Biomarkers Of Clinical Response Research Articles

Rituximab (Rituxan) Therapy for Severe Thyroid-Associated Ophthalmopathy Diminishes IGF-1R+ T Cells

Rituximab depletes CD20(+) B cells and has shown potential benefit in thyroid-associated ophthalmopathy (TAO). The impact of rituximab on T cell phenotype in TAO is unexplored. The objective of the study was to quantify the abundance of IGF-I receptor-positive (IGF-1R(+)) CD4 and CD8 T cells in active TAO before and after treatment with rituximab. This was a retrospective case series assessing IGF-1R(+) T cells before and after treatment with rituximab with an 18-month follow-up. The study was conducted at a tertiary care medical center. Study participants included eight patients with severe TAO. Two infusions of rituximab (1 g or 500 mg each) were administered 2 weeks apart. Quantification of IGF-1R(+) T cells using flow cytometry was measured. Eight patients with moderate to severe TAO [mean pretreatment clinical activity score (CAS) 5.1 ± 0.2 (SEM)] were treated. Four to 6 weeks after treatment, CAS improved to 1.5 ± 0.3, whereas the proportion of IGF-1R(+) CD3(+) T cells declined from 41.9% to 28.3% (P = .004). The proportion of IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) T cells declined 4-6 weeks after treatment (from 45.6% to 21.5% and from 32.0% to 15.8%, P = .003 and P = .001, respectively). In two patients, IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) subsets approximated pretreatment levels after 16 weeks. Frequency of IGF-1R(+) T cells in patients with TAO declines within 4-6 weeks after rituximab treatment. This phenotypic shift coincides with clinical improvement. Thus, assessment of the abundance of IGF-1R(+) T cells in response to rituximab may provide a biomarker of clinical response. Our current findings further implicate the IGF-1R pathway in the pathogenesis of TAO.

Killer-Cell Immunoglobulin-Like Receptor Expression on Lymphocyte Subsets in Multiple Sclerosis Patients Treated with Interferon-β: Evaluation as Biomarkers for Clinical Response

Both the adaptative and the innate immune systems interplay in multiple sclerosis (MS) pathogeny. Killer-cell immunoglobulin-like receptors (KIRs) are key regulators of the immune response, with activating and inhibitory isoforms. In this study we analysed whether the expression of KIR isoforms is implicated in MS pathogenesis and in the therapeutic response to interferon (IFN)-β. Peripheral blood samples were collected from 78 IFN-β-treated MS patients and 46 healthy controls (HC). KIR expression was evaluated by flow cytometry on natural killer (NK) and T cells. The expression of KIRs on NK cells and T lymphocytes did not differ between MS patients and HC. IFN-β therapy decreased the expression of KIR2DL1/2DS1 and increased that of KIR2DL2/3 on NK cells. This therapy also reduced KIR2DL1/2DS1, KIR2DL2/2DL3 and KIR3DL2 expression on CD8(+) T cells. The baseline evaluation of the percentage of circulating CD16(+) NK cells was predictive of the clinical response to IFN-β; however, response to this therapy did not appear related to KIR expression. This study shows that expression of KIR isoforms on NK and T lymphocytes correlated in different ways with IFN-β therapy, suggesting that KIR dynamics may be associated with the pathways involved in the mechanisms of action of IFN-β.

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome

Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1–5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration.

Regulation of CD4+NKG2D+ Th1 Cells in Patients with Metastatic Melanoma Treated with Sorafenib: Role of IL-15Rα and NKG2D Triggering

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4(+)NKG2D(+) T cells. Hence, the increase of blood CD4(+)NKG2D(+) T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4(+)NKG2D(+) subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHC class I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D(+) Th1 cells.

RNA-Sequencing Of Paired Pre-Treatment and Relapse Samples Reveals Differentially Expressed Genes Associated With Lenalidomide Resistance In Multiple Myeloma

BackgroundImmunomodulatory (IMiDs) drugs' cytotoxicity in MM cells is mediated through their binding to cereblon (CRBN) an adaptor protein of the Cul4a-DDB1-ROC1 ubiquitin E3 ligase complex. Loss of CRBN is clearly associated with resistance to IMiDs (Zhu et al. Blood. 2011) however this does not appear to be the sole mechanism through which MM cells may acquire resistance to this class of drugs. In addition, the acquisition of CRBN mutations within the Thalidomide binding domain (exons 10-11) have not been well studied. Similarly and while splice variants of CRBN were recently reported (Ghandi et al. Blood. 2012), no correlations has been made between the presence of these isoforms and IMiDs resistance in primary samples. Methods and ResultsIn this study, we first validated CRBN as biomarker of clinical response to lenalidomide. At the mRNA level, using qRT-PCR (n=26, amplicons with 2 sets of primers overlapping exons 8-9 and exons 10-11) low CRBN in CD138 sorted bone plasma cells was significantly associated with shorter PFS (p=0.008) and lack of response to lenalidomide. We next compared CRBN mRNA (qRT-PCR) expression in paired samples (n=17 patients - 34 pairs) collected immediately pre-treatment and at the time of disease progression post-lenalidomide. In 10/17 (58.8%), a significant reduction (2-ΔΔCT < 0.8) in the CRBN amplicon levels was observed between the paired pre- and post-treatment samples. These data suggest that in nearly half of the patients, CRBN-independent mechanisms may be mediating resistance to IMiDs. In order to elucidate these CRBN-independent mechanisms of resistance to lenalidomide and identify potential targets that may overcome it, RNA-seq analysis was performed in 12 paired patients samples obtained sequentially from the same patient prior to lenalidomide treatment initiation and after development of resistance. Transcriptome sequence data was generated by RNA-seq performed for each sample on Ion Torrent Proton sequencer with a minimum of 70 million reads per sample. Poly(A) is captured with poly(T) magnetic beads, fragmented and copied to cDNA libraries with reverse transcriptase and primers. Filtered Fastq files are processed with TopHat-Fusion alignment against hg19 as reference genome. Cufflinks and Cuffdiff algorithms were used to detect differentially expressed transcripts and spliced isoforms. A total of 830 genes were identified as differentially expressed (p value and FDR <0.05) between the pre- and post-lenalidomide paired samples. Selected differentially expressed genes were also measured by RT-PCR analysis to confirm the validity of the RNA-seq analysis. In order to gain insight into the cellular and molecular functions of this identified gene set, we compared it to the expression of 395 gene sets curated in the Molecular Signatures Database (MSigDB), using the Gene set enrichment analysis (GSEA) algorithm. Among the most enriched gene sets in this analysis were KRAS.KIDNEY_UP.V1_UP; KRAS.LUNG_UP.V1_UP; KRAS.600.LUNG.BREAST_UP.V1_UP; RAF_UP.V1_DN; MEK_UP.V1_DN; and STK33_DN suggesting a role for the RAS-RAF-MAPK pathway in the acquired resistance to IMiDs. Other gene sets of interest included AKT_UP_MTOR_DN.V1_DN, E2F3_UP.V1_UP; EIF4E_UP and RPS14_DN.V1_DN; consistent with a role for the translational machinery activity (mTOR-4EBP1-eIF4E pathway) in IMiDs resistance. Of interest variant splice isoforms of CRBN were visualized using the Integrative Genomics Viewer (IGV) tool including isoforms lacking exon 10, which contains a portion of the IMiD-binding domain. However these CRBN splice variants are unlikely to be implicated in resistance to IMiDs since they were not enriched in the relapsed paired samples. ConclusionsStudy of the transcriptome of paired pre- and post-IMIDs in myeloma primary cells confirms that acquired resistance to this class of drugs is associated with the direct loss of CRBN as well as through the modulation of other CRBN-independent pathways. Disclosures:No relevant conflicts of interest to declare.

FRI0247 Serum level of interleukin 33, a novel independent predictive biomarker of clinical response to rituximab in rheumatoid arthritis: results from the smart trial

BackgroundInterleukin-33 (IL33) is a recently identified member of the interleukin-1 family that binds to its ST2 receptor expressed on various cells including mast cells, T-cells and also B1-cells in mice...

Sera cytokine levels to predict survival in men with progressive castration-resistant prostate cancer.

5083 Background: Serum cytokines have been proposed as immunologic biomarkers of clinical responses based on their role in tumor biology. Recently, two whole blood mRNA signatures, which included immunomodulatory gene transcripts, were found to be predictive of survival in CRPC. This study explores serum cytokines, measured with analytically valid assays, as prognostic biomarkers in CRCP. Methods: Serum was collected from 75 progressive CRPC patients treated at MSKCC. A panel of 10 cytokines (M-CSF, IFN-γ, TNF-α, IL-1 β, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-13) was measured in a CLIA-certified laboratory by clinically validated ELISAs. To create a risk group classification based on the 10 cytokines, a regression tree methodology was used with the intent to maximize the survival differences between risk groups. In addition to the cytokine risk groups, PSA, LDH, albumin, hemoglobin and CTC enumeration were also independently prognostic. The Cox model was developed to determine the factors that jointly predicted survival. The concordance probability estimate (CPE) was used to determine the discriminatory power of this model. Results: Among the 10 cytokines, M-CSF (stimulates monocytes and macrophages) and IL-10 (suppresses T-cell-mediated anti-tumor responses), were most predictive of overall survival in a 3 risk-group model. The relative risk for log CTC was 1.78 (95% CI 1.43 – 2.21). The combination of the cytokine risk groups and CTC enumeration provided the best discriminatory power for predicting survival, yielding a discrimination index measured by the CPE equal to 0.77 (se = 0.03). Conclusions: A risk group classification, based on two serum cytokines, M-CSF and IL-10, and log CTC measured by analytically valid assays, predicted survival in patients with progressive CRPC. These cytokines may reflect the biology within the tumor microenvironment, and may also serve as biomarker for clinical benefit. Independent validation in a similar cohort of patients is ongoing. [Table: see text]

NOTCH1 as a potential prognostic biomarker for anti-VEGF therapy in patients with metastatic colorectal cancer.

11038 Background: There are no validated biomarkers for clinical response or survival benefit in patients treated with bevacizumab (Bv) in advanced metastatic colorectal cancer (mCRC). The aim of this study was to evaluate the predictive value of putative biomarkers in mCRC. Methods: One hundred and five mCRC patients who received Bv combined with FOLFOX or FOLFIRI were retrospectively evaluated for clinical and pathological characteristics. VEGFR1, VEGFR2, VEGFR3, PlGF, DLL4 and NOTCH1 expression were assessed by immunohistochemistry on formalin-fixed, paraffin-embedded neoplastic tissue of either primary or metastatic tissue in a tissue microarray. High levels of expression were defined as less than or equal to or more than the median. Survival curves were calculated by the Kaplan-Meier method and compared by the log-rank test. For multivariate analysis the Cox proportional hazards model was used. Results: Grade 1 or 2 (p=0.01), non-mucin-producing histology (p=0.04) and presence of liver metastasis (p=0.001) were associated with a higher response rate. There was no difference between the expression of markers and the response rate. ECOG 0 or 1 (p=0.002), grade 1 or 2 (p=0.02), liver metastasis (p=0.003), no lymph node metastasis (p=0.01) no peritoneal metastasis (p=0.02) and resection of metastasis (p&lt;0.001) were correlated with higher progression-free survival (PFS). There was also a strong correlation between ECOG 0 or 1 (p=0.001), grade 1 or 2 (p=0.006), no lymph node metastasis (p=0.004), liver metastasis (p&lt;0.001) and resection of metastasis (p&lt;0.0001) with better overall survival. There was a trend between high expression of NOTCH1 (p=0.06) and worst PFS.High expression of VEGFR2 (p=0.07) was slightly associated with a better overall survival, while high expression of NOTCH1 was associated with a worse overall survival (p=0.01). Using multivariate analysis, NOTCH1 proved to be an independent variable for adverse overall survival (HR 2.01, IC 1.07 – 3.77, p=0.02). Conclusions: High NOTCH1 expression assessed by immunohistochemistry is capable of predicting poor survival in advanced colorectal cancer patients treated with bevacizumab.

NRAS mutation: A potential biomarker of clinical response to immune-based therapies in metastatic melanoma (MM).

9019 Background: NRAS mutant (mut) MM comprises a distinct, molecularly defined cohort of this disease that appears to have a poor prognosis compared to other genetic subsets. In contrast to BRAF mut MM, there are no effective small molecule inhibitors specifically targeting NRAS. Immune based therapy (IT) has become a mainstay in MM treatment, especially in BRAF wild type (WT) patients (pts). Biomarkers to predict which pts will benefit from IT have not been validated. The goal of this study was to evaluate whether genetic subtype (specifically NRAS) has a role in predicting benefit from IT in BRAF WT MM. Methods: We identified 173 pts from 3 institutions who underwent clinical genotyping (exome or PCR based) for NRAS and BRAF mutation from 1/09 – 8/12 and were treated with IT (defined as IL-2, ipilimumab (ipi), or anti-PD-1 (nivolumab, MK3475)/ PD-L1 (MPDL3280A)). Only BRAF WT pts were included. Primary endpoints were response rate (RR) and clinical benefit rate (CBR), defined as RR + stable disease lasting &gt;24 wks to IT (best response as assessed by treating clinicians in any line of IT). Secondary endpoints were overall survival (OS) and progression-free survival (PFS) from first line IT. Results: Of the 173 pts, 59 had NRAS mut MM compared to 114 WT/WT (no mutation in NRAS or BRAF). Improved clinical outcomes were seen in the NRAS mut compared to WT/WT cohort in terms of RR (32% vs. 18%, p=0.042), and CBR (49% vs. 30%, p=0.012). Improvements in PFS and OS did not reach statistical significance. By specific IT, NRAS mutation predicted benefit compared to WT/WT for anti-PD-1/PD-L1 (RR 78% vs. 19%, p=0.002; CBR 78% vs. 29%, p=0.013, n=30) and ipi (RR 18% vs. 12%, p=0.315; CBR 41% vs. 22%, p=0.018, n=137). No significant differences were observed with IL-2 (RR 33% vs. 28%, p=0.730; CBR 33% vs. 39%, p=0.741, n=33). Conclusions: This study demonstrates that pts with NRAS mut MM achieve increased clinical benefit from IT compared to pts with BRAF/NRAS WT MM. A larger, prospective analysis is necessary to validate and expand on these results, including those with BRAF mut and KIT mut MM. However, our data suggest that NRAS mutation status may be a biomarker of response to IT in MM and that molecularly targeted immunotherapy may be feasible.

7P - Detection and Quantitation of Circulating Tumor Cells, Cancer Stem Cells and Endothelial Progenitor Cells Using Multiparameter Flow Cytometry

Advances in medical technology and an increased understanding of tumor cell biology have led to the discovery of promising cellular biomarkers including circulating tumor cells (CTCs), cancer stem cells (CSCs) and endothelial progenitor cells (EPCs). CTCs and CSCs provide an early, reliable indication of disease progression and survival for patients on systemic therapy for metastatic breast cancer, whereas levels of EPCs are reported to correlate with tumor stage and have been evaluated as biomarkers of the efficacy of anticancer/ antiangiogenic treatment. The aim of this study was to establish practical methods for detection and quantitation of CTCs, CSCs and EPCs using multiparameter flow cytometry. In brief, immediately after enrichment by density gradient centrifugation, cells were stained with 2 sets of fluorescently conjugated antibodies; against CD34, VEGFR2 and CD45 for EPC and against EpCAM, CD44, CD24 and CD45 for CTCs and CSCs. The labeled cells were analyzed on a flow cytometer by collecting 1,000,000 events. For CTCs detection and quantitation, spiking experiments with breast cancer cell line MCF-7 demonstrated a linear performance with an accepted reproducibility. CTCs and CSCs were defined as CD45-EpCAM+ and CD45-CD44 + CD24-, respectively. These protocols could be used for detection of cancer stem cells in enzyme digested tissues from breast cancer patients. EPCs (CD45dim CD34 + VEGFR2+) were significant higher in breast cancer patients compared with the cancer-free controls. These simple and sensitive multiparameter flow cytometry protocols could be used for detection and quantitation of CTCs, CSCs and EPCs in breast cancer patients. However, clinical validation studies should be performed before their implementation as potential surrogate biomarkers of clinical response and prognostic markers. Disclosure: All authors have declared no conflicts of interest.

HLA DR and DQ alleles and haplotypes associated with clinical response to glatiramer acetate in multiple sclerosis

ObjectiveClinical response to immunomodulatory therapies in multiple sclerosis (MS) is variable among patients. Currently, there are no validated biomarkers of clinical response to any of the approved treatments for MS. The objective of this study was to determine if HLA-class II alleles predict the clinical response to glatiramer acetate (GA). MethodsThis was a prospective study of 64 MS patients with relapsing-remitting disease. Patients were HLA-typed and classified as GA-responders or non-responders after 2 years of treatment based on a clinical criterion. Statistical models were used to determine whether HLA-DR and DQ alleles and haplotypes predict the clinical response to GA. ResultsTests of association of response singled out four alleles and two haplotypes with nominal p<0.01. The presence of alleles DR15 or DQ6 or the absence of DR17 and DQ2 alleles was associated with favorable clinical response. The presence of the DR15−DQ6 haplotype and the absence of the DR17–DQ2 haplotype were also associated with favorable treatment response. A best fitting two-haplotype model resulted in the identification of three prognostic categories (good, neutral, and poor). A DR15-DQ6 positive but DR17-DQ2 negative combination was strongly predictive of a favorable clinical response (71%). Conversely, a DR15-DQ6 negative but DR17-DQ2 positive combination was strongly predictive of poor clinical response to GA (17%). ConclusionHLA-DR and DQ typing may prove to be useful biomarkers of predicting response to GA in MS and may help select patients appropriate for this treatment.

GSK-3 Spatial Regulation Has an Essential Role in Lenalidomide Activity and Represents a Biomarker of Clinical Response in CLL

AbstractAbstract 1793Thalidomide (THAL) and IMID® immunomodulary drugs lenalidomide (LEN) and (POM) have proven beneficial in the treatment of a variety of hematological malignancies. Pre-clinical studies demonstrate multiple direct and indirect anti-tumor activities including anti-angiogeneic, proapoptotic, anti-proliferative and immunomodulatory effects. Recent studies have identified cerebron (CRBN) as a potential direct physical target of THAL and IMiD compounds and CRBN expression is reportedly required for IMiD compound activity. However, the precise link between CRBN and IMiD compound mechanism of action (MOA) have not been clearly defined. We applied Drosophila as a drug discovery platform to assess the MOA of THAL and IMiD compounds in vivo. THAL or POM fed Drosophila demonstrate morphological phenotypes that replicate wingless (wg) mutants indicating that drugs are inhibitors of Wg/Wnt signaling. In this model system, THAL and IMiD compounds disrupt membrane localization of GSK-3 indicating that the bioactivity of IMiD compounds is achieved through spatial regulation and potentiation of Sgg/GSK-3 in Drosophila. Using epistasis analysis we show that Drosophila expressing genetic mutants lacking GSK-3 activity and myeloma cells in which GSK-3a and GSK-3b have been knocked down by siRNA fail to respond to POM. In both Drosophila and myeloma cells therefore it appears that GSK-3 activity is required for biological responses. To test the clinical validity of GSK-3 as a biomarker, we obtained patient tumor samples from a Phase II clinical trial of single agent LEN for previously untreated CLL (Chen CI et al., J Clin Oncol, 2011; 29:1175). Twenty five patients were enrolled onto this study and received LEN at a starting dose of 2.5 mg days 1–21 of a 28 day cycle with monthly escalation to a target dose of 10 mg. The primary clinical endpoint for the trial was objective response to lenalidomide (complete response (CR) and partial response (PR)) evaluated as defined in the revised 1996 NCI Working Group guidelines. Peripheral blood samples for correlative studies were collected on days 1 (pre-dosing) and 8 of cycles 1 and 2. CRBN expression was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 patient samples regardless of LEN response. Thus CRBN expression does not appear to be a useful predictive biomarker of response in this population of previously untreated patients. However, GSK-3 localization was correlated with response. Paired analysis of CD19 selected CLL cells comparing day 1 vs day 8 revealed focal membrane localization of GSK-3 on day 1 and subcellular redistribution on day 8 in 11 out of 12 evaluable responders (PR or better). By contrast, in the CLL cells from all 6 evaluable non-responders, GSK-3 expression appeared diffusely distributed before and after treatment. In preliminary studies using confocal immunofluorescence microscopy we determined that CRBN and GSK-3 co-localize in day 1 CLL samples of responders but not in those of non-responders. In summary, our results indicate that THAL and the IMiD compounds target GSK-3 function by spatial regulation and identify GSK-3 localization as a potential clinical biomarker of IMiD response. Disclosures:Trudel:Celgene: Honoraria; GlaxoSmithKline: Research Funding; Janssen: Honoraria. Mercurio:Celgene: Equity Ownership, Research Funding. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Webb:Celgene: Employment, Equity Ownership. Chen:Johnson & Johnson: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Manoukian:Celgene: Research Funding.

Immune response during interferon beta-1b treatment in patients with multiple sclerosis who experienced relapses and those who were relapse-free in the START study

We measured immune markers in subjects with multiple sclerosis (MS) treated with IFNβ-1b for 12months. IL-17 levels were significantly higher at Month 6 (p=0.036) in relapsing subjects while BDNF levels were significantly higher at Month 3 (p=0.028) in relapse-free subjects. Change from baseline in IL-4 levels inversely correlated with disability score whereas change from baseline in IL-10/IFN-gamma ratio inversely correlated with occurrence of relapses. CXCR3+CD8+ T-cells tended to be higher but declined with treatment in relapse-free compared with relapsing subjects. Findings show the potential of cytokine and neurotrophic factors as biomarkers of clinical response to IFNβ-1b.

Using companion and coupled diagnostics within strategy to personalize targeted medicines.

Regulatory authorities expect the pharmaceutical and biotechnology industries to accelerate their development of companion diagnostics and companion therapeutics towards the goal of personalized medicine, and expect health services to fund, prescribers to adopt and patients to accept these new therapeutic technologies. Expected benefits from more systematic development of combination products (companion diagnostic and its companion therapeutic) are expected to include safer and improved clinical and cost-effective use of medicines, more efficient patient selection for clinical trials, more cost-effective treatment pathways for health services, and a more profitable approach for drug developers. This review discusses challenges to timely development of companion diagnostics and provides case studies of single and multiple protein and genetic biomarkers of clinical response and risk of adverse drug effects.

Maximum-Tolerated Dose, Optimum Biologic Dose, or Optimum Clinical Value: Dosing Determination of Cancer Therapies

Maximum-Tolerated Dose, Optimum Biologic Dose, or Optimum Clinical Value: Dosing Determination of Cancer Therapies

Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study

Methotrexate is activated by the sequential addition of glutamic acid residues to form methotrexate polyglutamates (MTXPG(1-5)). MTXPG(1-5) inhibit enzymes of the folate-purine-pyrimidine pathways, and longer-chain MTXPG(3-5) species are more active. To determine the pattern of erythrocyte MTXPG(1-5) in patients initiated on oral methotrexate for psoriasis, and to investigate the potential utility of MTXPGs as markers of compliance and/or clinical response. This was a single-centre, prospective study of 55 adult patients with chronic plaque psoriasis initiated on weekly oral methotrexate. Erythrocyte MTXPG(1-5) concentrations were measured (at weeks 4, 8, 12, 24 and 52) using high-performance liquid chromatography. Methotrexate responders achieved ≥ 50% improvement in Psoriasis Area and Severity Index or physician's global score of 'clear'/'nearly clear' at 24 weeks. MTXPG levels were measured in 14-33 patients at each time point. All MTXPG(1-5) species were detected at week 4 of therapy. Steady state for long-chain MTXPG(3-5) and total MTXPG(1-5) was achieved by week 24. MTXPG(3) emerged as the predominant MTXPG species (from week 12 onwards) and reflected overall polyglutamate status (correlating strongly with MTXPG(2-5) , MTXPG(3-5) and MTXPG(4-5) ; R = 0·76-0·95, P < 1·55 × 10(-5)). Age, renal function and sex were not significant determinants of MTXPG(3) concentration. No significant association was identified between MTXPG and adverse events or responder status. This is the first study to demonstrate the prospective accumulation of MTXPG(1-5) in patients with psoriasis. The detection of MTXPGs early in therapy and the establishment of a steady state with continuous treatment may offer measuring of MTXPG as a test to monitor patient compliance with therapy. Larger studies are required to determine the role of MTXPG as a potential biomarker of clinical response.

Predictive neural biomarkers of clinical response in depression: A meta-analysis of functional and structural neuroimaging studies of pharmacological and psychological therapies

We performed a systematic review and meta-analysis of neural predictors of response to the most commonly used, evidence based treatments in clinical practice, namely pharmacological and psychological therapies. Investigations of medication-free subjects suffering from a current major depressive episode who underwent positron emission tomography (PET) or functional or structural magnetic resonance imaging (MRI) scans prior to the initiation of treatment were reviewed. Results of 20 studies from 15 independent samples were included in the functional imaging meta-analysis and 9 studies from 6 independent samples in the structural neuroimaging meta-analysis. Regional activations with prognostic value include the well replicated finding that increased baseline activity in the anterior cingulate is predictive of a higher likelihood of improvement. As well, increased baseline activation in the insula and striatum is associated with higher likelihood of a poorer clinical response. Structural neuroimaging studies indicated that a decrease in right hippocampal volume is a statistically significant predictor of poorer treatment response. Overall, the predictive information that is measurable with brain imaging techniques is both multimodal and regionally distributed as it contains functional as well as structural correlates which encompass several brain regions within a frontostriatal–limbic network. To develop clinically relevant, prognostic markers will require high predictive accuracy at the level of the individual. Predicting clinical response will help to stratify patients and to identify at an early stage those patients who may require more intensive or combined therapies. We propose that structural and functional neuroimaging show significant potential for the development of prognostic markers of clinical response in the treatment of depression.

P.1.003 Brain-derived neurotrophic factor as a biomarker of clinical response in bipolar disorder: 16-week follow-up study

Simultaneous infusion of the selective 5-HT2A-receptor antagonist M100907, as well as asenapine, completely blocked the DOI-induced increase in dopamine and serotonin release and partially the increased noradrenaline release, indicating a similar mechanism by M100907 and asenapine. When given alone, infusion of M100907 or asenapine, in the concentrations used, did not affect monoamine release. Intracortical administration of the selective a2adrenoceptor agonist clonidine significantly decreased dopamine, noradrenaline and serotonin release in the mPFC (maximal effects; 61.0±8.0, 50.4±7.6, and 67.0±4.1% of baseline, respectively; p< 0.01–0.001). Simultaneous infusion of the a2-adrenoceptor antagonist idazoxan blocked the clonidine-induced reduction of dopamine and noradrenaline release. The effect of idazoxan on serotonin was less pronounced and not statistically significant. Simultaneous infusion of asenapine did not acutely block the clonidine-induced decrease in transmitter release. However, following termination of the clonidine infusion the transmitter release remained at a low level. During this interval, i.e. the following hour after the clonidine infusion, intracortical infusion of asenapine restored the catecholamine release to baseline level, indicating an a2-adrenoceptor blocking effect of asenapine which was somewhat delayed. When given alone, the infusion of idazoxan or asenapine, in the concentrations used, did not affect monoamine release. Our results propose that local application of asenapine in the mPFC indeed exhibits a pharmacological significant 5-HT2Aand, to a weaker extent, a2-receptor antagonistic activity. Whereas its 5-HT2A blocking property preferentially influenced the release of serotonin and dopamine but only to a lesser extent noradrenaline, blockage of a2-adrenoceptors preferentially influenced dopamine and noradrenaline release, albeit the effect was somewhat delayed. Thus, 5-HT2A-receptor antagonism and a2-adrenoceptor blockage induced by asenapine in the mPFC may contribute to enhance prefrontal monoamine release in vivo and, secondarily, its effect on positive and negative symptoms as well as pro-cognitive and antidepressant effects [1,2].

Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K i values, 3–26 μM), hENT1/2 and hCNT2 weakly (K i values, 0.5–3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [3H]gemcitabine, [14C]azacitidine, and [3H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC 50 values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ≫ hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ⋙ hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was observed for both drugs in the presence of inhibitors of nucleoside transport, thus suggesting the importance of hNTs in manifestation of toxicity. In summary, all seven hNTs transported azacitidine, with hCNT3 showing the highest rates, whereas hENT1 and hENT2 showed modest transport and hCNT1 and hCNT3 poor transport of decitabine. Our results show for the first time that azacitidine and decitabine exhibit different human nucleoside transportability profiles and their cytotoxicities are dependent on the presence of hNTs, which could serve as potential biomarkers of clinical response.

P-179 - Brain-derived neurotrophic factor as a biomarker of clinical response in bipolar disorder: 16 week follow-up study

Despite therapeutic advances, we still have difficulties in predicting response to treatment in bipolar disorder (BD). Brain-Derived Neurotrophic Factor (BDNF) has been put forward as a potential peripheral marker of treatment response. To prospectively study the relation between clinical response to treatment and serum BDNF levels. To investigate a) a possible association between serum BDNF levels and clinical response along 16 week follow-up and b) the role of val66met polymorphism in clinical response in a sample of drug-free patients with BD going through a mood episode. This is a naturalistic, open-label prospective nested case control study matched for age, gender and ethnicity. Patients were 18 years or older, required BD diagnosis, undergoing a current manic, mixed or depressive episode and be off-medication for at least 2 weeks. Clinical assessment and blood withdrawn were conducted along follow-up. At the end of the study, patients were classified according to clinical response. 25 of 36 (69.4%) of the patients were female and the mean age was 37.8 (SD 11.8) years old. Baseline serum BDNF levels did not show any difference between patients and controls ( p = 0.075).There was a significant negative correlation between differences in serum BDNF levels and in CGI score along follow-up ( r = −.372, p = 0.028). Serum BDNF levels were significant higher in responders compared to non-responders at week 4, 8 and 16 ( p = 0.026, p = 0.009, p = 0.001 respectively). Val66met polymorphism did not seem to interfere in clinical response. Changes in serum BDNF levels may help in monitoring treatment response.