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Clinical Cancer Research

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Clinical Cancer Research

A six-gene prognostic colorectal cancer hypoxia score was generated from expression data from in vitro experiments and microarray datasets and was validated in two distinct patient cohorts. The approach followed by the authors is original and biologically sound but could be limited by potential biases and other methodologic limitations. Clin Cancer Res; 20(8); 2019–22. ©2014 AACR.

The cooperation of tumor-infiltrating lymphocytes and tertiary lymphoid tissue in early-stage colorectal carcinoma further corroborates the strong immune influences on tumor progression and patient outcome. Immune factors in the tumor microenvironment may warrant inclusion in pathology reports and staging systems for prognostication and prediction of therapeutic response. Clin Cancer Res; 20(8); 2023–5. ©2014 AACR.

The protein phosphatase 2A (PP2A) tumor suppressor is inactivated in different leukemias through the activity of its endogenous inhibitors (e.g., SET), which are aberrantly regulated by oncogenic tyrosine kinases. Like other effective and nontoxic PP2A-activating drugs (PAD), OP449 inhibits SET and impairs leukemogenesis. This further supports the immediate use of PADs in patients with leukemia. Clin Cancer Res; 20(8); 2026–8. ©2014 AACR.

On August 26, 2011, the U.S. Food and Drug Administration (FDA) approved crizotinib (XALKORI Capsules, Pfizer Inc.) for treatment of patients with locally advanced or metastatic non–small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK) positive as detected by an FDA-approved test. The Vysis ALK Break-Apart FISH Probe Kit (Abbott Molecular, Inc.) was approved concurrently. In two multicenter, single-arm trials, patients with locally advanced or metastatic ALK-positive NSCLC previously treated with one or more systemic therapies received crizotinib orally at a dose of 250 mg twice daily. In 119 patients with ALK-positive NSCLC by local trial assay, the objective response rate (ORR) was 61% [95% confidence intervals (CI), 52%–70%] with a median response duration of 48 weeks. In 136 patients with ALK-positive NSCLC by the to-be-marketed test, the ORR was 50% (95% CI, 42%–59%) with a median response duration of 42 weeks. The most common adverse reactions (≥25%) were vision disorder, nausea, diarrhea, vomiting, edema, and constipation. Accelerated approval was granted on the basis of the high ORRs and durable responses. On November 20, 2013, crizotinib received full approval based on an improvement in progression-free survival in patients with metastatic ALK-positive NSCLC previously treated with one platinum-based chemotherapy regimen. Clin Cancer Res; 20(8); 2029–34. ©2014 AACR.

Dabrafenib and trametinib were approved for use as monotherapies in BRAF-mutant metastatic melanoma by the U.S. Food and Drug Administration (FDA) in 2013, and most recently, their use in combination has received accelerated FDA approval. Both drugs target the mitogen-activated protein kinase (MAPK) pathway: dabrafenib selectively inhibits mutant BRAF that constitutively activates the pathway, and trametinib selectively inhibits MEK1 and MEK2 proteins activated by RAF kinases. The phase III study of dabrafenib in BRAFV600E metastatic melanoma reported rapid tumor regression in most patients and a 59% objective RECIST response rate. The median progression-free survival (PFS) and overall survival (OS) were improved compared with dacarbazine. Toxicities were well tolerated and different from those reported for vemurafenib, the first FDA-approved BRAF inhibitor. Efficacy has been demonstrated in other BRAF-mutant genotypes. The phase III study of trametinib in BRAF inhibitor–naïve patients with BRAFV600E or BRAFV600K also showed benefit with a prolonged median PFS and OS compared with chemotherapy. Trametinib is ineffective in patients who have progressed on BRAF inhibitors. A phase II trial of combined dabrafenib and trametinib demonstrated higher response rates and longer median PFS than dabrafenib monotherapy, with less cutaneous toxicity. Here, we review the clinical development of both drugs as monotherapies and in combination, and discuss their role in the management of BRAF-mutant melanoma. Clin Cancer Res; 20(8); 2035–43. ©2014 AACR.

Interleukin-15 (IL-15) is a proinflammatory cytokine involved in the development, survival, proliferation, and activation of multiple lymphocyte lineages utilizing a variety of signaling pathways. IL-15 utilizes three distinct receptor chains in at least two different combinations to signal and exert its effects on the immune system. The binding of IL-15 to its receptor complex activates an "immune-enhancing" signaling cascade in natural killer cells and subsets of T cells, as well as the induction of a number of proto-oncogenes. Additional studies have explored the role of IL-15 in the development and progression of cancer, notably leukemia of large granular lymphocytes, cutaneous T-cell lymphoma, and multiple myeloma. This review provides an overview of the molecular events in the IL-15 signaling pathway and the aberrancies in its regulation that are associated with chronic inflammation and cancer. We briefly explore the potential therapeutic opportunities that have arisen as a result of these studies to further the treatment of cancer. These involve both targeting the disruption of IL-15 signaling as well as IL-15–mediated enhancement of innate and antigen-specific immunity. Clin Cancer Res; 20(8); 2044–50. ©2014 AACR.

Janus-activated kinases (JAK) are the mediators of a variety of cytokine signals via their cognate receptors that result in activation of intracellular signaling pathways. Alterations in JAK1, JAK2, JAK3, and TYK2 signaling contribute to different disease states, and dysregulated JAK–STAT signaling is associated with hematologic malignancies, autoimmune disorders, and immune-deficient conditions. Genetic alterations of JAK2 occur in the majority of patients with myeloproliferative neoplasms and occur in a subset of patients with acute leukemias. JAK-mediated signaling critically relies on STAT transcription factors, and on activation of the MAPK and PI3K/Akt signaling axes. Hyperactive JAK at the apex of these potent oncogenic signaling pathways therefore represents an important target for small-molecule kinase inhibitors in different disease states. The JAK1/2 inhibitor ruxolitinib and the JAK3 inhibitor tofacitinib were recently approved for the treatment of myelofibrosis and rheumatoid arthritis, respectively, and additional ATP-competitive JAK inhibitors are in clinical development. Although these agents show clinical activity, the ability of these JAK inhibitors to induce clinical/molecular remissions in hematologic malignancies seems limited and resistance upon chronic drug exposure is seen. Alternative modes of targeting JAK2 such as allosteric kinase inhibition or HSP90 inhibition are under evaluation, as is the use of histone deacetylase inhibitors. Combination therapy approaches integrating inhibition of STAT, PI3K/Akt, and MAPK pathways with JAK kinase inhibitors might be critical to overcome malignancies characterized by dysregulated JAK signaling. Clin Cancer Res; 20(8); 2051–9. ©2014 AACR.

The availability of agents directly targeting tumorigenic and angiogenic pathways has significantly improved the outcomes of patients with advanced renal cell carcinoma (RCC) in recent years. However, all patients eventually become resistant and a substantial percentage experience immediate disease progression with first-line targeted therapy. In addition, patients have variable clinical benefit and/or tolerance to different agents, including drugs within the same class. Thus, the choice of therapy for an individual patient remains empiric at present. Upon this landscape, several molecular biomarkers have been investigated with the purpose of guiding therapy. This review discusses prognostic biomarkers correlating with the outcome of patients independent of therapy, and predictive biomarkers of treatment response, including circulating biomarkers (such as VEGF and VEGF-related proteins, cytokine and angiogenic factors, and lactate dehydrogenase), and tissue-based biomarkers (such as single-nucleotide polymorphisms). Many potential prognostic and predictive molecular biomarkers have now been identified in RCC, although none has yet entered into clinical practice, and all require prospective validation in appropriately designed randomized studies. In the near future, however, validated biomarkers may become integral to management strategies in RCC, enabling tailored treatment for individual patients to improve clinical outcomes. Clin Cancer Res; 20(8); 2060–71. ©2014 AACR.

Hepatocellular carcinoma (HCC) is a major health problem. Most patients with HCC experience a recurrence after resection/ablation or are diagnosed at advanced stages. Sorafenib remains the only approved systemic drug for these patients. Molecular therapies targeting signaling cascades involved in hepatocarcinogenesis have been explored in phase III clinical trials. However, none of the drugs tested have shown positive results in the first-line (brivanib, sunitinib, erlotinib, and linifanib) or second-line (brivanib, everolimus) setting after sorafenib progression. Reasons for failure are heterogeneous and include lack of understanding of critical drivers of tumor progression/dissemination, liver toxicity, flaws in trial design, or marginal antitumoral potency. These trials are also challenging time to progression as a surrogate endpoint of survival. Trials ongoing testing drugs head-to-head versus sorafenib in "all comers" might have difficulties in achieving superior results in the first line. Novel trials are also designed testing drugs in biomarker-based subpopulations of patients with HCC. Most common mutations, however, are undruggable, such as p53 and CTNNB1. Two types of studies are proposed: (i) phase II pivotal proof-of-concept studies testing drugs blocking potential oncogenic addiction loops, such as the one testing MEK inhibitors in RAS+ patients or amplification of FGF19 as a target; and (ii) phase II to III studies using biomarker-based trial enrichment for defining HCC subpopulations, such as the case of enriching for MET-positive tumors. These strategies have been deemed successful in breast, melanoma, and lung cancers, and are expected to change the landscape of trial design of HCC. Clin Cancer Res; 20(8); 2072–9. ©2014 AACR.

Purpose: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples.

Experimental Design: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer.

Results: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA.

Conclusions: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice. Clin Cancer Res; 20(8); 2080–91. ©2014 AACR.

Purpose: The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A.

Experimental Design: In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis, and clonogenic assays. Efficacy of target inhibition by OP449 was evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model.

Results: We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1–mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells.

Conclusions: We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. Clin Cancer Res; 20(8); 2092–103. ©2014 AACR.

Purpose: When evaluated in patients with ovarian and other cancer, vintafolide (EC145), a potent folate-targeted vinca alkaloid conjugate, displayed a toxicity profile that seemed to be nonoverlapping with many standard-of-care cancer therapeutics. It was, therefore, hypothesized that combining vintafolide with certain approved anticancer drugs may afford greater therapeutic efficacy compared with single-agent therapy. To explore this concept, vintafolide was evaluated in combination with pegylated liposomal doxorubicin (PLD; DOXIL), cisplatin, carboplatin, paclitaxel, docetaxel, topotecan, and irinotecan against folate receptor (FR)–positive models.

Experimental Design: FR-expressing KB, M109, IGROV, and L1210 cells were first exposed to graded concentrations of vintafolide, either alone or in combination with doxorubicin (active ingredient in PLD), and isobologram plots and combination index values generated. The vintafolide combinations were also studied in mice bearing various FR-expressing tumors.

Results: Vintafolide displayed strong synergistic activity against KB cells when combined with doxorubicin, and no less-than-additive effects resulted when tested against M109, IGROV, and L1210 cells. In contrast, when either desacetylvinblastine hydrazide (DAVLBH; the vinca alkaloid moiety in vintafolide) or vindesine (the vinca alkaloid most structurally similar to DAVLBH) were tested in combination with doxorubicin, less-than-additive antitumor effects were observed. In vivo, all vintafolide drug combinations produced far greater antitumor effect (complete responses and cures) compared with the single agents alone, without significant increase in overall toxicity. Importantly, these benefits were not observed with combinations of PLD and DAVLBH or vindesine.

Conclusions: On the basis of these encouraging preclinical results, clinical studies to evaluate vintafolide drug combination therapies are now under way. Clin Cancer Res; 20(8); 2104–14. ©2014 AACR.

Purpose: Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal–regulated kinase 1/2 (ERK1/2) activation in primary breast cancer.

Experimental Design: Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment.

Results: Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors.

Conclusions: Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer. Clin Cancer Res; 20(8); 2115–25. ©2014 AACR.

Purpose: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid–specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [18F]4-fluorobenzylcarbonyl–Val–Ala–Asp(OMe)–fluoromethylketone ([18F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).

Experimental Design: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [18F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [18F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.

Results: Accumulation of [18F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in V600EBRAF colon cancer. In the latter setting, [18F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.

Conclusions: These studies illuminate [18F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine. Clin Cancer Res; 20(8); 2126–35. ©2014 AACR.

Purpose: Metabolic phenotyping has provided important biomarker findings, which, unfortunately, are rarely replicated across different sample sets due to the variations from different analytical and clinical protocols used in the studies. To date, very few metabolic hallmarks in a given cancer type have been confirmed and validated by use of a metabolomic approach and other clinical modalities. Here, we report a metabolomics study to identify potential metabolite biomarkers of colorectal cancer with potential theranostic value.

Experimental Design: Gas chromatography–time-of-flight mass spectrometry (GC–TOFMS)–based metabolomics was used to analyze 376 surgical specimens, which were collected from four independent cohorts of patients with colorectal cancer at three hospitals located in China and City of Hope Comprehensive Cancer Center in the United States. Differential metabolites were identified and evaluated as potential prognostic markers. A targeted transcriptomic analysis of 29 colorectal cancer and 27 adjacent nontumor tissues was applied to analyze the gene expression levels for key enzymes associated with these shared metabolites.

Results: A panel of 15 significantly altered metabolites was identified, which demonstrates the ability to predict the rate of recurrence and survival for patients after surgery and chemotherapy. The targeted transcriptomic analysis suggests that the differential expression of these metabolites is due to robust metabolic adaptations in cancer cells to increased oxidative stress as well as demand for energy, and macromolecular substrates for cell growth and proliferation.

Conclusions: These patients with colorectal cancer, despite their varied genetic background, mutations, pathologic stages, and geographic locations, shared a metabolic signature that is of great prognostic and therapeutic potential. Clin Cancer Res; 20(8); 2136–46. ©2014 AACR.

Purpose: Tumor-infiltrating T lymphocytes (TIL) play a key role in the clinical outcome of human colorectal cancer; however, the dynamics of their recruitment along colorectal cancer clinical progression have not been fully elucidated. Tertiary lymphoid tissue (TLT) is an ectopic organized lymph node–like structure that typically forms at sites of chronic inflammation and is involved in adaptive immune responses. Its occurrence in cancer is sporadically documented and its role and clinical relevance is largely unknown.

Experimental Design: The occurrence of TLT, the correlation with TILs, and the clinical relevance were evaluated retrospectively, in a cohort study involving a consecutive series of 351 patients with stage II and III colorectal cancer. The role of TLT in lymphocyte recruitment was assessed in a preclinical model of colorectal cancer.

Results: In both human colorectal cancer and in a murine model of colorectal cancer, we identified organized TLT, highly vascularized (including high endothelial venules), and correlated with the density of CD3+ TILs. Intravenous injection in mice of GFP splenocytes resulted in homing of lymphocytes to TLT, suggesting an active role of TLT in the recruitment of lymphocytes to tumor areas. Accordingly, TLT density and TIL infiltration correlated and were coordinated in predicting better patient's outcome among patients with stage II colorectal cancer.

Conclusions: We provide evidence that TLT is associated with lymphocyte infiltration in colorectal cancer, providing a pathway of recruitment for TILs. TLT cooperates with TILs in a coordinated antitumor immune response, when identifying patients with low-risk early-stage colorectal cancer, thus, representing a novel prognostic biomarker for colorectal cancer. Clin Cancer Res; 20(8); 2147–58. ©2014 AACR.

Purpose: Hypoxia is considered a major microenvironmental factor influencing cancer behavior. Our aim was to develop a hypoxia-based gene score that could identify high and low risk within stage II and III colon cancer patients.

Experimental Design: Differential gene expression of CaCo-2 colon cancer cells cultured in chronic hypoxia versus normoxia was tested for correlation with prognostic variables in published microarray datasets. These datasets were further used to downsize and optimize a gene score, which was subsequently determined in paraffin-embedded material of 126 patients with colon cancer treated in our center.

Results: In the CaCo-2 cells, 923 genes with a 2-fold change and Limma corrected P ≤ 0.0001 were found differentially expressed in hypoxia versus normoxia. We identified 21 genes with prognostic value and overlapping in three different training sets and (n = 224). With a fourth published dataset (n = 177), the six-gene Colon Cancer Hypoxia Score (CCHS) was developed. Patients with low CCHS showed a significant better disease-free survival at three years (77.3%) compared with high CCHS patients (46.4%; log-rank, P = 0.006). This was independently confirmed in an external patient cohort of 90 stage II patients (86.9% vs. 52.2%; P = 0.001).

Conclusions: Hypoxia-driven gene expression is associated with high recurrence rates in stage II and III colon cancer. A six-gene score was found to be of independent prognostic value in these patients. Our findings require further validation and incorporation in the current knowledge on molecular classification of colon cancer. Clin Cancer Res; 20(8); 2159–68. ©2014 AACR.

Purpose: Available tools for prostate cancer diagnosis and prognosis are suboptimal and novel biomarkers are urgently needed. Here, we investigated the regulation and biomarker potential of the GABRE~miR-452~miR-224 genomic locus.

Experimental Design: GABRE/miR-452/miR-224 transcriptional expression was quantified in 80 nonmalignant and 281 prostate cancer tissue samples. GABRE~miR-452~miR-224 promoter methylation was determined by methylation-specific qPCR (MethyLight) in 35 nonmalignant, 293 prostate cancer [radical prostatectomy (RP) cohort 1] and 198 prostate cancer tissue samples (RP cohort 2). Diagnostic/prognostic biomarker potential of GABRE~miR-452~miR-224 methylation was evaluated by ROC, Kaplan–Meier, uni- and multivariate Cox regression analyses. Functional roles of miR-224 and miR-452 were investigated in PC3 and DU145 cells by viability, migration, and invasion assays and gene-set enrichment analysis (GSEA) of posttransfection transcriptional profiling data.

Results: GABRE~miR-452~miR-224 was significantly downregulated in prostate cancer compared with nonmalignant prostate tissue and had highly cancer-specific aberrant promoter hypermethylation (AUC = 0.98). Functional studies and GSEA suggested that miR-224 and miR-452 inhibit proliferation, migration, and invasion of PC3 and DU145 cells by direct/indirect regulation of pathways related to the cell cycle and cellular adhesion and motility. Finally, in uni- and multivariate analyses, high GABRE~miR-452~miR-224 promoter methylation was significantly associated with biochemical recurrence in RP cohort 1, which was successfully validated in RP cohort 2.

Conclusion: The GABRE~miR-452~miR-224 locus is downregulated and hypermethylated in prostate cancer and is a new promising epigenetic candidate biomarker for prostate cancer diagnosis and prognosis. Tumor-suppressive functions of the intronic miR-224 and miR-452 were demonstrated in two prostate cancer cell lines, suggesting that epigenetic silencing of GABRE~miR-452~miR-224 may be selected for in prostate cancer. Clin Cancer Res; 20(8); 2169–81. ©2014 AACR.

Purpose: The norepinephrine transporter (NET) is a critical regulator of catecholamine uptake in normal physiology and is expressed in neuroendocrine tumors like neuroblastoma. Although the norepinephrine analog, meta-iodobenzylguanidine (MIBG), is an established substrate for NET, 123I/131I-MIBG has several clinical limitations for diagnostic imaging. In the current studies, we evaluated meta-[18F]-fluorobenzylguanidine ([18F]-MFBG) and compared it with 123I-MIBG for imaging NET-expressing neuroblastomas.

Experimental Design: NET expression levels in neuroblastoma cell lines were determined by Western blot and 123I-MIBG uptake assays. Five neuroblastoma cell lines and two xenografts (SK-N-BE(2)C and LAN1) expressing different levels of NET were used for comparative in vitro and in vivo uptake studies.

Results: The uptake of [18F]-MFBG in cells was specific and proportional to the expression level of NET. Although [18F]-MFBG had a 3-fold lower affinity for NET and an approximately 2-fold lower cell uptake in vitro compared with that of 123I-MIBG, the in vivo imaging and tissue radioactivity concentration measurements demonstrated higher [18F]-MFBG xenograft uptake and tumor-to-normal organ ratios at 1 and 4 hours after injection. A comparison of 4 hours [18F]-MFBG PET (positron emission tomography) imaging with 24 hours 123I-MIBG SPECT (single-photon emission computed tomography) imaging showed an approximately 3-fold higher tumor uptake of [18F]-MFBG, but slightly lower tumor-to-background ratios in mice.

Conclusions: [18F]-MFBG is a promising radiopharmaceutical for specifically imaging NET-expressing neuroblastomas, with fast pharmacokinetics and whole-body clearance. [18F]-MFBG PET imaging shows higher sensitivity, better detection of small lesions with low NET expression, allows same day scintigraphy with a shorter image acquisition time, and has the potential for lower patient radiation exposure compared with 131I/123I-MIBG. Clin Cancer Res; 20(8); 2182–91. ©2014 AACR.

Purpose: This phase I/II study evaluated safety, efficacy, and pharmacokinetics of escalating, multiple doses of siltuximab, a chimeric anti-interleukin (IL)-6 monoclonal antibody derived from a new Chinese hamster ovary (CHO) cell line in patients with advanced/refractory solid tumors.

Experimental Design: In the phase I dose-escalation cohorts, 20 patients with advanced/refractory solid tumors received siltuximab 2.8 or 5.5 mg/kg every 2 weeks or 11 or 15 mg/kg every 3 weeks intravenously (i.v.). In the phase I expansion (n = 24) and phase II cohorts (n = 40), patients with Kirsten rat sarcoma-2 (KRAS)-mutant tumors, ovarian, pancreatic, or anti-EGF receptor (EGFR) refractory/resistant non–small cell lung cancer (NSCLC), colorectal, or H&N cancer received 15 mg/kg every 3 weeks. The phase II primary efficacy endpoint was complete response, partial response, or stable disease >6 weeks.

Results: Eighty-four patients (35 colorectal, 29 ovarian, 9 pancreatic, and 11 other) received a median of three (range, 1–45) cycles. One dose-limiting toxicity occurred at 5.5 mg/kg. Common grade ≥3 adverse events were hepatic function abnormalities (15%), physical health deterioration (12%), and fatigue (11%). Ten percent of patients had siltuximab-related grade ≥3 adverse events. Neutropenia (4%) was the only possibly related adverse event grade ≥3 reported in >1 patient. Serious adverse events were reported in 42%; most were related to underlying disease. The pharmacokinetic profile of CHO-derived siltuximab appears similar to the previous cell line. No objective responses occurred; 5 of 84 patients had stable disease >6 weeks. Hemoglobin increased ≥1.5 g/dL in 33 of 47 patients. At 11 and 15 mg/kg, completely sustained C-reactive protein suppression was observed.

Conclusions: Siltuximab monotherapy appears to be well tolerated but without clinical activity in solid tumors, including ovarian and KRAS-mutant cancers. The recommended phase II doses were 11 and 15 mg/kg every 3 weeks. Clin Cancer Res; 20(8); 2192–204. ©2014 AACR.

Purpose: Lurbinectedin (PM01183) binds covalently to DNA and has broad activity against tumor cell lines. This first-in-human phase I study evaluated dose-limiting toxicities (DLT) and defined a phase II recommended dose for PM01183 as a 1-hour intravenous infusion every three weeks (q3wk).

Experimental Design: Thirty-one patients with advanced solid tumors received escalating doses of PM01183 following an accelerated titration design.

Results: PM01183 was safely escalated over 200-fold, from 0.02 to 5.0 mg/m2. Dose doubling was utilized, requiring 15 patients and nine dose levels to identify DLT. The recommended dose was 4.0 mg/m2, with one of 15 patients having DLT (grade 4 thrombocytopenia). Clearance was independent of body surface area; thus, a flat dose of 7.0 mg was used during expansion. Myelosuppression, mostly grade 4 neutropenia, occurred in 40% of patients but was transient and manageable, and none was febrile. All other toxicity was mild and fatigue, nausea and vomiting were the most common at the recommended dose. Pharmacokinetic parameters showed high interindividual variation, though linearity was observed. At or above the recommended dose, the myelosuppressive effect was significantly associated with the area under the concentration-time curve from time zero to infinity (white blood cells, P = 0.0007; absolute neutrophil count, P = 0.016). A partial response was observed in one patient with pancreatic adenocarcinoma at the recommended dose.

Conclusion: A flat dose of 7.0 mg is the recommended dose for PM01183 as a 1-hour infusion q3wk. This dose is tolerated and active. Severe neutropenia occurred at this dose, although it was transient and with no clinical consequences in this study. Clin Cancer Res; 20(8); 2205–14. ©2014 AACR.

Purpose: GVHD after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4+CD25+FoxP3+ regulatory T cells (Tregs). Because Tregs express high levels of the interleukin (IL)-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T effector T-cell populations, thereby preventing GVHD.

Experimental Design: We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related (n = 12) or unrelated (n = 4) donor grafts received ULD IL-2 post hematopoietic stem cell transplantation (HSCT; 100,000–200,000 IU/m2 x3 per week), starting <day 30 and continuing for 6 to 12 weeks.

Results: No grade 3/4 toxicities were associated with ULD IL-2. CD4+CD25+FoxP3+ Tregs increased from a mean of 4.8% (range, 0%–11.0%) pre IL-2 to 11.1% (range, 1.2%–31.1%) following therapy, with the greatest change occurring in the recipients of matched related donor (MRD) transplants. No IL-2 patients developed grade 2–4 acute GVHD (aGVHD), compared with 4 of 33 (12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2 of 13 (15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (P = 0.022).

Conclusions: Hence, ULD IL-2 is well tolerated, expands a Treg population in vivo, and may be associated with a lower incidence of viral infections and GVHD. Clin Cancer Res; 20(8); 2215–25. ©2014 AACR.

Purpose: Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206.

Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA).

Results: In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% confidence interval, 0%–17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drug-related toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%–45%) and limited inhibition of downstream targets.

Conclusions: Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored. Clin Cancer Res; 20(8); 2226–35. ©2014 AACR.