Post-treatment Plasma Samples Research Articles

Cell-free DNA as a biomarker for taxane treatment in advanced prostate cancer.

5070 Background: Antitumor efficacy of chemotherapeutic drugs docetaxel and cabazitaxel against hormone-sensitive and castration-resistant prostate cancer (CRPC) is well recognized. The identification and validation of minimally invasive biomarkers of taxane response remain of paramount importance. Methods: Serial pre-, during and post-treatment plasma samples were collected prospectively from two large Phase III clinical trials, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Chemotherapy-naïve patients (pts) were treated with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2) in FIRSTANA, and pts previously treated with docetaxel received cabazitaxel (20 or 25 mg/m2) as second-line chemotherapy in PROSELICA. Plasma cell-free DNA (cfDNA) was extracted, low-pass whole genome sequencing (lp-WGS) libraries were prepared using the QIAGEN QiaSeq FX DNA library kit, and samples sequenced on the Illumina NovaSeq 6000. Targeted next-generation sequencing (NGS) on a custom-made AmpliSeq panel of 30 genes, pre-selected for putative roles in taxane resistance, was also performed. lp-WGS copy number (CN) profiles were generated using ichorCNA (v0.1.0) and targeted NGS variant calls derived using IonReporter software. Results: Overall, lp-WGS data was generated from 265 samples (99 pts; 51 treated on the FIRSTANA study, 48 treated on the PROSELICA study), acquired at three time-points (pre-, during and at progression). Average cfDNA input was 10 ng and mean coverage achieved was ~2X (SD 1.2X). We observed changes in lp-WGS tumor purity over time as a result of therapy; samples from non-responding pts exhibited significantly higher tumor purity values post-treatment compared with samples from responding pts (p = 0.02). Targeted NGS results were available from 294 pts (153 from FIRSTANA, 141 from PROSELICA), and changes in tumor purity were also associated with treatment response (p = 0.04). CN frequency of key CRPC genes was similar to previously reported datasets; several aberrant loci associated with response to taxane therapy. Conclusions: cfDNA lp-WGS may have clinical utility in the management of lethal prostate cancer. Funding: Sanofi. Clinical trial information: NCT01308580, NCT01308567.

Assessing the impact of clonal hematopoiesis in disease monitoring using targeted cell-free DNA (cfDNA) sequencing technology.

e14530 Background: Somatic variants found in plasma cell-free DNA (cfDNA) may derive from either solid tumors or clonal hematopoiesis (CH). Little is known about how this may impact plasma-based longitudinal disease monitoring using targeted sequencing of circulating tumor DNA (ctDNA). Methods: To assess the potential impact of CH in disease monitoring, we evaluated monitoring algorithms by targeted sequencing with and without matched peripheral blood mononuclear cells (PBMC). Samples were collected from a prospective observational study, where 62 late stage lung adenocarcinoma subjects were treated with first-line chemo or chemoradiation therapy. Pre-treatment plasma cfDNA and matched PBMC were analyzed with the AVENIO ctDNA Surveillance Kit (For Research Use Only, not for use in diagnostic procedures), a sequencing panel of 198 kilobases targeting cancer genes. Median input amounts of 25 ng cfDNA and 50 ng PBMC DNA were sequenced to median deduplicated depths of 4582 and 6134, respectively. Results: A median of 120 single nucleotide variants were detected per cfDNA sample, with 93.1% of these identified in matched PBMC. Most PBMC-matched cfDNA variants were germline SNPs, with allele frequency (AF) ~ 50% or 100%. A median of 1 (range 0-5) PBMC-matched cfDNA variants per sample were detected with an AF < 10%, consistent with CH. The number of these variants was positively associated with age (p-value = 0.0039) and the most frequently mutated gene was TP53. The remaining somatic variants (i.e., in cfDNA and not PBMC) had an AF range 0.03-40.9%. These PBMC-informed variants (median of 7 per sample) were used in longitudinal monitoring in the first post-treatment plasma sample to assess early response to therapy. Association between ctDNA level and progression-free survival using the same monitoring algorithm yielded nearly identical results on somatic variants derived from filtering approaches independent of matched PBMC (HR 0.32; 95% CI 0.16 - 0.65; log-rank P = 0.0009) and the PBMC-informed method (HR 0.31; 95% CI 0.14 - 0.66; log-rank P = 0.0013). Conclusions: A targeted panel focused on solid tumors by design has limited impact from CH. For disease monitoring applications in a non-MRD setting, measuring multiple variants instead of a single variant further enables robust classifiers that can moderate the impact of variants, if any, from CH.

Abstract 2740: High-throughput and sensitive quantification of circulating tumor DNA

Abstract Circulating tumor DNA (ctDNA) has a great potential to serve as non-invasive biomarkers for monitoring disease progression and treatment response. However, its broad clinical applications have been hindered by sensitivity, throughput and complexity of existing ctDNA detection methods. Here we report an adaptation of a multiplex PCR pre-amplification (PreAmp) into the microfluidic multiplex PCR (MMP-Seq) technology for high-throughput and sensitive quantitation of ctDNA. We have demonstrated that PreAmp MMP-Seq enabled sensitive (down to 0.6% allele frequency), reproducible (R2=0.97), accurate and quantitative mutation detection from as low as 1.6 ng DNA input. An Empirical Bayesian Model has been established to estimate background error rates for every base of amplicons in the ctDNA panel using data from 43 normal plasmas. 0.1-0.3% of median error rates were observed across all positions. 149 longitudinal plasma samples from 12 ovarian and 10 pancreatic cancer patients of PhI DMUC5754A anti-MUC16 ADC trials were then profiled using PreAmp MMP-Seq. 79% (11/14) driver mutations found in tissue samples were also identified in matching baseline plasma samples at frequencies well above estimated error rates. Moreover, we found that changes of corresponding ctDNA concentration in post-treatment plasma samples were correlated with changes in tumor burdens and circulating protein marker levels. In conclusion, the MMP-seq PreAmp workflow provides an inexpensive, reproducible and flexible solution for mutation detection in plasma DNA samples. Citation Format: Yinghui Guan, Oleg Mayba, Shan Lu, Thomas Sandmann, Younjeong Choi, Walter Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W. Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Yulei Wang. High-throughput and sensitive quantification of circulating tumor DNA [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 2740. doi:10.1158/1538-7445.AM2017-2740

Circulating tumor DNA analysis for outcome prediction in localized esophageal cancer.

4055 Background: Blood-based biomarkers are not used in routine clinical practice in patients with esophageal carcinomas (ECs). Circulating tumor DNA (ctDNA) is an attractive biomarker that could be applied to ECs. We performed a study to explore pre- and post-treatment ctDNA analysis using the next generation sequencing-based CAPP-Seq method as a prognostic biomarker for localized EC. Methods: We prospectively enrolled 29 patients with localized EC treated with chemoradiotherapy (CRT) between June 2011 and October 2015. 12 (43%) patients were treated with CRT alone and 17 (57%) were treated with CRT followed by esophagectomy. Our cohort included patients with stage IB (1; 3.4%), II (7; 24.1%), and III (21; 72.4%) disease. Eight (27.6%) harbored squamous cell carcinoma (SCC) and 21 (72.4%) adenocarcinoma (AC). All patients received pre-treatment evaluation by thoracic CT, PET/CT, and esophagoduodenoscopy. ctDNA levels were quantitated in pre-treatment and post-treatment plasma samples using CAPP-Seq. Results: Median follow-up time was 21 months. We detected ctDNA pre-treatment in 72.4% of cases (N = 21) with a median concentration of 2.69 haploid genome equivalents per mL (hGE/mL; range 0.34-107.3). Pre-treatment ctDNA concentrations were strongly correlated with metabolic tumor volumes (MTV; R2= 0.74; p = 1.7e-07) and were significantly higher in SCC than AC patients (28.2 vs. 2.1 mean hGE/mL; p = 0.002). Overall survival (OS) at 2 years for pretreatment ctDNA+ vs. ctDNA- patients was 47% vs. 86% (HR = 6.0; 95% CI = 0.74-49.2; p < 0.05) and trended toward significance when accounting for stage, histology, and age (p = 0.09). A single post-treatment plasma sample was collected within 3 months of treatment and was available for 19 patients. Post-treatment ctDNA was detected in 3 (15.7%) patients with a median concentration of 11.5 hGE/mL (range 2.2–11.9). Post-treatment ctDNA detection was strongly predictive of poor event-free survival (p < 0.0001) and time to distant metastasis (p < 0.0001). Conclusions: Our data suggest that pre- and post-treatment ctDNA levels may be prognostic for patients with localized EC and could potentially guide risk-adapted adjuvant therapy approaches.

Bioavailability and biochemical effects of diclofenac sodium 0.1% ophthalmic solution in the domestic chicken (Gallus gallus domesticus).

To determine if topical ophthalmic diclofenac sodium 0.1% solution alters renal parameters in the domestic chicken, and to determine if the drug is detectable in plasma after topical ophthalmic administration. Thirty healthy domestic chickens. Over 7days, six birds were treated unilaterally with one drop of artificial tear solution (group 1), 12 birds were treated unilaterally (group 2) and 12 bilaterally (group 3) with diclofenac sodium 0.1% ophthalmic solution. Treatments were provided every 12h in all groups. Pre- and post-treatment plasma samples from all birds were evaluated for changes in albumin, total protein, and uric acid. Post-treatment samples of all birds, collected 15min post-administration, were analyzed by high-performance liquid chromatography with mass spectrometry for diclofenac sodium detection. A randomly selected renal sample from each group was submitted for histopathologic review. Changes in pre- and post-treatment plasma albumin were significant (P<0.05) in groups 2 and 3, but not for group 1. Pre- and post-treatment changes in total protein and uric acid were not significant for any group. Diclofenac sodium was not detectable (limit of detection=0.10ng/mL) in plasma samples from birds in group 1. Post-treatment concentration of diclofenac in group 3 was statistically greater than group 2 (P=0.0008). Histopathologic changes did not identify diclofenac-induced acute renal tubular necrosis. Ophthalmic diclofenac sodium 0.1% administered topically every 12h in one or both eyes for 7days is detectable in systemic circulation in the domestic chicken, but does not cause overt significant changes in plasma uric acid or total protein.

Abstract B32: Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia

Abstract Introduction: The adenoma-carcinoma sequence is well-recognized as a stepwise pattern of mutational activation of oncogenes and inactivation of tumor suppressor genes resulting in sporadic colorectal cancer (CRC) development from colorectal adenomas (CRA) within the chromosomal instability pathway. MicroRNAs (miRNA) play an important role in oncogenesis by regulating gene expression and are known to be actively released from cells. They are found in body fluids such as plasma, saliva, feces and urine. We have previously demonstrated differential expression of plasma miRNAs in patients with CRC as compared to that of controls. Methods: Plasma was isolated from 10 patients: 5 patients with advanced colorectal adenoma (&amp;gt;0.6cm diameter) and 5 patients with stage II or III colorectal cancer prior to treatment and 4-6 weeks following endoscopic removal or surgical resection, respectively. RNA was extracted from plasma (Qiagen® miRNeasy), RT-PCR performed and 768 miRNAs were screened using microfluidic array technology (Applied BioSystems®) in pre- and post-treatment samples for each patient. Data were analyzed using paired t-tests after normalizing raw cycle threshold data to endogenous RNU6 for pre- and post-treatment samples. In addition, miRNA expression data from both pre- and post-treatment CRA and CRC samples were compared to that of plasma from 10 individuals without neoplasia (controls) (ANOVA). Results: Significant differential expression of plasma miRNAs was observed between pre- and post-treatment samples in both CRA and CRC groups. Interestingly, there was generalized miRNA upregulation in CRA when comparing pre-treatment samples to post-treatment samples. Conversely there was miRNA down-regulation in pre-treatment CRC plasma as compared to post-resection samples. Utilizing significantly dysregulated miRNAs, as well as p-values, AUC, fold-change and biological significance, miRNA panels were selected to facilitate detection of CRA recurrence (following complex polypectomy for large polyps) or for detection of recurrent CRC following resection. The resulting panel was able to differentiate between pre- and post-treatment samples for CRA (miR-186 and miR-623) with AUC 0.94 (95% CI 0.76 – 1.00) and for CRC (miR-324-5p, miR-30d and miR-766) with AUC 0.88 (95% CI 0.63 – 1.00). In addition, while miRNA expression of pre-treatment CRA and CRC samples differed significantly from controls, post-treatment CRA and CRC samples did not. Conclusion: Comparison of pre- and post-treatment plasma samples reveals differing patterns of changes in miRNA expression in benign colorectal neoplasms (CRA) as opposed to invasive neoplasia (CRC). The observed miRNA down-regulation prior to surgical resection of CRC might indicate decreased expression of miRNAs inhibiting oncogenes prior to cancer treatment, while the miRNA upregulation seen in pre-treatment CRA plasma could indicate inhibition of tumor suppressors. Such observations will perhaps elucidate the role of miRNA in carcinogenesis and may provide for a relatively non-invasive method of detecting such lesions. Further validation is needed to develop a miRNA panel that will allow monitoring for evidence of recurrent disease. Citation Format: Jane Carter, Vanessa States, Uri Netz, Jianmin Pan, Shesh Rai, Susan Galandiuk. Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B32.

Post-treatment plasma EBV-DNA positivity predicts early relapse and poor prognosis for patients with extranodal NK/T cell lymphoma in the era of asparaginase.

Circulating Epstein-Barr virus (EBV) DNA is a biomarker of EBV-associated malignancies. Its prognostic value in early stage NK/T-cell lymphoma (NKTCL) in the era of asparaginase was investigated. 68 patients were treated with a median of 4 cycles of asparaginase-based chemotherapy followed by a median of 54.6 Gy (range 50-60 Gy) radiation. The amount of EBV-DNA was prospectively measured in both pretreatment and post-treatment plasma samples by real-time quantitative PCR. At the end of treatment, complete response (CR) rate was 79.4%, and overall response rate (ORR) was 88.2%. Patients with negative pretreatment EBV-DNA had a higher CR rate (96.0% vs. 69.8%, p = 0.023). The 3-year progression-free survival (PFS) rate and overall survival (OS) rate was 71% and 83%, respectively. In multivariate survival analysis, post-treatment EBV-DNA positivity and treatment response (non-CR) were prognostic factors for both worse PFS and OS (p < 0.05). Local tumor invasion was also a prognostic factor for worse OS (p = 0.010). In patients with CR, post-treatment EBV-DNA positivity correlated with inferior PFS and OS (both p < 0.0001). In patients with positive pretreatment EBV-DNA, negative post-treatment EBV-DNA correlated with better PFS and OS (both p < 0.0001). These findings indicate that post-treatment EBV-DNA positivity can predict early relapse and poor prognosis for patients with early stage NKTCL in the era of asparaginase, and may be used as an indicator of minimal residual disease.

MiR125 and miR200a as potential circulating miRNA biomarkers in metastatic urothelial carcinoma patients treated with docetaxel.

364 Background: Docetaxel is frequently used as second-line therapy in advanced bladder cancer. The response rate is 10-15% with a median PFS of 2.5 months and without data showing improvement of QoL, however, some selected patients derive significant benefit. Understanding the mechanisms underlying the sensitivity of docetaxel in bladder cancer will help to customize treatment in second line. Circulating miRNAs have been shown to be predict outcome in GU malignancies (for example, prostate cancer). Using circulating miRNA can identify patients who will respond to therapy non-invasively. The main aim of our study was identify circulating miRNAs predicting docetaxel response in urothelial carcinoma (UC). Methods: We obtained pre- and post-treatment plasma samples from patients included in the zactima phase II clinical trial assigned to the placebo + docetaxel arm. RNA from plasma was extracted and RT-PCR was performed on both the pre- and post-treatment samples looking at miRNAs known to be important in docetaxel response1(miR125, miR27a, miR34a, miR429, miR141, miR200a, miR200c, miR 146a, and miR9). Samples were normalized by the mean global miRNA levels. We divided patients based on progression free survival (PFS) as good responders (&gt;4 months PFS or 6 cycles) versus poor responders (&lt;2 months PFS or progression at first evaluation). Differences in miRNA levels were assessed using the student t test (p&lt;0.05). Results: miR125 and miR200a were shown to be significantly different between the poor and good responders (p=0.056 and p=0.028, respectfully) in the pre-docetaxel samples. Higher expression of both miRNAs correlated with a good response in our small patient cohort. Additional analysis comparing pre- and post-miRNA levels will be presented. Conclusions: miR125 and miR200a are potential extracellular biomarker to docetaxel treatment in metastatic UC.

Abstract 5228: Characterization of miR-21 as a potential circulating biomarker for osteosarcoma

Abstract MicroRNAs (miRNAs) are a class of small noncoding RNAs that are implicated in various important biological processes by regulation of gene expression. Aberrant miRNA expression is suggested to be associated with various human disorders including cancer. In this study, we studied if miRNAs can be used as biomarkers in osteosarcoma (OS). OS is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, OS patients who respond poorly towards chemotherapy or develop relapses have a dismal outcome. Thus, it is of clinical significance if a biomarker approach can be developed to monitor tumor burden and detect early relapses in the patients, so that they can be treated as early as possible to improve the survival. To achieve this goal, we performed miRNA profiling on a cohort of OS plasma samples collected from the Texas Children's Hospital. miR-21 and other miRNAs were identified to be elevated in OS patients relative to a group of healthy controls. Circulating miR-21 has been detected in other types of cancer and in the tumor it is known to negatively regulate tumor suppressor genes involved in proliferation, apoptosis and invasion; however, its role in OS is still unclear. Hence, we first validated that miR-21 was elevated in a set of independent osteosarcoma plasma samples but not in the control samples. Using serial plasma samples from a group of patients, we found that the circulating miR-21 level was lower in the posttreatment plasma samples than in the pretreatment samples. These results suggest that circulating miR-21 may correlate with tumor burden of the patients. In addition, miR-21 was expressed in many OS tumors as detected by in situ hybridization on an OS tissue microarray. When tested on two OS cell lines, miR-21was significantly increased in the culture media between 24 and 48 hours after plating in a cell number dependent manner, suggesting that miR-21 could be released from the tumor cells. Taken together, our results suggest that miR-21 is expressed in OS and its level in plasma may reflect tumor burden in OS patients. Citation Format: Manjula Nakka, Yiting Li, Colin McGee, Ching Lau, Tsz Kwong Man. Characterization of miR-21 as a potential circulating biomarker for osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5228. doi:10.1158/1538-7445.AM2014-5228

Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer.

Human papillomavirus type 16 (HPV-16) is a major causative factor in oropharyngeal squamous cell carcinoma (OPSCC). The detection of primary OPSCC is often delayed owing to the challenging anatomy of the oropharynx. To investigate the feasibility of HPV-16 DNA detection in pretreatment and posttreatment plasma and saliva and its potential role as a marker of prognosis. This is a retrospective analysis of a prospectively collected cohort. Among a cohort of patients with oropharyngeal and unknown primary squamous cell carcinoma with known HPV-16 tumor status from the Johns Hopkins Medical Institutions and Greater Baltimore Medical Center (from 1999 through 2010), 93 patients were identified with a complete set of pretreatment and posttreatment plasma or saliva samples, of which 81 patients had HPV-16-positive tumors and 12 patients had HPV-16-negative tumors. Real-time quantitative polymerase chain reaction was used to detect HPV-16 E6 and E7 DNA in saliva and plasma samples. Main outcomes included sensitivity, specificity, negative predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status, as well as the association of posttreatment HPV DNA status with clinical outcomes, including recurrence-free survival and overall survival. The median follow-up time was 49 months (range, 0.9-181.0 months). The sensitivity, specificity, negative predictive value, and positive predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status were 76%, 100%, 42%, and 100%, respectively. The sensitivities of pretreatment saliva or plasma alone were 52.8% and 67.3%, respectively. In a multivariable analysis, positive posttreatment saliva HPV status was associated with higher risk of recurrence (hazard ratio [HR], 10.7; 95% CI, 2.36-48.50) (P = .002). Overall survival was reduced among those with posttreatment HPV-positive status in saliva (HR, 25.9; 95% CI, 3.23-208.00) (P = .002) and those with HPV-positive status in either saliva or plasma but not among patients with HPV-positive status in plasma alone. The combined saliva and plasma posttreatment HPV-16 DNA status was 90.7% specific and 69.5% sensitive in predicting recurrence within 3 years. Using a combination of pretreatment plasma and saliva can increase the sensitivity of pretreatment HPV-16 status as a tool for screening patients with HPV-16-positive OPSCC. In addition, analysis of HPV-16 DNA in saliva and plasma after primary treatment may allow for early detection of recurrence in patients with HPV-16-positive OPSCC.

The use of multiplex platforms for absolute and relative protein quantification of clinical material

When introducing multiplex platforms to measure protein content in precious clinical material there is an increased risk of cross reactivity, loss of sensitivity as well as accuracy. In this paper, four multiplex platforms and one singleplex platform were compared by running pre- and post-treatment plasma samples from CML patients. We found a variation of absolute protein concentrations between platforms. For some of the analytes and platforms, relative differences between pre- and post-treatment samples correlated. We conclude that absolute concentrations measured by different platforms should be compared with caution and comparing relative differences could be more accurate.

Minimal Residual Disease (MRD) Detection By Deep Sequencing In Newly Diagnosed Multiple Myeloma Patients Treated With Carfilzomib, Lenalidomide and Dexamethasone

BackgroundMultiple myeloma (MM) therapies are becoming increasingly effective with deeper responses. Indeed, recent prospective clinical trials based on carfilzomib, lenalidomide and dexamethasone (CRd) show complete response (CR)/near (n)-CR rates of over 75%. Consequently, newer techniques are needed to detect minimal residual disease (MRD). We are conducting a prospective Phase II clinical trial studying response to CRd in newly diagnosed MM patients. We assessed treatment responses obtained by traditional IMWG response criteria, MRD measurement by 8-color multiparameter flow cytometry (MFC) based on a minimum of 3 million events (maximum detection rate: 0.0005%), and MRD measurement by high throughput sequencing in 14 newly diagnosed MM patients who achieved very good partial response (VGPR)/CR/nCR. MethodsBone marrow (BM) and/or plasma samples were obtained from 14 newly diagnosed MM patients receiving CRd therapy. Samples were subjected to deep sequencing using the LymphoSIGHT™ platform, which has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood (Faham et al, Blood 2012). Briefly, using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA obtained from CD138+ BM cell lysate at baseline, as well as post-treatment plasma samples. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency within the B-cell repertoire in the CD138+ cell lysate BM sample. The presence of the myeloma-specific clonotype was then quantified in plasma samples obtained post-treatment. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. For patients who achieved VGPR/CR/nCR, we compared the results from MFC of the BM with those obtained by sequencing the cell free DNA in plasma samples. ResultsWe detected a high frequency myeloma-specific gene rearrangement in 13 of 14 (93%) CD138+ BM cell lysate samples obtained at diagnosis. We assessed for MRD by flow cytometry and deep sequencing for the seven patients who were in VGPR/CR/nCR based on traditional protein response criteria. One patient was MRD positive by flow cytometry of the marrow and negative by deep sequencing in blood. Another patient was MRD positive by deep sequencing but negative by flow cytometry. Additionally, in the diagnostic BM sample of one patient, we observed two distinct high-frequency IGH clones that were related through a process of somatic hypermutation. The specific mutation pattern observed is consistent with a branched model of evolution where the two observed clones did not evolve from each other but rather from a common ancestor. We are currently analyzing samples collected at baseline and post-treatment in an additional 23 MM patients enrolled on the CRd trial, and results will be presented. ConclusionsThe development of sensitive, non-invasive MRD assays is becoming increasingly important to assess the impact of modern anti-myeloma therapies. One novel approach for MRD detection, termed the LymphoSIGHT™ platform, relies on high-throughput sequencing of VDJ rearrangements at the immunoglobulin locus. Based on our CRd clinical trial for newly diagnosed MM patients, we found sequencing of cell free tumor DNA in bone marrow aspirates and peripheral blood (plasma) to be technically feasible. Among 6 patients who obtained VGPR/CR/nCR and were found to be MRD negative by MFC of the BM, we found one patient to be MRD positive by sequencing in the peripheral blood (plasma compartment). One patient was MRD positive by flow cytometry but negative by deep sequencing. These preliminary results suggest that a sequencing-based MRD blood test may be more sensitive than standard protein response criteria and complementary to MFC-based BM tests. Analysis of additional samples will be presented. Disclosures:Faham:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Research Funding.

Relationship between specific microRNAs in plasma and juvenile idiopathic arthritis

Objective To investigate the potential possibilities of specific microRNA in plasma as novel biomarkers for the early diagnosis in juvenile idiopathic arthritis(JIA). Methods This research was be segmented into 4 stages.1. Screened candidate microRNAs: 5 candidate plasma microRNAs were detected by biochips of microRNAs, corresponding 5 JIA patients with onset of oligoarthritis, 5 JIA patients with onset of polyarthritis and 3 age-matched and sex-matched healthy controls individual.2. The feasibility of the microRNAs as novel biomarkers was validated by Real-time quantitative polymerase chain reaction (RT-PCR) in plasma on 80 JIA patients (43 oligoarthritis, 37 polyarthritis), 29 juvenile ankylosing spondylitis(JAS) patients and 30 healthy control individuals.3. The change of microRNAs was observed by RT-PCR in plasma from another 9 JIA patients before and 3 months after anti-rheumatic drug treatment.4. The correlation between the levels of candidate plasma microRNAs and clinical parameters of JIA patients was analyzed. Results Plasma concentrations of miR-16, miR-146a and miR-223 in JIA patients, including oligoarthritis and polyarthritis, were significantly higher than those in healthy subjects (all P 0.05). More importantly, it was found that the expression of miR-16 was considerably reduced in the post-treatment plasma samples when compared to the pre-treatment samples(P=0.061). In addition, the levels of miR-16 in JIA plasma inversely corrected with tender joint. Conclusions Plasma levels of miR-16 were well-discriminated between JIA patients and healthy subjects or JAS patients. It is suggested that plasma miR-16 might serve as a novel and potential biomarker for screening JIA. Furthermore, it might serve as a novel biomarker for joint inflammation but not specifically for differentiating the subtypes of JIA. Key words: MicroRNA; Juvenile idiopathic arthritis; Biomarker

Analysis of plasma biomarker and tumor genetic alterations from a phase II trial of lenvatinib in patients with advanced endometrial cancer.

5591 Background: Lenvatinib is an oral receptor tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT, and PDGFRβ. A phase 2 study in patients with advanced endometrial cancer following 1 or 2 prior platinum-based treatments was performed and is presented in a separate abstract. Here we report potential predictive markers of clinical benefit. Methods: Pre- and post-treatment plasma samples collected from 122 of 133 treated subjects were analyzed for 50 circulating cytokine and angiogenic factors (CAFs) using ELISA and multiplex assay platforms; 107 archival tumor tissues obtained from 133 enrolled subjects were analyzed for gene mutation (mut) (81 samples) and gene expression profiling (GEP) (64 samples). For GEP analysis, the NanoString nCounter platform was used to measure the expression level of approximately 300 genes identified preclinically as relevant. Correlation with clinical outcome measures including maximal tumor shrinkage (MTS), objective response rate (ORR), PFS, and OS was performed. Results: Clinical correlation identified baseline levels of 7 CAFs related to angiogenesis (Ang-2, IL-8, HGF, VEGFA, PlGF, Tie-2, and TNFa) that associated with survival. Only baseline levels of Ang-2 associated with greater MTS (R=0.36 [Spearman], P&lt;0.001), ORR (61% vs 18%), mPFS (9.5 vs 3.7 mos), and mOS (23 vs 8.9 mos) as determined using a defined cut-off value for baseline Ang-2. In gene mut analysis, no significant correlations were observed among the genes tested. Mut in PIK3CA showed a trend toward shorter OS (p = 0.085). In GEP analysis, expression levels of approximately 90 genes correlated with clinical outcome. Combination of GEP and CAF signatures identified signaling pathways, including Ang-2, that associated with OS. Clinical and preclinical GEP analysis identified overlapping gene signatures involving MAPK and PI3K signaling pathways that contributed to lenvatinib resistance. Conclusions: Baseline circulating Ang-2 levels may potentially predict outcomes in patients with advanced endometrial cancer and require further examination. This may provide a basis for stratification of patients in future clinical trials. Clinical trial information: NCT01111461.

ASPIRATION: Phase II study of continued erlotinib beyond RECIST progression in Asian patients (pts) with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC).

TPS7614 Background: First-line erlotinib (an EGFR tyrosine-kinase inhibitor) significantly increased progression-free survival (PFS) vs chemotherapy in phase III trials of pts with EGFR mutation-positive NSCLC. Discontinuation of erlotinib on RECIST disease progression (PD) may lead to rapid disease flare-up; continued erlotinib beyond RECIST PD may extend clinical benefit by slowing progression of this life-threatening disease. We describe ASPIRATION, a large, Asian, multicenter, single-arm, open-label, phase II trial (NCT01310036), which will increase understanding of first-line erlotinib and erlotinib continuation beyond RECIST PD in pts with EGFR-mutated NSCLC. Methods: Pts (n=204) ≥18 yrs with stage IV/recurrent NSCLC, ≥1 measurable lesion (≥10mm), ECOG performance status (PS) 0-2 and positive EGFR mutation status established by local pathology laboratory (that underwent voluntarily QA/QC) are eligible. All pts receive erlotinib 150mg/day. Tumor response is evaluated using RECIST (v1.1). The primary endpoint is PFS. At investigator's discretion, pts may continue on erlotinib beyond RECIST PD, e.g. if they have slow PD (&gt;6 months of partial response/stable disease), asymptomatic minimal PD, or new brain metastasis controlled locally. Pts should not continue erlotinib if they have extracranial PD with symptoms; rapid PD and/or worsening of PS; or life-threatening complications. Pts continuing erlotinib who present with second RECIST PD will discontinue. Secondary endpoints include objective response rate, disease control rate, overall survival, and safety. For the exploratory biomarker study, pre-treatment tumor tissue blocks are collected; remaining tissue (after EGFR mutation testing for eligibility) will be analyzed centrally to study the association of biomarkers and clinical outcomes. Pre-treatment and post-treatment plasma and serum samples will be obtained at various time points for biomarker assays, including EGFR mutations and other candidate NSCLC biomarkers. Recruitment began in Apr 2011; the estimated final data collection for the primary endpoint is Dec 2014.

Preoperative treatment with pazopanib (GW786034), a multikinase angiogenesis inhibitor in early-stage non-small cell lung cancer (NSCLC): A proof-of-concept phase II study

7557 Background: Pazopanib is an oral angiogenesis inhibitor targeting VEGFR, PDGFR and c-kit. The present phase II open-label trial (NCT00367679; VEG105290) was designed to determine the clinical/biological activity and the safety of pazopanib given preoperatively in patients with stage I-II NSCLC. Methods: After initial biopsy and prior to scheduled surgery, eligible patients received pazopanib 800 mg QD for 2–6 weeks followed by a washout period of 7 days prior to surgery. The primary endpoint was tumor volume reduction assessed by high resolution CT (HRCT) images performed prior to and after pazopanib treatment. Secondary endpoints were response according to RECIST criteria and safety. The sequence of centrally reviewed scans was blinded. Analysis of surgical specimens as well as pre-and post-treatment plasma samples allowed comparative biological analyses to be performed. The target accrual was 33 patients between February 2007 and February 2008. Results: 26 patients were enrolled by November 30 2007, by which time 23 had pre and post treatment HRCT available. Median age was 65 yrs (51–79), 62% were female. Histologies were adenocarcinoma 62%, squamous cell 15%, and other NSCLC 23%. Stage IA: 16 patients; stage IB: 9 patients; stage IIA: 1 patient. Median duration of therapy was 18 days. Twenty pts (87%) had a reduction in tumor volume after pazopanib treatment. Tumor changes ranged from -86% to +17%. Three patients had a partial response. Grade 3 toxicities were seen in 3 patients: including ALT increase (2), hypertension (1), dyspnea (1), pneumonia (1), urinary tract infection (1), and lymphopenia (1). No grade 4 toxicities were seen. One patient discontinued due to Aes. Conclusions: Short-term pazopanib treatment demonstrated clear single-agent activity in early stage NSCLC with a favorable tolerability profile in this population. Further clinical development in this setting is planned. Final results will be presented at the meeting. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline General Electric, Pneum Rx GlaxoSmithKline, Pneum Rx GlaxoSmithKline

Elevated osteopontin (OPN) plasma levels are highly prognostic in advanced non-small cell lung cancer (NSCLC): Analysis of SWOG S0003

7198 Background: OPN is a secreted glycoprotein with a diverse array of functions, including induction of uPA &amp; increased cell migration. OPN has been shown to be elevated in a number of tumor types, &amp; its downregulation reduces tumorigenicity &amp; metastasis in tumor models. High levels have also been associated with tumor hypoxia/angiogenesis, as are vascular endothelial growth factor (VEGF) &amp; plasminogen activator inhibitor (PAI-1). We hypothesized that secreted levels of these biomarkers would correlate with clinical outcome after treatment. Methods: Plasma concentrations of OPN, VEGF &amp; PAI-1 were measured by ELISA in 160 NSCLC patients enrolled on the Southwest Oncology Group (SWOG) trial S0003 (paclitaxel/carboplatin ± the hypoxic cytotoxin tirapazamine). Post-treatment plasma samples were available in 56 patients. Results: Baseline OPN plasma levels correlated significantly with patient overall survival (OS). High interpatient variability was observed, with levels ranging from undetectable to 2560 ng/ml, (median: 606.5 ng/ml). When dichotomized, median OS was 11 months for patients below median OPN levels &amp; 7 months for those above (p = 0.004). Survival decreases with increasing OPN concentration. Furthermore, OPN levels correlated with response rate (RR) (median responders: 497; median non-responders: 698 ng/ml. Wilcoxon rank-sum p = 0.03). No association between baseline levels of either VEGF or PAI-1 with RR or OS was observed. However, plasma levels of both PAI-1 &amp; VEGF were significantly inter-related &amp; trended together (p &lt; 0.0001), &amp; both decreased significantly after treatment (p = 0.0004 &amp; 0.04, respectively). Median decrease: OPN: 17%, PAI: 44%, VEGF: 42%. No significant differences were observed between study arms, suggesting that OPN is prognostic in NSCLC, but not predictive for response to tirapazamine. Conclusions: 1) There is a great need for development of tumor biomarkers which can be serially assessed pre- &amp; post-therapy. 2) High OPN plasma levels were significantly associated with reduced RR &amp; OS for patients on this trial. OPN is a strong candidate for inclusion in a panel of prognostic (&amp; perhaps predictive) markers for NSCLC. Supported by the Hope Foundation &amp; R01-CA107228. No significant financial relationships to disclose.

Role of Epstein-Barr virus DNA measurement in plasma in the clinical management of nasopharyngeal carcinoma in a low risk area

Objective: To evaluate the role of quantitative measurement of Epstein-Barr virus (EBV) DNA in the clinical management of nasopharyngeal carcinoma (NPC) in a low tumour risk area (western Europe). Methods:...

Clinical utility of genotyping resistance test on determining the mutation patterns in HIV-1 CRF01_AE and subtype B patients receiving antiretroviral therapy in Hong Kong

Background HIV-1 genotypic resistance test (GRT) has been widely used to monitor HIV infection but only few reports revealed the mutation patterns of non-B HIV-1 subtypes. Objective To evaluate the concordance of GRT and clinical treatment outcomes on different HIV-1 subtypes and monitor the mutation patterns and frequencies. Study design Pre- and post-treatment plasma samples from 123 patients (39 treatment naïve and 84 treatment experienced) were tested by ViroSeq HIV-1 Genotyping System followed by analysis using the Stanford HIV database. The mutation patterns and frequencies developed in the pol gene were compared among subtypes. Results HIV-1 subtypes among patients in Hong Kong were mainly subtype B and CRF01_AE. Primary mutation was not detected among all pre-treatment samples. For post-treatment samples, primary mutations were only detected in the treatment failure group. The mutation patterns and frequencies were similar between CRF01_AE and subtype B viruses. However, the frequencies of L74V/I and K103N in the reverse transcriptase region were different between CRF01_AE and subtype B viruses. VirtualPhenotype was unable to analyze an in-frame insertion of arginine and isoleucine at protease codon 35 of one CRF01_AE isolate. Conclusions This is the first report to demonstrate the high degree of concordance of longitudinal genotyping data and clinical treatment outcome in patients harboring different HIV-1 subtypes. Our findings shed light to the emergence of resistance mutations and its testing in CRF01_AE, which is relevant to other prevailing places in Asia.

Induction of inflammation as a possible mechanism of probiotic effect in atopic eczema–dermatitis syndrome

The immunomodulating mechanisms of Lactobacillus GG (LGG) and other probiotics are poorly understood. We studied in vivo the immunologic effects of probiotics in infants with atopic eczema-dermatitis syndrome (AEDS) and cow's milk allergy (CMA). Two hundred thirty infants with AEDS and suspected CMA received, concomitant with elimination diet, either LGG, a mixture of 4 probiotic strains (MIX), or placebo for 4 weeks. All available paired pretreatment and posttreatment plasma samples (n = 132) were analyzed for concentrations of IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma, soluble intercellular adhesion molecule 1, soluble E-selectin, TGF-beta1, TGF-beta2, and C-reactive protein. In infants with IgE-associated AEDS, treatment with LGG induced higher C-reactive protein levels than in the placebo group (geometric mean, 0.83 microg/mL [95% CI, 0.56-0.81] vs 0.42 microg/mL [95% CI, 0.27-0.65]; P = .021). Concomitantly, IL-6 levels increased after treatment with LGG ( P = .023) but not with MIX or placebo. Soluble E-selectin levels were higher after probiotic than after placebo treatment in infants with IgE-mediated CMA (LGG geometric mean, 86.7 ng/mL [95% CI, 75.2-100]; MIX geometric mean, 91.6 ng/mL [95% CI, 74.8-111.9]; and placebo geometric mean, 64.9 ng/mL [95% CI, 53-79.3]; analysis of covariance, P = .035; LGG vs placebo, P = .023; MIX vs placebo, P = .020). Use of MIX induced an increase in plasma IL-10 levels ( P = .016). Probiotics induced systemically detectable low-grade inflammation, which might explain the clinical effects of probiotics in AEDS and CMA.