Molecular Stratification Research Articles

Comparative Study of Tumor Biopsy and Ctdna to Support Patient Selection in Phase I Trials: The Gustave Roussy Single Center Experience in Aggressive B-Cell Lymphomas

Patient's selection in early phase clinical trials is guided by stringent clinical criteria and can be supported or improved by precise tumor characterization and/or molecular stratification. Most of the precision medicine data are generated using historical tumor biopsies often collected before anticancer drugs treatment, which may thus only partially reflect the tumor's molecular landscape at inclusion in early phase clinical trials. The gold standard fresh tumor biopsy is not always feasible without risk for the patient. For this reason, ctDNA could be a simple, safe and attractive technique for obtaining critical information on the tumor genomic landscape. This study was designed to explore if whether NGS results obtained with from ctDNA could replace fresh tumor biopsies in patients (pts) at inclusion in early phase clinical trials. Data generated on both tissue samples collected in a short period of time in 52 pts treated for a B-cell aggressive lymphoma in the DITEP department at Gustave Roussy prior to inclusion were compared (18/52 pts had one course of chemotherapy between the 2 tissue samples). Characteristics at sampling were: mean age = 66 y.o., male/female: 32/20, Ann Arbor staging: 1/2/3/4 = 4 (7.7%), 5 (9.6%), 8 (15.4%) and 35 (67.3%) pts. The median number of prior chemotherapy was 2 (Min=1; Max=7). NGS tests were done using a panel of 39 genes and more recently 44 genes. 34 genes were found mutated in at least one pt. 150 mutations of pathologic significance were identified and 105 mutations out of 150 (70.0%) were concordant between the fresh biopsy and the blood (95%CI=62.0%; 77.2%). No patient had a mutation detected in the ctDNA analysis that was not found in the fresh tumor biopsy. In 8/52(15%) patients, no mutation was detected in both tumor biopsy and blood. Of the 44-remaining pts, 10 had no mutation detected in the blood and 34 pts (77.3%) carried at least one mutation detectable in both the tumor and blood. In 25/44patients (56.8%) all mutations identified in the tumor could be also detected in the blood (95%CI=41.0%; 71.7%), which are "informative ctDNA patients" if we consider that tumor biopsy is the "gold standard" as it is in clinical trials. Among the 44 pts that have at least one mutation detected in the tumor, the median VAF detection in the tumor biopsy was 41% (range 2-98%) and in the blood it was 7% (range 0-83%), but if we focus on the 105 mutations detected in both the biopsy and in the blood, the median VAF observed is 43% in the tumor (range 2-98%) and 14% in the blood (range 1-83%). Our results suggest that 85% of the pts included in phase I trials have at least one detectable mutation in the tumor biopsy, but approximately 50% of the pts have concordant mutations found in both ctDNA and fresh tumor samples. These results also suggest that ctDNA could be a useful tool in those "informative ctDNA" pts for residual disease assessment without sequential biopsies. Disclosures Ribrag: MSD: Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel, accommodations, and expenses ; BMS: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, and expenses ; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; AZ: Membership on an entity's Board of Directors or advisory committees; ArgenX: Research Funding; Epizyme: Consultancy, Research Funding.

RNA-Seq Can Help Identify IGH Disease Clones for MRD Monitoring in Childhood B-Lymphoblastic Leukemia

Background As a single platform, RNA sequencing (RNA-Seq) can accurately identify majority of oncogenic driver fusion genes and aneuploidy. This provides refined molecular subtyping and risk stratification; RNA-Seq is increasing used upfront for genetic subtype assignment in on-going contemporary ALL trials. The IGH gene is highly expressed in our diagnostic ALL samples using RNA-Seq. We investigated the usefulness of RNA-Seq in identifying IGH disease sequences for MRD monitoring in 258 childhood B-lymphoblastic leukemia (B-LL) samples. Methods RNA-seq was performed on a total of 259 diagnostic bone marrow or peripheral blood samples from children with B-LL treated in the Ma-Spore ALL 2003 and ALL 2010 studies. The reads were aligned to germline IGH gene V, D and J segments, and reads covered IGH VHDJH or DJH junctional regions are clustered into clones. Overall, the distribution of read count of the IGH background clones follows Zipf's law, i.e. the read count of the IGH background clones is negative linearly correlated with the number of IGH clones with that read count in the log-log scale (Figure 1A). IGH disease clones are outliers with exceedingly high read count. For each sample, a linear model is fitted between log transformed read count (using only read count ≤10) and log transformed number of IGH clones. The expected number of IGH clones (E-value) of a read count can be predicted by the fitted linear model. A clone with E-value <0.01 is defined as a disease clone (Figure 1B). Disease clones identified by RNA-Seq are compared with conventional Sanger sequencing. Results In 90.3% of the patients, 497 IGH disease clones (median 2, range 0-7 clones/patient) are identified. The number of IGH disease clones significantly correlated with genetic subtype (P=1.9×10-8; Table 1). Specifically, high hyperdiploid patients have the most IGH disease clones (median 3) with 67% (22/33) have >2 IGH disease clones, due to the gain of chromosome 14. In contrast, DUX4 high expressers have the least (median 1) IGH disease clones with 19% (8/43) have no IGH disease clone, due to the rearrangements involving one of the IGH locus. Of the 348 IGH disease clones identified by Sanger sequencing, 90.8% are also identified by RNA-Seq; and in the 199 Sanger sequences that yielded sensitive markers, 191 (96.0%) are also defined as IGH disease clones by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets by Sanger sequencing, the disease clones identified by RNA-Seq were used collectively for MRD monitoring by targeted deep sequencing of IGH (IGH-Seq) and showed high correlation with conventional RQ-PCR MRD using non-IGH targets (R=0.93; P=1.3x10-14;Figure 1C). In addition, IGH-Seq showed improved sensitivity with quantifiable MRD in 20% more samples compared to RQ-PCR (78% vs 58%; Figure 1C). In the samples that are positive below sensitivity or negative by RQ-PCR, IGH-Seq obtained quantifiable MRD for 60% (9/15) or 25% (5/20) of the samples (Figure 1C). Conclusion In conclusion, IGH disease clone sequences are highly expressed and can be distinctly identified from whole transcriptome RNA-Seq in >90% of children with B-LL. These can be used for molecular MRD monitoring using RQ-PCR or NGS-based IGH-Seq. In addition to identifying the constellation of oncogene fusions and chromosomal aneuploidies that drive B-LL and RNA-Seq can identify IGH MRD sequences for molecular MRD monitoring. Disclosures No relevant conflicts of interest to declare.

Molecular Treatment Stratification for Newly Diagnosed High-Risk Myeloma, Including Plasma Cell Leukemia - Feasibility Results of the Ukmra Optimum: MUK9 Trial (NCT03188172)

Background High-risk myeloma patients have unsatisfactory outcomes with current treatments and are in urgent need of improved diagnostic and therapeutic strategies. We have recently validated specific markers predicting high-risk disease in newly diagnosed MM (NDMM), in particular double-hit with presence of ≥2 consensus high-risk markers t(4;14), t(14;16), t(14;20), del(1p), gain(1q), del(17p) (Shah V, et al., Leukemia 2018) and diagnostic GEP SKY92 high risk signature (Sherborne A, et al., IMW 2017). Diagnostic tests for these markers were implemented in the UK multi-center OPTIMUM: MUK9 trial to prospectively stratify therapy for high-risk NDMM. Trial design OPTIMUM: MUK9 is a phase 2 trial for transplant eligible NDMM, consisting of two inter-related protocols: a molecular screening protocol (MUK9A) and an interventional trial (MUK9B) for high-risk MM identified in MUK9A. Patients with suspected or confirmed MM fit for intensive therapy enrolled in MUK9A have central molecular profiling at ICR, London, of CD138-selected BM MM cells for translocations, copy number aberrations (qRT-PCR; MLPA P425, MRC Holland) and SKY92 signature status (MMprofiler; SkylineDx). If clinically indicated SOC therapy (VTD, max. 2 cycles) can be given whilst central results are generated. Patients found to have high-risk MM by double-hit and/or SKY92 are offered enrolment into MUK9B. All other patients receive SOC (VTD, HD-MEL+ASCT) for which clinical data is collected. Patients diagnosed with plasma cell leukemia (PCL) can be enrolled directly in MUK9B. MUK9B treatment consists of quintuplet daratumumab, cyclophosphamide, bortezomib, lenalidomide, dexamethasone (Dara-CVRd) induction (up to 6 cycles), bortezomib-augmented single HD-MEL+ASCT, Dara-VRd consolidation 1 (6 cycles), Dara-VR consolidation 2 (12 cycles) and Dara-R maintenance (until PD). Dose adjustments are permitted in order to maximize tolerability of long-term therapy. Patient reported outcomes (PRO) are recorded at baseline and throughout treatment. Response and MRD are centrally assessed (Birmingham, Leeds). Primary endpoint for MUK9A is feasibility of central molecular testing within 56 days turnaround time, which we report on here. Primary endpoint of MUK9B is treatment efficacy, comparing MUK9B PFS to near-concurrent molecularly matched high-risk patient outcomes from UK NCRI Myeloma XI using a Bayesian design. Secondary endpoints include safety, PFS2, MRD and OS and study of molecular evolution in high-risk disease. Results The protocol recruited 29/Sep/17 - 31/Jul/19 at 39 UK sites, achieving the recruitment target of 105 high-risk patients treated on MUK9B ahead of projections. At the time of analysis (12/Jul/19), 430 patients with suspected or confirmed NDMM have been recruited to MUK9A across 39 UK NHS hospitals. Of these, 376 (87%) patients were confirmed to have symptomatic MM (60.9% male; median age 61y (range 29-79)) as per updated IMWG diagnostic criteria (2014), including 9 (2%) PCL patients, with the remainder diagnosed as SMM/MGUS (31; 7%) or other (14; 3%). For 371 of the 376 symptomatic MM patients BM was received by the central laboratory and was of sufficient quality for profiling in 331 (89%) patients. Repeat samples were requested for all others and a sufficient sample received for 20/45 (44%). Central results were successfully reported within the pre-specified 56 day interval for all patients (median 17 days; IQR 13-22). Of 346 patients with a reported result, 128 (37.0%) have high-risk MM, with molecular characteristics mirroring Myeloma XI patients (Figure 1). PCL patients show expected characteristics as listed in Table 1. Basic demographics were not different between high-risk vs. non-high-risk. 101 high-risk patients have or are planning to enter MUK9B, 10 pending decision; 17 high-risk patients did not enter MUK9B, the majority due to ineligibility. 92 patients have started Dara-CVRD therapy. There are currently no safety concerns, the majority of patients are completing induction successfully; 1 patient stopped induction therapy due to adverse events. Updated results will be presented. Discussion Our data demonstrate feasibility of multi-center molecular stratified trial delivery for high-risk NDMM patients. These early trial results strongly support accelerated trial strategies for MM patient groups with high unmet need and rational drug development specifically for high-risk MM. Disclosures Jenner: Abbvie, Amgen, Celgene, Novartis, Janssen, Sanofi Genzyme, Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hall:Celgene, Amgen, Janssen, Karyopharm: Other: Research funding to Institution. Walker:Janssen, Celgene: Other: Research funding to Institution. Croft:Celgene: Other: Travel expenses. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Flanagan:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Drayson:Abingdon Health: Consultancy, Equity Ownership. Owen:Celgene, Janssen: Consultancy; Celgene: Research Funding; Janssen: Other: Travel expenses; Celgene, Janssen: Honoraria. Pratt:Binding Site, Amgen, Takeda, Janssen, Gilead: Consultancy, Honoraria, Other: Travel support. Cook:Celgene, Janssen-Cilag, Takeda: Honoraria, Research Funding; Janssen, Takeda, Sanofi, Karyopharm, Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen, Bristol-Myers Squib, GlycoMimetics, Seattle Genetics, Sanofi: Honoraria. Brown:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Kaiser:Celgene, Janssen: Research Funding; Abbvie, Celgene, Takeda, Janssen, Amgen, Abbvie, Karyopharm: Consultancy; Takeda, Janssen, Celgene, Amgen: Honoraria, Other: Travel Expenses.

A Phase I/II Study of Ibrutinib and Ixazomib in Relapsed/Refractory Mantle Cell Lymphoma: PrE0404

A Phase I/II Study of Ibrutinib and Ixazomib in Relapsed/Refractory Mantle Cell Lymphoma: PrE0404

CBMT-03. EVALUATION OF PET TRACERS TO DIFFERENTIATE IDH1 MUTANT GBM

Abstract OBJECTIVE PET/CT offers the unique potential to noninvasively evaluate biomarker expression and aberrant metabolism. 18F-FDG has driven PET/CT to the forefront of cancer imaging, as altered glucose metabolism is a hallmark of oncogenesis. However, 18F-FDG is suboptimal for GBM due to high physiologic uptake in normal brain. The development of alternative tracers has reignited the field of PET/CT in GBM and offers hope for diagnosis and molecular staging in GBM. We hypothesize that fundamental differences in metabolism and oncogene expression present in IDH1 mutatant gliomas can be shown using PET imaging, as has been suggested in some correlative clinical studies. Our objective was therefore to establish a causal link between IDH1 mutation in GBM and uptake of targeted PET tracers in a unique proneural GBM transgenic model characterized by p53, IDH1 mutations and PDGF expression. METHODS We examined the uptake of 3 blood-brain-barrier (BBB) penetrant tracers that have been used in GBM: (a) 18F-FDG, a surrogate for increased glycolysis; (b) 18F-Fluciclovine, an amino acid derivative transported into cells through the energy-independent L-type amino acid transporter (LAT) system and is approved by the FDA for prostate cancer; and (c) 11C-ER176, a tracer that binds the TSPO receptor that is generally expressed on activated microglia as well as GBM. RESULTS We did not observe significant differences in 18F-FDG uptake between wt and IDH mutant GBM cells. However, we found that IDH mutant GBM cells demonstrated significantly increased 18F-Fluciclovine (47%) and 11C-ER176 (53%) versus IDH wt cells. CONCLUSION We have established a causal link between IDH mutation status and uptake of (a) 18F-Fluciclovine, a promising FDA approved PET tracer and (b) 11C-ER176, a second generation TSPO ligand. Thus, we are performing further studies in orthotopic syngeneic GBM models to determine if PET imaging can non-invasively demonstrate molecular characterization and therapeutic stratification in glioma.

RBTT-06. TESSA JOWELL BRAIN MATRIX STUDY: A BRITISH FEASIBILITY STUDY OF MOLECULAR STRATIFICATION AND TARGETED THERAPY TO OPTIMIZE THE CLINICAL MANAGEMENT OF PATIENTS WITH GLIOMA

Abstract In 2016 there were 5250 brain cancer deaths in the UK. Standard treatment is surgical resection followed by chemo-radiotherapy. In most cases of diffuse glioma, complete tumour resection is not feasible. Many chemotherapy drugs have untested penetration through the blood brain barrier, potentially leading to sub-therapeutic concentrations in the tumour. There is need to refine current treatment strategies in relation to the understanding of tumour biology, and rapidly introduce and evaluate novel therapeutic approaches and agents through delivering rigorous clinical trials. The TESSA JOWELL BRAIN MATRIX Study will evaluate the feasibility of delivering precision medicine for brain cancer patients within the NHS. A multicentre, platform feasibility study of 1200 patients with diffuse glioma will build on the 100,000 genome project to develop and evaluate an infrastructure to collect and integrate: 1) real time comprehensive integrated molecular analysis, including whole genome sequencing and epigenetic classification; 2) serial sampling and annotation of tumours; 3) collection of matched clinical data; 4) assessment of patient quality of life; 5) centralised radiological review and response assessment as per RANO criteria. Once developed this will allow rapid introduction of therapeutic trials to specific patient groups. Secondary objectives include: understanding the association between extent of resection and molecular stratification to refine the role of surgery; optimisation and harmonisation of protocols to best collect, manage and store tissue, clinical data, and radiological images in order to provide a resource for researchers, both within and outside of the study. Improve patient recruitment by identifying and removing recruitment barriers and improve the information and consent processes for patients. Promote the development of a national network with expertise in brain cancer. Enrolment of the first patient is expected in late 2019. For further information, please contact the Brain Matrix Trial Office BrainMatrix@trials.bham.ac.uk.

Optimization of in silico tools for predicting genetic variants: individualizing for genes with molecular sub-regional stratification.

Genes are unique in functional role and differ in their sensitivities to genetic defects, but with difficulties in pathogenicity prediction. This study attempted to improve the performance of existing in silico algorithms and find a common solution based on individualization strategy. We initiated the individualization with the epilepsy-related SCN1A variants by sub-regional stratification. SCN1A missense variants related to epilepsy were retrieved from mutation databases, and benign missense variants were collected from ExAC database. Predictions were performed by using 10 traditional tools with stepwise optimizations. Model predictive ability was evaluated using the five-fold cross-validations on variants of SCN1A, SCN2A, and KCNQ2. Additional validation was performed in SCN1A variants of damage-confirmed/familial epilepsy. The performance of commonly used predictors was less satisfactory for SCN1A with accuracy less than 80% and varied dramatically by functional domains of Nav1.1. Multistep individualized optimizations, including cutoff resetting, domain-based stratification, and combination of predicting algorithms, significantly increased predictive performance. Similar improvements were obtained for variants in SCN2A and KCNQ2. The predictive performance of the recently developed ensemble tools, such as Mendelian clinically applicable pathogenicity, combined annotation-dependent depletion and Eigen, was also improved dramatically by application of the strategy with molecular sub-regional stratification. The prediction scores of SCN1A variants showed linear correlations with the degree of functional defects and the severity of clinical phenotypes. This study highlights the need of individualized optimization with molecular sub-regional stratification for each gene in practice.

<p>Poor Prognosis With Coexistence Of <em>EGFR</em> T790M Mutation And Common <em>EGFR</em>-Activating Mutation In Non- Small Cell Lung Cancer</p>

PurposePrevious studies have shown that the presence of EGFR T790M mutation may reduce the treatment efficacy of tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. However, little is known about the clinical features and outcomes of EGFR T790M mutation in pretreated patients with NSCLC.Patients and methodsThe clinical features of EGFR-activating and T790M mutations were assessed in a large cohort of patients with EGFR-TKI-naïve NSCLC (all/EGFR mutations, n=16,347/7,687). The correlation between the pretreatment T790M mutation status and clinical outcomes was evaluated using univariate and multivariate analyses.ResultsPretreatment T790M mutation was reported in 1.39% of the patients and coexisted with an EGFR-activating or uncommon mutation. The dual EGFR T790M and common EGFR-activating mutations were more likely to be detected in lung adenocarcinoma, whereas single T790M mutation was more prevalent in non-adenocarcinomas. The presence of de novo T790M mutation correlated with reduced recurrence-free survival (RFS) in patients with NSCLC (odds ratio [OR] 3.37, 95% confidence interval [CI] 1.67–6.79, P = 0.001). After molecular stratification, T790M mutation was shown to exert adverse effects on the RFS of EGFR 19-del group (OR 2.89, 95% CI 1.10–7.91, P = 0.028) and EGFR L858R group (OR 3.43, 95% CI 1.33–8.88, P = 0.013). Furthermore, pretreatment T790M mutation promoted tumor metastasis to different sites.ConclusionT790M-positive tumors presented special clinical features, and the coexistence of T790M and common EGFR-activating mutations was associated with poor prognosis in patients with NSCLC.

Combination of mTOR inhibition and paclitaxel as a personalised strategy in the context of MYC-amplified high-grade serous ovarian cancer

Abstract Background The extreme genomic heterogeneity of high-grade serous ovarian carcinoma (HGSOC) is a major barrier to precision medicine approaches. We have developed mutational signature analysis from shallow whole genome sequencing (sWGS) for molecular stratification and therapeutic prediction. PARP inhibitor therapy has significant survival advantage for women with germline BRCA1/2 mutations, but treatments and predictive biomarkers for women without homologous recombination deficiency have not been developed. MYC amplification is the most common driver in HGSOC, present in 42% of the TCGA cases. The best responder from the phase 1 trial of AZD2014 (dual m-TORC1/2 inhibitor) and paclitaxel in HGSOC had a mutation in MYC and we have hypothesised that biosynthetic effects of MYC are mediated through the mTOR pathway and blocked by AZD2014 perturbation. Methods Primary ovarian cancer spheroids were purified from 28 human ascites and profiled with sWGS. Ex-vivo therapeutic response was measured to four standard of care chemotherapeutic agents and eight inhibitors targeting nodal points of the PI3K and DNA damage repair pathways. RNA seq data from the TCGA HGSOC cohort was used to establish the genes whose expression was highly correlated with the expression of MYC. Results Ex-vivo assays were predictive of first-line clinical response to chemotherapy agents (P Conclusions Our results suggest that therapeutic strategies combining paclitaxel and inhibition of mTOR pathway can be used as personalised treatment in the context of MYC-amplified HGSOC. We are currently examining gene expression after AZD2014 pertubation in HGSOC models and performing the genomic analysis of cases from the phase 1 trial of AZD2014 and paclitaxel. Legal entity responsible for the study The authors. Funding Academy of Medical Sciences, Addenbrookes Charitable Trust, Cancer Research UK, University of Cambridge. Disclosure F.C. Martins: Research grant / Funding (self), The Clinical Lectureship from the Experimental Medicine Initiative is partly funded by AstraZeneca: AstraZeneca. All other authors have declared no conflicts of interest.

Using next generation sequencing technology to analyze gene mutations in patients with acute myeloid leukemia and the impact on prognosis

Objective: To analyze the distribution of gene mutations in newly diagnosed acute myeloid leukemia (AML) patients, based on next generation sequencing technology (NGS) and to evaluate their value in AML risk stratification. Methods: The study analyzed 453 newly diagnosed AML(excluded acute promyelocytic leukemia, APL) patients from seven hospitals in Shanghai, from January 1st 2014 to December 31th 2017. RNA and DNA were extracted from pretreatment bone marrow mononuclear cells and targeted sequencing of AML genes were performed. The data of different groups was compared. Results: A total of 453 newly diagnosed AML patients were enrolled in the study, including 247 males and 206 females with a median age of 49.5 (range,11-85) years. A total of 540 mutations/fusion genes were detected in 289 patients, 29.1% (132/259) of whom with two or more mutations/fusion genes. In all patients, NPM1 was the most common mutation(12.8%), followed by ETO and TET2 mutation (11.92% and 11.04%, respectively) . And WT1 over-expression accounted for 10.6%. Patients over the age of 50 were with a higher frequency of mutations associated with epigenetic modification, 11.93% for ASXL1, 13.99% for DMNT3A, 6.58% for IDH1/IDH2, and 13.17% for TET2. The frequency of DMNT3A mutations was three times higher than that of patients under 50 years of age (P=0.017). In this study, a relatively low proportion of genetic mutations was observed in low-risk karyotype group. In the medium-risk karyotype group, the relatively high mutation frequencies were observed in NPM1, TET2, FLT3-ITD, DNMT3A, ASXL1, and CEBPA genes. In the poor-risk karyotype group, the mutation frequencies of ASXL1, TET2, DNMT3A and PHF6 genes were more than 10%, especially ASXL1 and PHF6 mutation frequencies were significantly higher than other molecular risk stratification groups (P<0.05). Of the 254 patients (56%) with normal karyotype AML (NK-AML), 56 patients were detected to have gene mutations about epigenetic modification. The median OS of this group was worse than that of patients without related mutations, while the median LFS had no significant difference. In patients with NK-AML older than 50 years, the OS and LFS of patients with epigenetic modification related gene mutations was 12 months and 10 months, versus 18 months and 12 months of patients without mutations. Conclusions: The gene mutations frequencies in AML patients with different age and molecular risk stratification groups are different. Epigenetics gene mutation frequencies, such as DNMT3A, ASXL1, IDH1/IDH2 and TET2,are higher in patients older than 50 years. A shorter OS can be observed in older patients(>50 years) with epigenetics gene mutation.

Molecular stratification of idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (INS) describes a group of pathologies of the renal glomerulus that result in the classic triad of heavy proteinuria, oedema and hypoalbuminaemia. The disease has historically been defined by evidence of distinctive histological changes in the absence of clinical evidence of a distinct pathological driver. However, the current classification is not based on any systematic mechanistic understanding of biological processes, and therefore current treatment regimens are broad, iterative and nonspecific. Over the past 20 years delineation of the underlying biology of the target cell in INS - the glomerular podocyte - has transformed our understanding of the mechanisms that contribute to breakdown of the glomerular filtration barrier and the development of INS. It is increasingly clear that nephrotic syndrome caused by monogenic mutations is distinct from immune-driven disease, which in some cases is mediated by circulating factors that target the podocyte. The combination of systems biology and bioinformatics approaches, together with powerful laboratory models and ever-growing patient registries has potential to identify disease 'signatures' that reflect the underlying molecular mechanism of INS on an individual basis. Understanding of such processes could lead to the development oftargeted therapies.

Observation after surgery for low grade glioma: long-term outcome in the light of the 2016 WHO classification.

To provide detailed long-term data after initial observation for patients after histological confirmation of low grade (WHO II) gliomas according to molecular stratification. A series of 110 patients with watchful waiting strategy after initial surgery for LGG and re-surgery at tumor progression were analyzed. Progression-free survival, time to malignant transformation, post-recurrence survival, and overall survival were estimated with the Kaplan-Meier method. Prognostic factors were identified by the Log Rank test and Cox multivariate proportional hazards model. The cohort comprised 18 IDH wild type (IDHwt) and 53 IDH mutated (IDHmut) astrocytomas, and 39 IDH mutated and 1p 19q co-deleted (IDHmut/codel) patients. The median follow-up was 126 (95% CI 109-143) months. Surgery was gross total resection in 58, subtotal resection in 28, and biopsy in 24 patients. Progression-free survival rates at 5, 10 and 15 years was 38% 18% and 1%. The corresponding malignant transformation rates were 17%, 39% and 71%. The initial extent of resection influenced progression-free survival, time to malignant transformation and overall survival. Molecular subtype IDHmut/codel was the strongest prognostic factor for overall survival and for time to malignant transformation. The strongest determinant of the patients' course after initial watchful waiting was the molecular tumor status. Extensive resection may increase time to progression and malignant transformation. Observation may be justified in selected patients.

Relevance of calcification and contrast enhancement pattern for molecular diagnosis and survival prediction of gliomas based on the 2016 World Health Organization Classification

ObjectivesThe significance of conventional neuroimaging features for predicting molecular diagnosis and patient survival based on the updated World Health Organization (WHO) classification remains uncertain. We assessed the relevance of neuroimaging features (ring enhancement [RE], non-ring enhancement [non-RE], overall gadolinium enhancement [GdE], and intratumoral calcification [IC]) for molecular diagnosis and survival in glioma patients. Patients and methodsWe evaluated 234 glioma patients according to the updated WHO classification. Isocitrate dehydrogenase (IDH), H3F3A, BRAF hotspot mutations, TERT promotor mutation, and chromosome 1p/19q co-deletion were examined. RE, non-RE, GdE, and IC were evaluated as significant neuroimaging findings. Kaplan-Meier analyses were performed to evaluate overall survival (OS) and the correlations of prognostic factors were evaluated by log-rank tests. Univariate and multivariate analyses were performed to detect prognostic factors for OS. ResultsA total of 207 patients were eligible. In 110 patients presenting RE, 102 (93%) were glioblastoma (GBM), IDH-wild type. In 97 patients without RE, presence of GdE or IC were not significantly different between IDH-mutant and -wild type tumors, whereas presence of GdE was a significant indicator of higher WHO grades. IC was the only significant finding for 1p/19q co-deleted tumors. TERT promoter mutation was observed in 7/17 patients with diffuse astrocytic glioma, IDH-wild type; recently-defined as “molecular GBM.” IC, RE, and GdE were observed with lower prevalence in molecular GBMs. While presence of RE, GdE, and absence of IC were significant factors of OS in overall cohort, presence of GdE was not significant in OS in cases without RE, and IDH-mutant tumors. IC was a significant predictor of favorable OS in cases without RE and IDH-wild type tumors. Multivariate analysis also validated these findings. ConclusionGdE alone is not a significant predictor of IDH mutation status, but the pattern of enhancement is a significant predictor with RE demonstrating high sensitivity and specificity for GBM, IDH-wild type. Predicting “molecular GBM” by conventional neuroimaging is difficult. Moreover, GdE is not a significant factor of survival analyzed with pattern of enhancement or molecular stratifications. IC is an important radiographic finding for predicting molecular diagnosis and survival in glioma patients.

878P - Prognostic value of tumour suppression genes (TP53, PTEN, Rb) in metastatic hormone sensitive prostate cancer

BackgroundLoss of tumour suppressor genes (TSGs), TP53, PTEN and Rb are common in advanced prostate cancer. They are associated with worse outcomes in hormone-resistant prostate cancer. However, the prognostic value of TSGs loss in metastatic hormone-sensitive prostate cancer (mHSPC) is unknown. Therefore we sought to evaluate the prognostic value of TSGs loss on progression-free survival (PFS) in mHSPC. MethodsWe performed a retrospective analysis of patients with mHSPC treated at Mayo Clinic Arizona between 2015 and 2018. Clinical characteristics, first-line treatment, and TSGs alterations were recorded. TSG-altered (TSG-alt) was defined as an alteration in≥1 TSG (TP53, PTEN or Rb). Demographics and clinical characteristics were compared between groups by chi-square test for frequency data or Kruskal Wallis rank-sum test for continuous measures. PFS was estimated based on the Kaplan-Meier method and compared between groups by the log-rank test. Results59 patients with mHSPC were included. 41 (69%) patients received first-line treatment with abiraterone and 18 (31%) patients with docetaxel. 32 patients (54%) had TSG-alt. 20 patients (39%) had damage to 1 TSG, 11 patients (19%) had damage to 2 TSGs, and 1 patient had damage to all 3. Clinical characteristics, prior locoregional treatment, and metastatic disease on diagnosis were similar between TSG-alt and TSG-normal groups. The rate of damaging alterations to the genes was: TP53 21 patients (35.6%), PTEN 21 patients (35.6%), and Rb 3 patients (5.1%). Median PFS was 33.2 mos (95% CI 23.1-NE) for TSG-normal versus 19.7 mos (95% CI 14.4-30.0) for TSG-alt, p=0.005. There was no difference in PFS between having 1 TSG alteration (18.4 mos; 95% CI 14.7 - NE) vs≥2 TSG alterations (21.8 mos; 95% CI 10.6 - NE) or between first-line treatments with abiraterone (20.2 mos; 95% CI 14.7-35.1) vs docetaxel (19.6 mos; 95% CI 8.0 - NE) in those with TSG-alt. ConclusionsLoss of TSGs function is common in patients with mHSPC, and is prognostic for early progression during first-line treatment. The presence of≥1 TSG-alt indicates an adverse prognostic impact in mHSPC. Confirmation of these findings may establish the need for inclusion of molecular stratification when defining high risk mHSPC patients. Legal entity responsible for the studyMayo Clinic Cancer Center. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Liquid biopsy in oncology: a consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology

The proportion of cancer patients with tumours that harbour a potentially targetable genomic alteration is growing considerably. The diagnosis of these genomic alterations can lead to tailored treatment at the onset of disease or on progression and to obtaining additional predictive information on immunotherapy efficacy. However, in up to 25% of cases, the initial tissue biopsy is inadequate for precision oncology and, in many cases, tumour genomic profiling at progression is not possible due to technical limitations of obtaining new tumour tissue specimens. Efficient diagnostic alternatives are therefore required for molecular stratification, which includes liquid biopsy. This technique enables the evaluation of the tumour genomic profile dynamically and captures intra-patient genomic heterogeneity as well. To date, there are several diagnostic techniques available for use in liquid biopsy, each one of them with different precision and performance levels. The objective of this consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology is to evaluate the viability and effectiveness of the different methodological approaches in liquid biopsy in cancer patients and the potential application of this method to current clinical practice. The experts contributing to this consensus statement agree that, according to current evidence, liquid biopsy is an acceptable alternative to tumour tissue biopsy for the study of biomarkers in various clinical settings. It is therefore important to standardise pre-analytical and analytical procedures, to ensure reproducibility and generate structured and accessible clinical reports. It is essential to appoint multidisciplinary tumour molecular boards to oversee these processes and to enable the most suitable therapeutic decisions for each patient according to the genomic profile.

Identification of distinctive long noncoding RNA competitive interactions and a six-methylated-gene prognostic signature in acute myeloid leukemia with -5/del(5q) or -7/del(7q).

Acute myeloid leukemia (AML) with -5/del(5q) or -7/del(7q) has special clinical and biological characteristics, but its molecular mechanisms and risk stratification remain unknown. The RNA sequencing and DNA methylation of 23 patients with -5/del(5q) or -7/del(7q) and 128 patients with other subtypes of acute myeloid leukemia were obtained from The Cancer Genome Atlas (TCGA). The regulatory mechanisms of competitive endogenous RNA (ceRNA) network and DNA methylation on gene expression were explored. To find robust and specific risk stratification for this AML subtype, a prognostic model was established and evaluated through four independent data sets. We identified 966 differentially expressed long noncoding RNA, 2274 differentially expressed genes, and 47 differentially expressed microRNAs, and constructed a ceRNA network. After the integrated analysis of differentially methylated and expressed genes, 19 genes showed the opposite trend between the methylation variation and gene expression. An six-methylated-gene prognostic signature which highly correlated with overall survival was established, and the performance was validated by leave-one-out cross validation method and permutation test. Furthermore, the excellent prognostic value of this model was supported by an independent cohort, while specificity of this model was validated by three independent data sets, suggesting it as a predictive classifier with high efficiency for distinguishing those with -5/del(5q) or -7/del(7q) from other AML subtypes. The ceRNA network may provide new ideas for the diagnosis and treatment for patients with -5/del(5q) or -7/del(7q).The six-methylated-gene prognostic signature was a robust, specific, and clinically practical risk stratification for the outcome of patients with AML having -5/del(5q) or -7/del(7q).

OS12.3 L3MBTL3 is a novel suppressor of medulloblastoma tumorigenesis

Abstract BACKGROUND Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements but are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed and molecular stratification of patients with medulloblastoma has yet to be routinely implemented in the clinic. TheNotchpathway governs cell proliferation in many biological contexts, including SHHand Group#3medulloblastoma tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of Notch for the modulation of Notch signals, and L3MBTL3, a methyllysine reader.L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of Notch/RBPJ target genes to repress their expression.Deletions ofthe L3MBTL3locus are observed in patientswith WNTand Group#3medulloblastomaand expression of L3MBTL3 in the SHHmedulloblastoma-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in medulloblastoma. METHODS To further investigate the putative role of L3MBTL3 as a suppressor of medulloblastoma tumorigenesis, we used our novel L3mbtl3KO mouse in combination with a genetically engineered ND2:SmoA1mouse model of SHHmedulloblastoma in a survival analysis. RESULTS Our survival analysis validated in vivoour hypothesis that L3mbtl3is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P= 0.0322; Log-rank test). Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of Notch/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in medulloblastomathrough aberrant “de-repression” of Notch/RBPJ target genes. CONCLUSION L3MBTL3 is a novel suppressor of medulloblastoma tumorigenesis. Our discovery providesinsights into the role of the L3MBTL3 inmedulloblastomathat could be harnessed in the future for the therapeutic benefit of patientswith medulloblastoma.

Plasmacytoid DCs From Patients With Sjögren's Syndrome Are Transcriptionally Primed for Enhanced Pro-inflammatory Cytokine Production

Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each n = 31) and performed RNA-sequencing, after which data-driven networks and modular analysis were used to identify robustly reproducible transcriptional “signatures” of differential and co-expressed genes. Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of in vitro activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon in vitro stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.

Novel Poly(Adenosine Diphosphate-Ribose) Polymerase (PARP) Inhibitor, AZD2461, Down-Regulates VEGF and Induces Apoptosis in Prostate Cancer Cells

Background:Prostate cancer (Pca) is a heterogeneous disease, and current treatments are not based on molecular stratification. Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors have recently been found to be remarkably toxic to cells with defects in homologous recombination, particularly cells with BRCA-mutated backgrounds. Therefore, this preliminary study was designed to evaluate whether PTEN expression status could have an impact on the sensitivity of invasive Pca cells to the PARP inhibitor, AZD2461.Methods:MTT viability test, Annexin V‐FITC/propidium iodide double staining, and caspase3 activity assay were used to evaluate the apoptosis and relative expression of PTEN and VEGF in PC-3 and DU145 cell lines using real-time PCR. Results:MTT results showed that the inhibitory effects of AZD2461 were higher in PC-3 than DU145 cells (with IC50 of 36.48 and 59.03 µM at 48 hours of treatment, respectively). Flow cytometric analysis also showed the same results. When exposed to 40 µM of AZD2461, PC-3 (38.8%) and DU145 (28%) cells underwent apoptosis (p < 0.05). Treatment of cells by AZD2461 also caused a significant increase in apoptosis through caspase3 activation in both cell lines. VEGF mRNA levels in PC-3 cells significantly decreased compared to adjusted untreated cells (p < 0.05) in all measured times while displaying different alteration patterns in DU145 cells (p < 0.05). Conclusion:AZD2461 suppresses the growth of prostate tumor cells since AZD2461 monotherapy could prove to be efficacious, especially against cells not expressing PTEN besides activating the possible apoptosis-independent cell death pathways.

Robustness of sepsis-3 criteria in critically ill patients

BackgroundEarly recognition of sepsis is challenging, and diagnostic criteria have changed repeatedly. We assessed the robustness of sepsis-3 criteria in intensive care unit (ICU) patients.MethodsWe studied the apparent incidence and associated mortality of sepsis-3 among patients who were prospectively enrolled in the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) cohort in the Netherlands, and explored the effects of minor variations in the precise definition and timing of diagnostic criteria for organ failure.ResultsAmong 1081 patients with suspected infection upon ICU admission, 648 (60%) were considered to have sepsis according to prospective adjudication in the MARS study, whereas 976 (90%) met sepsis-3 criteria, yielding only 64% agreement at the individual patient level. Among 501 subjects developing ICU-acquired infection, these rates were 270 (54%) and 260 (52%), respectively (yielding 58% agreement). Hospital mortality was 234 (36%) vs 277 (28%) for those meeting MARS-sepsis or sepsis-3 criteria upon presentation (p < 0.001), and 121 (45%) vs 103 (40%) for those having sepsis onset in the ICU (p < 0.001). Minor variations in timing and interpretation of organ failure criteria had a considerable effect on the apparent prevalence of sepsis-3, which ranged from 68 to 96% among those with infection at admission, and from 22 to 99% among ICU-acquired cases.ConclusionThe sepsis-3 definition lacks robustness as well as discriminatory ability, since nearly all patients presenting to ICU with suspected infection fulfill its criteria. These should therefore be specified in greater detail, and applied more consistently, during future sepsis studies.Trial registrationThe MARS study is registered at ClinicalTrials.gov (identifier NCT 01905033).