Potential Pharmacodynamic Biomarkers Research Articles

A CRUK first-in-human phase I trial of a CDC7 Inhibitor, LY3143921 hydrate, in patients with advanced solid tumors.

TPS3167 Background: CDC7 is a protein with key roles in DNA replication initiation, the intra-S-phase checkpoint and M-phase completion. CDC7 is over-expressed in malignant compared to non-malignant cells, particularly those with TP53 mutations, making it an attractive therapeutic target. LY3143921 hydrate is an orally administered ATP-competitive CDC7 inhibitor. Pre-clinical studies in colorectal cancer (CRC) and squamous non-small cell lung cancer (sqNSCLC) demonstrate favourable anti-cancer activity, particularly in squamous NSCLC and in CRC with TP53 null and missense mutations. We hypothesise that solid tumours mutated in TP53 will be sensitive to LY3143921 therapy. Methods: This is a first-in-human, phase I trial of LY3143921 hydrate (LY3143921) monotherapy given twice daily, continuously on a 21 day schedule until disease progression, patient (pt) withdrawal or unacceptable toxicity (NCT03096054). Eligible pts have histologically proven advanced/metastatic solid tumours for which no further standard therapy exists and WHO PS 0-1. Pts have regular clinical assessment and tumour imaging every 2 cycles. Phase Ia (dose escalation) is recruiting in a 3+3 design following 3 initial single patient cohorts (starting dose 30 mg OD), enriching for patients with malignancies associated with p53 mutations (CRC, sqNSCLC, high grade serous ovarian, squamous cell oesophageal, squamous cell head & neck, urothelial, pancreatic and triple negative breast cancer). Recruitment to cohort 6 (180 mg BD) is ongoing. On determination of the maximum tolerated dose (MTD) and recommended phase II dose and schedule (RP2D), 2 expansion cohorts (≤ 25 pts each) of patients with CRC and sqNSCLC will be evaluated. Primary objectives: assess safety and tolerability of LY3143921, determine MTD and RP2D. Secondary objectives: evaluate preliminary efficacy and PK profile of LY3143921. All pts will have archival tumour tissue retrospectively analysed, while patients in phase Ib will also have pre- and on-treatment tumour biopsies. Evaluation of potential predictive and pharmacodynamic biomarkers including p53 mutations, phosphorylated MCM2, cyclin B1 and molecular subgroups of target tumours will be included. Clinical trial information: NCT03096054.

Circulating miRNA Signature as a Potential Biomarker for the Prediction of Analgesic Efficacy of Hydromorphone.

No practical biomarkers currently exist for the prediction of the analgesic efficacy of opioids. Previously, we reported circulating miRNA signatures differentially regulated by µ-opioid receptor (MOR) agonists in healthy subjects. We hypothesized that these miRNAs could be potential pharmacodynamic biomarkers to estimate MOR stimulation, and predict the efficacy of opioids; i.e., patients with low MOR stimulation may be more vulnerable to strengthening of the MOR signal upon hydromorphone treatment. To test this hypothesis, plasma samples were obtained from 25 patients with cancer pain prior to the initiation of hydromorphone treatment and the circulating miRNA levels were evaluated, focusing on four miRNAs (i.e., hsa-miR-423-3p, hsa-let-7a-5p, hsa-miR-26a-5p, and hsa-let-7f-5p) and four miRNAs (i.e., hsa-miR-144-3p, hsa-miR-451a, hsa-miR-215, and hsa-miR-363-3p) that were most clearly up and downregulated by hydromorphone and oxycodone. The patients were classified into two classes with putative high and low MOR signal, estimated based on the plasma miRNA signature. A significant correlation was observed between the analgesic efficacy and the putative MOR signal level, and patients with low MOR signal achieved better pain control (i.e., ΔVAS < 0) through hydromorphone. These results suggested that plasma miRNA signatures could serve as clinical biomarkers for the prediction of the analgesic efficacy of hydromorphone.

Therapeutic potential of the new TRIB3-mediated cell autophagy anticancer drug ABTL0812 in endometrial cancer

ObjectivesThe PI3K/AKT/mTOR pathway is frequently overactivated in endometrial cancer (EC). We assessed the efficacy of ABTL0812, a novel first-in-class molecule presenting a unique mechanism of action inhibiting this pathway. MethodsWe investigated the effects of ABTL0812 on proliferation, cell death and modulation of intracellular signaling pathways in a wide panel of endometrioid and non-endometrioid cell lines, an inducible PTEN knock-out murine model, and two patient-derived xenograft murine models of EC. Then, TRIB3 expression was evaluated as potential ABTL0812 pharmacodynamic biomarker in a Phase 1b/2a clinical trial. ResultsABTL0812 induced an upregulation of TRIB3 expression, resulting in the PI3K/AKT/mTOR axis inhibition and autophagy cell death induction on EC cells but not in healthy endometrial cells. ABTL0812 treatment also impaired PTEN knock-out cells to progress from hyperplasia to cancer. The therapeutic effects of ABTL0812 were demonstrated in vivo. ABTL0812 increased TRIB3 mRNA levels in whole blood samples of eight EC patients, demonstrating that TRIB3 mRNA could be used as a pharmacodynamic biomarker to monitor the ABTL0812 treatment. ConclusionsABTL0812 may represent a novel and highly effective therapeutic agent by inducing TRIB3 expression and autophagy in EC patients, including those with poorer prognosis.

Plasma-based exploratory biomarker analysis to evaluate GT0918 in a phase I/II study in mCRPC patients.

279 Background: GT0918, a novel potent androgen receptor antagonist, has been evaluated in a ph 1/2 study in mCRPC patients for its safety and efficacy. Here using patient plasma samples, we explored cell-free DNA &amp; RNA-based biomarkers to assess their associations with clinical response of GT0918. Methods: Blood samples were prospectively collected from mCRPC patients at baseline, on- and after study during the trial. PredicinePLUS cfDNA+cfRNA assay, a 180-gene targeted NGS Panel, was used to detect genomic alterations in plasma. Statistical analyses were performed in R 3.5.1. Results: Genomic alterations were detected in all 50 blood samples. Top mutated genes are TP53 (52.5%) and AR (32.5%). When combing variants from genes in a signaling pathway, mutation rates in PI3K axis and homologous recombination pathway are 32.0% and 34.0% respectively. Importantly, AR hotspot mutations (W742C, T878A, and S889G) and amplifications are detected in multiple subjects at baseline, suggesting that it may be the driving events for resistance of prior therapies. AR splicing variants (AR-V3, AR-V7) and TMPRSS2-ERG fusion were also detected by cfRNA profiling. Interestingly, patient 001-001 harboring both AR W742C and T878A mutations detected in cfDNA with high mutation frequency didn’t carry corresponding mutations in cfRNA at the baseline but started to accumulate the mutations in C3D1. Patient 004-002 remained in the study with stable disease for 10 treatment cycles while having continuous increase of PSA. cfDNA profiling indicates frequency of prostate-specific SPOP mutation continued to decrease during the treatment. Conclusions: This is a preliminary analysis to explore potential predictive or pharmacodynamic biomarkers in mCRPC in response to GT0918 treatment. As an non-invasive assay, PredicinePLUS assay was able to detect genomic alterations in blood with a high sensitivity and provided useful molecular insights for treatment monitoring and drug sensitivity &amp; resistance mechanisms. Clinical trial information: NCT02826772.

A Clinical Trial of the STAT3 Inhibitor Pyrimethamine in Chronic Lymphocytic Leukemia

Despite advances in targeted therapy, chronic lymphocytic leukemia (CLL) remains a highly prevalent and essentially incurable malignancy. One molecular hallmark of CLL is the constitutive serine phosphorylation and transcriptional activation of the oncogenic transcription factor STAT3. Targeting STAT3 may have a high therapeutic index since normal cells can tolerate a loss of STAT3 function. To identify STAT3 inhibitors that could be rapidly introduced into proof-of-concept clinical trials, we screened a chemical library of drugs known to be safe in humans for specific inhibitors of STAT3-dependent transcription. Using this strategy, we identified the anti-parasitic agent pyrimethamine as a drug that could inhibit STAT3 at levels safely achieved for months at a time in humans. Pyrimethamine inhibited STAT3-dependent gene expression in CLL cells ex vivo, and decreased survival of CLL cells, but not peripheral blood mononuclear cells from healthy donors. To determine whether STAT3 inhibition would confer clinical benefit in patients with CLL, we conducted a phase I clinical trial of continuous daily pyrimethamine, in the era before targeted therapy, in relapsed CLL patients whose disease progressed despite standard therapies. We used a typical 3+3 dose escalation design with three cohorts, 12.5, 25 and 50 mg per day (mg/d). Samples for PK/PD analysis were drawn weekly in the first month and every other week in the second. Sixteen heavily pretreated patients enrolled on the phase 1 portion of this study. The median age was 63 (36-85) and the median time from diagnosis to study therapy was 74 months (range, 6-176 months). Six patients had 17p deletion and 4 had 11q deletion, while 12 of 13 evaluable patients had unmutated IGHV. The patients had a median of 6 prior therapies, including 8 patients who had received prior high dose methylprednisolone, five who had received prior alemtuzumab, and one each with prior allogeneic and autologous stem cell transplantation. Three patients each enrolled on cohorts 1 and 2, and ten patients enrolled on cohort 3. There were no dose limiting toxicities and no significant drug-related toxicities. No objective responses by IW-CLL 2008 criteria were observed. Half of patients achieved stable disease, with one patient dosed at 50 mg/d on therapy for 12 months, and two at 25 mg/d on therapy for 4 and 6 months. The remaining patients had progressive disease, and all but one patient discontinued therapy for progressive disease. The median time on therapy was 1.1 months (0.23-9.99), with median progression free survival of 1.5 months (0.92-5.52) and median overall survival of 22 months (11.75-NA).To determine whether the levels of pyrimethamine achieved in patients corresponded with levels known to inhibit STAT3 transcriptional function in vitro, we measured trough concentrations of pyrimethamine in plasma and in peripheral blood mononuclear cells at distinct time points for patients on the trial. Plasma concentrations and intracellular concentrations were highly correlated, and also correlated closely with dose level. Only in the highest dose cohort did the plasma concentrations reproducibly approach the threshold of 10 micromolar at which STAT3 inhibition occurred consistently in vitro.To determine whether pyrimethamine was exerting an on-target effect in inhibiting STAT3-dependent gene expression in vivo, we first defined a signature of five STAT3-dependent genes (AIM2, ATXN1, ENPP2, GAB1, and ID3) that are upregulated in CLL cells relative to normal B lymphocytes. We then determined expression of these genes from the patients' CLL cells at baseline and while on pyrimethamine. In addition, to further generate predictors of response, we also quantitated the cytotoxic effect of pyrimethamine on the pre-treatment cells of patients ex vivo. We then correlated these potential predictive and pharmacodynamic biomarkers with drug levels and clinical course.In conclusion, pyrimethamine is an inhibitor of STAT3 transcriptional function that can safely be given to patients with CLL. It may be necessary to increase the daily dose of pyrimethamine to beyond 50 mg/day to adequately determine whether this drug can exert an on-target therapeutic effect in CLL patients. Since STAT3 target genes include many immune-suppressive and anti-apoptotic genes, it is likely that pyrimethamine will be most beneficial when combined with other therapeutic modalities. DisclosuresBrown:Pharmacyclics: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Boehringer: Consultancy; Sun Pharmaceutical Industries: Research Funding; Gilead: Consultancy, Research Funding; Loxo: Consultancy; Verastem: Consultancy, Research Funding; Invectys: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Janssen: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Genentech: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy. Wu:Neon Therapeutics: Equity Ownership.

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

Abstract 4839: Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes

Abstract Among heterogeneous cell population in a tumor, cancer stem cells (or tumor-propagating cells) are defined as a cell fraction that shows self-renewal, multipotency, high tumorigenicity, and resistance to chemo/radiotherapy. Because cancer stem cells are supposed to drive initiation, metastasis, and recurrence of the disease, identification of their therapeutic targets would have a profound implication for eradication of cancer. G-quadruplex (G4) is an atypical four-stranded nucleic acid structure that can be formed at guanine-rich sequences, such as the telomeric TTAGGG repeats. Accumulating evidence supports that G4s are widely distributed through the genome in living cells and affect various intracellular events, including DNA replication, gene expression and translation, whereas pharmacologic stabilization of G4s has been implicated for cancer intervention. However, precise mechanisms for the efficacy and target cancer types remain elusive. Here we demonstrate that G4 stabilization by chemical compounds, called G4 ligands, preferentially inhibits the growth of glioma stem cells in culture and in vivo. While the established glioma stem cells maintain stemness under the serum-free sphere culture conditions, serum stimulation induces their differentiation into non-stem glioma cells. We found that these glioma stem cells are highly sensitive to G4 ligands, such as a natural compound telomestatin and its newly synthesized derivative, and a bisquinolinium compound Phen-DC3, all of which can stabilize G4s. Upon the ligand treatment, glioma stem cells activated the replication stress pathway more potently than non-stem glioma cells, which could partly explain the selectivity of the deleterious effect. Consistently, these ligands induced DNA damage response in glioma stem cells but not in non-stem glioma cells. In glioma stem cells, about 20% to 30% of DNA damage foci were telomeric (i.e., telomere dysfunction-induced foci: TIFs) whereas the rest were non-telomeric, suggesting that the G4 ligands recognized both telomeric and non-telomeric G4s. Temozolomide, a clinical alkylating agent for brain tumors, induced DNA damage response but did not preferentially attack telomeres. As a potential pharmacodynamic biomarker, the immunofluorescence intensities of the nuclear G4 foci were enhanced by G4 ligands. Furthermore, these ligands directly inhibited in vitro transcription and translation of mRNAs that contained G4-forming sequences, suggesting additional mode of action. While G4 ligands inhibit telomerase activity, classical telomerase inhibitors without G4-stabilizing activity had no preferential impact on glioma stem cells. Together, these observations suggest that G4 is a promising target for pinpointing intractable glioma stem cells. Citation Format: Sachiko Okabe, Takahiro Nakamura, Daiki Hasegawa, Reina Kojima, Keiji Okamoto, Ichiro Nakano, Kazuo Shin-ya, Kazuo Nagasawa, Hiroyuki Seimiya. Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4839.

Abstract 4833: Pharmacodynamic and tumor biomarker analysis of a PLK1 inhibitor, PCM-075, in a phase 1b/2 trial for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic disease that is characterized by the accumulation of immature myeloid precursor cells. Treatment options for patients are selected based on factors such as age, comorbidities, and cytogenetics. For patients deemed ineligible for standard intensive induction chemotherapy, treatments include either low-dose cytarabine (LDAC) or hypomethylating agents (e.g., azacitidine and decitabine) but when used as single agents, relapses are inevitable. Polo-like Kinase 1 (PLK1), a serine/threonine kinase that is a master regulator of cell-cycle progression, is overexpressed in a number of cancer types including AML. Depletion of PLK1 preferentially induces cell death in tumor versus normal cells. Inhibition of PLK1 in preclinical AML models results in significant efficacy. PCM-075, a highly selective PLK1 inhibitor, is currently in a phase 1b/2 trial (NCT03303339) in combination with standard-of care (LDAC or decitabine) for AML. Collection and processing of blood and bone marrow samples from patients is being performed pre- and post-treatment to assess 1) the inhibition of PLK1 kinase activity and downstream effects by measurement of pharmacodynamic (PD) and mechanism of action (MOA) biomarkers (blood), and 2) the genomic profile of blast cell population(s) from individual patients (blood and bone marrow). For each patient, blood is collected into CellSave, EDTA, and PAXgene blood RNA tubes at various time points as per the clinical protocol schedule of events. PBMCs isolated from CellSave blood tubes are used to assess the phosphorylation status of PD biomarkers using Western blot and phospho-flow cytometry. DNA extracted from blasts (EDTA tube) is used to detect somatic alterations and RNA extracted from the total cell population (PAXgene) is used to detect fusions and determine gene expression profiles by next-generation sequencing. To identify potential PD biomarkers that would enable assessment of PLK1 inhibition in AML patients treated with PCM-075, nine candidate substrates phosphorylated by PLK1 were screened in the AML cell line MV4-11 by Western blot using selected phospho-antibodies. Of these, phosphorylation of Translational Control Tumor Protein (TCTP) at Ser46 was both time- and dose-dependently inhibited by PCM-075. pTCTP detection by Western blot and phospho-flow cytometry was first optimized in PBMCs isolated from healthy donors and later in blast cells from AML patients resulting in a functional biomarker. Additional downstream MOA biomarkers of PCM-075 activity (DNA content, phospho-histone H3, cleaved caspase 3) and tumor DNA biomarkers that enable categorization of AML molecular subtypes have been validated. We present preliminary patient data from the ongoing phase 1b/2 trial and describe biomarker assays that will be used to evaluate target engagement of PCM-075 in AML patients. Citation Format: Maya Ridinger, Karena Kosco, Latifa Hassaine, Jeffrey N. Miner, Mark Erlander. Pharmacodynamic and tumor biomarker analysis of a PLK1 inhibitor, PCM-075, in a phase 1b/2 trial for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4833.

Optimization and application of an HPLC method for quantification of inosine-5′-monophosphate dehydrogenase activity as a pharmacodynamic biomarker of mycophenolic acid in Chinese renal transplant patients

BackgroundThe immunosuppressive agent mycophenolic acid (MPA) is a non-competitive, reversible inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH). Thus, IMPDH activity can serve as a potential pharmacodynamic biomarker to optimize dosing of MPA. MethodsPeripheral blood mononuclear cells were isolated from 2 mL blood samples and an in vitro enzymatic reaction was subsequently performed for 120 min. To determine IMPDH activity in Chinese healthy volunteers and renal transplant patients, a high performance liquid chromatography assay was established and validated by subtracting adenosine monophosphate (AMP) from blank samples for eliminating exogenous AMP interference. ResultsThe accuracy of our method ranged between −0.8% and 12.5%, and the precision ranged between 0.7% and 6.3%. The mean value of IMPDH activity across 11 healthy volunteers was 46.60 ± 14.28 μmol/s/mol AMP. A negative relationship between MPA concentration and IMPDH activity was observed in four renal transplant patients treated with MPA 13 days post-transplantation, while the inhibitory rate of IMPDH activity ranged from 24% to 42%. ConclusionA bioanalytical assay for IMPDH quantification was optimized and evaluated. The differences in the pharmacodynamics of MPA between Asians and Caucasians may provide some evidence for dosing differences among ethnicities.

Preclinical Activity of Abemaciclib Alone or in Combination with Antimitotic and Targeted Therapies in Breast Cancer.

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2- and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2-, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2-, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897-907. ©2018 AACR.

Phosphodiesterase-5 inhibition potentiates cerebrovascular reactivity in chronic traumatic brain injury.

BackgroundTraumatic cerebrovascular injury (TCVI), a common consequence of traumatic brain injury (TBI), presents an attractive therapeutic target. Because phosphodiesterase‐5 (PDE5) inhibitors potentiate the action of nitric oxide (NO) produced by endothelial cells, they are candidate therapies for TCVI. This study aims to: (1) measure cerebral blood flow (CBF), cerebrovascular reactivity (CVR), and change in CVR after a single dose of sildenafil (ΔCVR) in chronic TBI compared to uninjured controls; (2) examine the safety and tolerability of 8‐week sildenafil administration in chronic symptomatic moderate/severe TBI patients; and as an exploratory aim, (3) assess the effect of an 8‐week course of sildenafil on chronic TBI symptoms.MethodsForty‐six subjects (31 chronic TBI, 15 matched healthy volunteers) were enrolled. Baseline CBF and CVR before and after administration of sildenafil were measured. Symptomatic TBI subjects then completed an 8‐week double‐blind, placebo‐controlled, crossover trial of sildenafil. A neuropsychological battery and neurobehavioral symptom questionnaires were administered at each study visit.ResultsAfter a single dose of sildenafil, TBI subjects showed a significant increase in global CVR compared to healthy controls (P < 0.001, d = 0.9). Post‐sildenafil CVR maps showed near‐normalization of CVR in many regions where baseline CVR was low, predominantly within areas without structural abnormalities. Sildenafil was well tolerated. Clinical Global Impression (CGI) scale showed a trend toward clinical improvement while on sildenafil treatment.FindingsSingle‐dose sildenafil improves regional CVR deficits in chronic TBI patients. CVR and ΔCVR are potential predictive and pharmacodynamic biomarkers of PDE5 inhibitor therapy for TCVI. Sildenafil is a potential therapy for TCVI.

NFAT-regulated cytokine gene expression during tacrolimus therapy early after renal transplantation.

Despite pharmacokinetic monitoring of calcineurin inhibitors, the long-term outcome after transplantation (Tx) is still hampered by the side effects of these drugs. The aim of the present study was to characterize nuclear factor of activated T cells (NFAT)-regulated gene expression as a potential pharmacodynamic biomarker for further individualization of tacrolimus (Tac) therapy. In 29 renal allograft recipients, samples were drawn once pre-Tx, and before and 1.5h after Tac dosing at approximately 1week, 6weeks and 1year post-Tx. Tac concentrations were measured by immunoassay, while the expression of genes encoding NFAT-regulated cytokines [interleukin 2 (IL2), interferon gamma (IFNG), colony stimulating factor 2 (CSF2)] and cytochrome P450 3A5 (CYP3A5) genotyping were determined by real-time polymerase chain reaction. The cytokine response after Tac dosing varied up to 46-fold between patients and changed significantly with time post-engraftment. Tac concentrations 1.5h postdose (C1.5 ) >15μg l-1 were associated with strong cytokine inhibition and residual gene expression (RGE) ≤10%, while lower Tac C1.5 resulted in more variable responses (RGE 2.5-68.7%). Patients with ongoing subclinical acute rejection (n = 5) demonstrated limited cytokine inhibition (RGE 39.7-72.6%), while patients with polyoma virus viraemia (n = 3) had relatively strong inhibition of cytokines (RGE 2.5-32.5%). By contrast, there was no association between Tac exposure and rejection or viraemia. The findings of our study support the potential of NFAT-regulated gene expression measurements as a pharmacodynamic tool for additional monitoring of Tac therapy, especially in the context of overimmunosuppression and viraemia.

POLY-GP DIPEPTIDE REPEATS AND NEUROFILAMENT LIGHT CHAIN AS BIOMARKERS IN PRESYMPTOMATIC AND SYMPTOMATIC FRONTOTEMPORAL DEMENTIA CAUSED BY C9ORF72 REPEAT EXPANSIONS

A pathogenic repeat expansion in chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Therapies that target C9orf72 repeat expansions will enter clinical trials this year and robust monitoring markers are critically needed, but to date, lacking. C9orf72 repeat expansions are translated via repeat-associated non-ATG (RAN) translation into repeating dipeptide units. One of these dipeptide-repeats, poly(GP), is detectable in the cerebrospinal fluid (CSF) of presymptomatic carriers and patients with C9orf72-ALS and thus shows promise as a potential pharmacodynamic biomarker. In contrast, neurofilament light chain (NfL), a marker for axonal death, is elevated in neurodegenerative diseases in general, including C9orf72-FTD and C9orf72-ALS, but remains at normal levels during the presymptomatic phase. Thus, we aimed to compare poly(GP) and NfL in presymptomatic and symptomatic carriers and identify potential associated gray matter deficits. poly(GP) and NfL were measured in CSF samples from GENFI sites and an American cohort: 12 controls, 25 presymptomatic C9orf72 carriers and 66 C9orf72 patients, including 38 behavioral variant FTD, 2 ALS, 9 FTD-MND. Presymptomatic carriers were required to have no behavioral, cognitive or motor changes. Comparisons of poly(GP) and NfL levels between groups and associations with clinical characteristics (phenotype, cognitive scales, survival) and regional brain volumes (both region of interest and voxel-wise analyses) were explored. CSF poly(GP) was absent in controls and elevated in C9orf72 carriers, without statistical differences between presymptomatic carriers and patients (medians respectively 0.75 and 1.44 ng/ml, p=0.11, Figure 1). CSF NfL levels were higher in patients (1915 pg/ml) than in presymptomatic carriers (429 pg/ml, p<0.001, Figure 2), and controls (333 pg/ml, p<0.001), without difference between the latter two groups (p=0.79). CSF poly(GP) did not correlate with CSF NfL (rs=0.08, p=0.47) in the C9orf72 carriers.

Abstract CT018: Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial

Abstract Background: The tumor necrosis factor receptor GITR is expressed at high levels on regulatory T cells (Tregs) and up-regulated on activated T cells. Evidence from preclinical studies has shown the dual activity of GITR stimulation to counteract Tregs functionally and quantitatively and enhance T-cell effector functions, thus providing the rationale to develop GITR agonist antibodies for cancer immunotherapy. These agents have demonstrated potent therapeutic effects associated with both Treg reduction and modulation in tumor-bearing mice. Here we investigate Treg modulation as a biomarker of activity of the fully humanized agonist anti-GITR antibody TRX-518 in advanced solid cancer patients and the potential mechanisms underlying this effect. Methods: Patients were accrued to 9 cohorts (up to 6 patients/cohort) to receive a single dose of TRX518 (dose range: 0.0001-8 mg/kg). Pharmacodynamic analyses included flow cytometric evaluation of frequency and phenotype of circulating T cells at different time points up to 12 weeks after treatment. Relevant changes observed with these analyses were monitored in pre- and post-treatment tumor biopsies by immunofluorescence staining. Effects of GITR stimulation with TRX-518 on human Tregs were studied in in vitro cultures with CD4+ T cells from healthy donors. Results: Analysis of peripheral blood mononuclear cells from 37 advanced refractory solid cancer patients treated with &amp;gt;=0.005 mg/kg TRX-518 (cohorts 3-9) revealed frequent reduction in circulating Tregs after treatment with TRX-518. Proliferation potential of Tregs was not altered by TRX-518, as indicated by their persistent Ki67 expression. We then asked whether a subset of Tregs was preferentially affected and found that GITR+ Tregs and CD45RA-Foxp3hi effector Tregs (eTregs) were specifically targeted by TRX-518 in a dose-dependent manner. Accordingly, we observed that eTregs express GITR at higher levels than naïve CD45RA+Foxp3+ Tregs. In a subset of patients for whom pre- and post-treatment tumor biopsies were available (n=8; 3 melanoma, 2 lung, 2 colorectal and 1 bladder cancer patients), we tested whether intra-tumor Tregs, which are typically enriched in eTregs, were also affected. Noteworthy, we observed that changes in intra-tumor and circulating Tregs after TRX-518 were positively correlated. As a potential mechanism responsible for this effect, our initial in vitro analyses indicate that GITR stimulation with TRX-518 destabilizes Treg phenotype by down-regulating Foxp3 and enhancing expression of the prototype Th1-lineage transcription factor T-bet. This suggests that TRX-518 may favor Treg to effector-T-cell conversion, thus explaining the effect of Treg loss observed in patients. Conclusions: Circulating Treg reduction is a potential pharmacodynamic biomarker of TRX518 biological activity. This parameter may allow predictive correlation with changes in intratumoral Treg infiltration. We plan to further investigate this effect and its relevance for the association with clinical responses in our recently opened TRX518 multi-dose study. Clinical trial information: NCT01239134. Citation Format: Roberta Zappasodi, Yanyun Li, Mohsen Abu-Akeel, Jingjing Qi, Philip Wong, Cynthia Sirard, Michael Postow, David A. Schaer, Walter Newman, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd D. Wolchok, Taha Merghoub. Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT018. doi:10.1158/1538-7445.AM2017-CT018

A phase 1, multicenter, dose-escalation study of PRN1371, an irreversible covalent FGFR1-4 kinase inhibitor, in patients with advanced solid tumors, followed by expansion cohorts in patients with FGFR genetic alterations.

TPS2616 Background: FGFR inhibition is a promising and clinically proven therapeutic approach in a number of solid tumors where genetic alterations of FGFR drive oncogenesis. PRN1371 is a highly selective oral, irreversible inhibitor of FGFR1-4 that exhibits high potency in cancer cell lines harboring FGFR alterations, including mutations and fusions. Methods: Part A of this phase 1 clinical trial explores ascending doses of PRN1371 in adult patients with advanced solid tumors in a "3 + 3" design, where cohorts of three patients are studied at each level until additional patients need to be added to better assess safety, establish the maximum tolerated dose and define the recommended phase 2 dose (RP2D). PRN1371 is dosed once or twice daily in continuous, 28-day cycles until disease progression. Part B studies include two or three expansion cohorts of different tumor types, 10 patients each with FGFR1-4 gene mutations, fusions, or amplification at the RP2D. The on-target effect of serum phosphorus and FGF23 increases are measured as potential pharmacodynamic biomarkers. Elevated serum phosphorus is managed with oral phosphate binding medications and a low phosphate diet, with dose interruptions and use of acetazolamide if certain thresholds are exceeded. Circulating tumor DNA from patients at baseline and during follow up is analyzed for FGFR genetic alterations. Pre and on-treatment tumor biopsies in Part B will be tested for a panel of pharmacodynamic biomarkers of FGFR inhibition. Clinical trial information: NCT02608125.

Early proliferation of PD-1+ CD8 T cells in peripheral blood as predictive of response to PD-1 inhibition for patients with advanced NSCLC.

e20648 Background: Anti PD-1 and PDL-1 antibodies are now established immune targeted therapies for a subset of patients with advanced non-small cell lung cancer (NSCLC). There is a need for predictive biomarkers to better guide patient selection. We profiled peripheral blood samples from patients receiving PD-1 pathway targeted antibodies for lymphocyte subsets and correlated temporal changes with clinical response. Methods: NSCLC patients treated at our institution with PD-1 or PDL-1 inhibitors (nivolumab, pembrolizumab, and atezolizumab) consented to an IRB-approved biomarker profiling protocol. We collected baseline peripheral blood samples before first treatment and subsequently prior to each new treatment cycle until progression of disease or for a maximum of six cycles. Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed by flow cytometry for kinetics of proliferation of PD1+CD8+ T cells. Clinical response to anti PD-1 therapy was classified according to Response Evaluation Criteria in Solid Tumors (RECIST1.1) criteria and correlated with T cell proliferation kinetics. Results: We enrolled 27 patients with baseline characteristics: median age 66, male 70%, smokers 85%, adenocarcinoma 70%; 10 (37%) patients achieved partial response (PR), 7 (26%) had stable disease (SD) and 10 (10%) had disease progression (PD) as best response. There was a &gt; 1.5-fold increase in Ki67+CD8+ T cells expressing PD-1 within 4 weeks of treatment initiation in 80% of patients with PR compared with 30% in patients with SD and 30% in those with PD. Conclusions: Early proliferation of PD1+CD8+ T cells in peripheral blood is a potential pharmacodynamic biomarker of anti PD-1 immunotherapy that could serve as a tool to identify the NSCLC patient subset most likely to respond to PD-1 and PDL-1 inhibitors.

Metabolomics combined with pattern recognition and bioinformatics analysis methods for the development of pharmacodynamic biomarkers on liver fibrosis.

The major obstacle for the development of targeted therapies is the lack of pharmacodynamic (PD) biomarkers to provide an early readout of biological activities. As the modulation of metabolites may reflect the biological changes occurring in the targets, metabolomics is promising to be an efficient way to explore PD biomarkers. In the present study, a liver fibrosis rat model was established by intraperitoneal injection of CCl4 twice weekly for 6 weeks, the treatment of total aglycone extracts of Scutellaria baicalensis (TAES) was begun 4 weeks after the modeling, and gas chromatography-mass spectrometry (GC-MS) based metabolomics combined with pattern recognition and network analysis were carried out for the research on PD biomarkers of TAES on liver fibrosis. After 2 weeks of treatment, TAES shows positive effects on CCl4-induced liver fibrosis. In the metabolomics study, 63 urinary metabolites contributing to liver fibrosis were identified. Six metabolic pathways significantly enriched in metabolomics data were mapped onto a network to determine global patterns of metabolic alterations in liver fibrosis. By topological analysis, 6 metabolites with high centrality in the metabolic sub-network were selected as potential PD biomarkers. Within 24 h of the final administration, the 6 identified urine metabolic biomarkers with response to time variation of TAES were validated as PD biomarkers. This integrative study presents an attractive strategy to explore PD biomarkers, which may give insight into the actual pharmacological effect of target drugs, and the information from PD biomarkers can be combined with pharmacokinetics to select the optimal dose and a schedule of administration for the drugs.

Normalization of Patient-Identified Plasma Biomarkers in SMNΔ7 Mice following Postnatal SMN Restoration.

Introduction and ObjectiveSpinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the SMN1 gene and retention of the SMN2 gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers.MethodsSMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from SMN2 or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90.ResultsOf the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO.ConclusionThis study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.

Rac1 as a Potential Pharmacodynamic Biomarker for Thiopurine Therapy in Inflammatory Bowel Disease

Azathioprine and mercaptopurine (MP) are effective in treating patients with inflammatory bowel disease (IBD). Immunosuppressive effects of thiopurines involve T-cell apoptosis after inhibition of GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). This study aimed to assess whether expression and activity of Rac1 or phosphorylated ezrin-radixin-moesin (pERM) in patients with IBD could provide a useful biomarker for the pharmacodynamic thiopurine effect and might be related to clinical effectiveness. This was a 2-stage study: stage 1 concerned a cross-sectional cohort of patients with IBD clinically in remission and treated with (n = 10) or without stable weight-based thiopurine therapy (n = 11) and healthy controls (n = 6); stage 2 concerned a prospective study regarding IBD patients with clinically active disease who initiated MP therapy (n = 11) compared with healthy controls (n = 11). Expression and activity of Rac1 and ERM and pERM were determined. The median Rac1 expression was statistically significantly reduced by thiopurine maintenance therapy {0.54 [interquartile range (IQR) 0.47-0.88] versus 0.80 arbitrary units [IQR 0.64-1.46]} compared with patients without immunosuppressive therapy (P = 0.042), but not Rac1 activity and pERM. In responders to MP therapy (n = 6), both median active Rac1 [93 (IQR 81-151) to 76 ng Rac1/mg protein (IQR 62-98)] and Rac1 expression [16.2 (8.8-29.4) to 1.5 arbitrary units (0.9-5.3)] decreased (P = 0.028). In nonresponders (n = 3), Rac1 expression and activity increased. IBD patients treated with thiopurines had a lower expression of Rac1 compared with those not treated with thiopurine. Effective MP therapy led to decreasing concentrations of Rac1-GTP and Rac1 expression. Therefore, Rac1-GTP and expression of Rac1, but not phosphorylation of ERM, form potentially pharmacodynamic markers of therapeutic thiopurine effectiveness in patients with IBD.

Clinical and kinomic analysis identifies peripheral blood mononuclear cells as a potential pharmacodynamic biomarker in metastatic renal cell carcinoma patients treated with sunitinib.

BackgroundSunitinib is a protein tyrosine kinase (PTK) inhibitor that has immune-modulating properties. In this context, peripheral blood mononuclear cells (PBMC), mainly constituted by lymphocytes, could be a perfect surrogate tissue for identifying and assaying pharmacodynamic biomarkers of sunitinib. In this study, we investigated the changes in lymphocytes count as pharmacodynamic biomarker in metastatic renal cell carcinoma (mRCC) patients under sunitinib therapy. Thereafter, we studied the ex vivo effect of sunitinib and SU12262 (active metabolite) on PBMC from naïve mRCC patients using a high throughput kinomic profiling method.MethodsThe prognostic value of total lymphocytes count between Day 0 and Day 21 (expressed as a ratio D21/D0) was retrospectively investigated in 88 mRCC patients under sunitinib therapy. PTK PamChip® microarrays were used to explore prospectively the ex vivo effect of sunitinib and SU12662 on PTK activity in PBMC from 21 naïve mRCC patients.ResultsIn this retrospective study, D21/D0 lymphocytes ratio (Hazard Ratio, 1.83; CI95%, 1.24-2.71; p=0.0023) was independently associated with PFS. Interestingly, kinomic analysis showed that D21/D0 lymphocytes ratio and Heng prognostic model was statistically associated with the ex vivo sunitinib and SU12662 effect in PBMC.ConclusionThe present study highlights that D21/D0 total lymphocytes ratio could be a promising pharmacodynamic biomarker in mRCC patients treated with sunitinib. Additionally, it paves the way to investigate the kinomic profile in PBMC as a prognostic factor in a larger cohort of mRCC patients under sunitinib therapy.