRC. Radiosensitization may occur through a direct effect on tumor cells followed by a wide scale suppression of transcription factors and genes involved in DNA repair and proliferation. (Clin Cancer Res 2009;15(22):7069–76)

Purpose: This dedicated QTc phase I study, conducted in advanced-stage cancer patients, assessed the effect of a single supratherapeutic dose (800 mg) of vorinostat on the QTc interval.

Experimental Design: A randomized, partially blind, placebo-controlled, two-period, crossover study was conducted. Patients (n = 25) received single doses of 800 mg vorinostat and placebo in the fasted state. Holter electrocardiogram monitoring was done before each treatment and for 24 h postdose. Blood samples for vorinostat concentration were collected through 24 h postdose following vorinostat treatment only. Prescribed electrocardiogram and blood sampling times were designed to capture the expected C_max of vorinostat.

Results: Twenty-four of the 25 patients enrolled in the study were included in the QTc analysis. The upper bound of the two-sided 90% confidence interval for the QTcF interval for the placebo-adjusted mean change from baseline of vorinostat was <10 ms at every time point. No patient had a QTcF change from baseline value >30 ms. One patient had QTcF values >450 ms (seen after both vorinostat and placebo administration) and none had values >480 ms. Mean AUC_0- and C_max values attained were on the order of ~1.93- and ~1.41-fold higher, respectively, compared with the 400 mg clinical dose. Based on assessment of clinical and laboratory adverse experiences, single doses of 800 mg vorinostat were generally well tolerated.

Conclusions: Administration of a single supratherapeutic dose of the histone deacetylase inhibitor vorinostat is not associated with prolongation of the QTc interval. A dedicated QTc study in advanced cancer patients is a robust means for assessing risk for ventricular repolarization prolongation. (Clin Cancer Res 2009;15(22):7077–84)

Purpose: Carfilzomib (formerly PR-171) is a novel proteasome inhibitor of the epoxyketone class that is selective and structurally distinct from bortezomib. Proteasome inhibition by carfilzomib is mechanistically irreversible. Consequently, proteasome inhibition is more sustained with carfilzomib than with bortezomib.

Experimental Design: In a phase 1 trial evaluating the safety and efficacy of carfilzomib in relapsed or refractory hematologic malignancies, eight dose groups of three to six patients received 5 consecutive days of carfilzomib i.v. push at doses of 1.2, 2.4, 4, 6, 8.4, 11, 15, and 20 mg/m² within 14-day cycles.

Results: Twenty-nine patients enrolled that were relapsed or refractory after at least two prior therapies. Nonhematologic toxicities included fatigue, nausea, and diarrhea in more than one third of patients—mostly grade 1 or 2 in severity. At 20 mg/m², grade 3 febrile neutropenia and grade 4 thrombocytopenia were reported, establishing 15 mg/m² as the maximum tolerated dose. No grade 3 or 4 peripheral neuropathies were reported. Antitumor activity was observed at doses ≥11 mg/m²: one unconfirmed complete response (mantle cell), one partial response (multiple myeloma), and two minimal responses (multiple myeloma and Waldenström's macroglobulinemia).

Conclusion: This is the first clinical use of carfilzomib that shows tolerability and clinical activity in multiple hematologic malignancies using consecutive-day dosing. (Clin Cancer Res 2009;15(22):7085–91)

Purpose: Intracerebral microdialysis (ICMD) is an accepted method for monitoring changes in neurochemistry from acute brain injury. The goal of this pilot study was to determine the feasibility of using ICMD to examine the neuropharmacokinetics of temozolomide in brain interstitium following oral administration.

Experimental Design: Patients with primary or metastatic brain tumors had a microdialysis catheter placed in peritumoral brain tissue at the time of surgical debulking. Computerized tomography scan confirmed the catheter location. Patients received a single oral dose of temozolomide (150 mg/m²) on the first postoperative day, serial plasma and ICMD samples were collected over 24 hours, and temozolomide concentrations were determined by tandem mass spectrometry.

Results: Nine patients were enrolled. Dialysate and plasma samples were successfully collected from seven of the nine patients. The mean temozolomide areas under the concentration-time curve (AUC) in plasma and brain interstitium were 17.1 and 2.7 µg/mL x hour, with an average brain interstitium/plasma AUC ratio of 17.8%. The mean peak temozolomide concentration in the brain was 0.6 ± 0.3 µg/mL, and the mean time to reach peak level in brain was 2.0 ± 0.8 hours.

Conclusions: The use of ICMD to measure the neuropharmacokinetics of systemically administered chemotherapy is safe and feasible. Concentrations of temozolomide in brain interstitium obtained by ICMD are consistent with published data obtained in a preclinical ICMD model, as well as from clinical studies of cerebrospinal fluid. However, the delayed time required to achieve maximum temozolomide concentrations in brain suggests that current chemoradiation regimens may be improved by administering temozolomide 2 to 3 hours before radiation. (Clin Cancer Res 2009;15(22):7092–8)

Purpose: Increasing evidence indicates that enhanced intratumoral androgen synthesis contributes to prostate cancer progression after androgen deprivation therapy. This phase II study was designed to assess responses to blocking multiple steps in androgen synthesis with inhibitors of CYP17A1 (ketoconazole) and type I and II 5-reductases (dutasteride) in patients with castration-resistant prostate cancer (CRPC).

Experimental Design: Fifty-seven men with CRPC were continued on gonadal suppression and treated with ketoconazole (400 mg thrice daily), hydrocortisone (30 mg/AM, 10 mg/PM), and dutasteride (0.5 mg/d).

Results: Prostate-specific antigen response rate (≥50% decline) was 56% (32 of 57; 95% confidence interval, 42.4-69.3%); the median duration of response was 20 months. In patients with measurable disease, 6 of 20 (30%) responded by the Response Evaluation Criteria in Solid Tumors. Median duration of treatment was 8 months; 9 patients remained on therapy with treatment durations censored at 18 to 32 months. Median time to progression was 14.5 months. Grade 3 toxicities occurred in 32% with only one reported grade 4 (thrombosis) toxicity. Dehydroepiandrosterone sulfate declined by 89%, androstenedione by 56%, and testosterone by 66%, and dihydrotestosterone declined to below detectable levels compared with baseline levels with testicular suppression alone. Median baseline levels and declines in dehydroepiandrosterone sulfate, androstenedione, testosterone, and dihydrotestosterone were not statistically different in the responders versus nonresponders, and hormone levels were not significantly increased from nadir levels at relapse.

Conclusion: The response proportion to ketoconazole, hydrocortisone, and dutasteride was at least comparable with previous studies of ketoconazole alone, whereas time to progression was substantially longer. Combination therapies targeting multiple steps in androgen synthesis warrant further investigation. (Clin Cancer Res 2009;15(22):7099–105)