WHO Subgroups Research Articles

MDB-75. MOLECULAR PATHOLOGY, TREATMENT AND PROGNOSIS OF INFANT SONIC HEDGEHOG MEDULLOBLASTOMA: A GLOBAL MULTI-COHORT STUDY

Abstract BACKGROUND Clinical studies in infant medulloblastoma (iMB; <5 years) have, to date, focussed on modestly-sized or national trials cohorts and have not directly compared therapeutic approaches or placed these in the context of dedicated biomarker studies, to inform future treatment strategies. METHODS We assembled a multi-national cohort of molecularly and clinically-annotated iMBs (<5 years at diagnosis; n=646), the largest to date. We investigated molecular pathology, treatments received, and relationships to outcome within this population. RESULTS The SHH group (iMBSHH; n=267, 40%) predominated, encompassing SHH-1 (37.7%, median age 2.0 years), SHH-2 (47.0%, 1.4 years) and SHH-3 (14.4%, 3.0 years) WHO subgroups. MBEN histology was significantly enriched in SHH-2, and SUFU mutation and MYCN amplification in SHH-1 and SHH-3, respectively. Notably, TP53 mutations were identified in all subgroups; in SHH-1 and SHH-2 (each n=3) tumours lacked features typically associated with TP53-mutated SHH-3 (LCA histology and MYCN amplification) and did not have a worse survival. Upfront radiation-sparing treatments were used in 132/267 children and comprised regimens founded on intraventricular methotrexate (IVT-MTX; 54.5%), high-dose (HDCTx; 22.0%) or standard-dose (23.5%) chemotherapy. Across all radio-naive iMBSHH, non-DN/MBEN histology (HR 2.76, CI 1.26-6.01, p=0.011) and SHH-1 (vs SHH-2, HR 2.51, CI 1.25-5.01, p=0.009) conferred worse PFS in univariable analysis; subgroup was the only independently prognostic risk-factor (multivariable analysis; SHH-1 HR 2.65, CI 1.27-5.51, p=0.009). 5-year OS for HDCTx recipients was very favourable in SHH-1 (100%) and SHH-2 (93.3%), and subgroup-dependent for IVT-MTX (SHH-1; 76.4%, SHH-2; 100%, p=0.009). In CSI-treated iMBSHH (n=49), non-DN/MBEN histology (HR 10.23, CI 2.22-47.04, p=0.003) and MYCN amplification (HR 7.35, CI 2.01-26.87, p=0.003) conferred worse PFS. CONCLUSION iMBSHH outcomes in this cohort are dependent upon WHO subgroup, histology and therapy received. These findings provide an evidence-based foundation for selection of cohorts and therapies for prospective assessment in forthcoming iMBSHH clinical trials (e.g. SIOP-CONNECT-YC-MB-LR).

Comparison of cytomorphology and histomorphology in myelodysplastic syndromes.

Gold standard for the establishment of the diagnosis of myelodysplastic syndromes (MDS) are cytomorphological features of hematopoietic cells in peripheral blood and bone marrow aspirates. There is increasing evidence that bone marrow histomorphology not only aids in the diagnosis of MDS but can provide additional prognostic information, particularly through assessment of fibrosis and cellularity. However, there is only sparse data on direct comparison between histological and cytomorphological findings within the same MDS patient cohort. Therefore, we performed such an analysis under exceptionally well-standardized conditions. We reexamined biopsy material of 128 patients from the Düsseldorf MDS registry who underwent bone marrow trephine biopsy (in addition to bone marrow aspiration) at the time of diagnosis, addressing the following items: a. Analysis of concordance of diagnoses made by histology and cytomorphology b. Analysis of additional information by histology with regard to the diagnosis and prognosis. The respective biomaterials were available at our institution and had been processed according to unchanged protocols between 1992 and 2010. Fresh histopathological sections were obtained from the tissue blocks, stained under identical conditions and re-assessed by a designated expert pathologist (C.B.) without knowledge of the previous histopathological report or the respective cytomorphological diagnosis. The latter, likewise, was uniformly made by the same expert cytomorphologist (U.G.). Histopathology of bone marrow trephine biopsies reliably captured the diagnosis of MDS. Assignment to the diagnostic WHO subgroup was not entirely concordant with cytomorphology, mainly due to incongruences between the proportion of CD34-positive cells on histopathology and the cytomorphological blast count. Histopathology provided additional diagnostic and prognostic information with high diagnostic and prognostic significance, such as fibrosis. Likewise, histopathology allowed more reliable estimation of bone marrow cellularity.

Characteristics and Distribution of Erythroid Progenitors and Precursors Correlate with Erythropoiesis Stimulating Agents (ESAs) Response in Lower Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are an heterogenous group of clonal myeloid disorders characterized by dysplastic ineffective hematopoiesis, peripheral blood cytopenias and a variable risk of progression to acute myeloid leukemia (AML) evaluated by IPSS-R score. Early stage erythropoiesis is dependent on growth factors like EPO, whereas late erythroid differentiation is iron-dependent. The entire process requires a fine regulation between apoptosis and cell survival. Anemia is the most common cytopenia in IPSS-R lower risk MDS (LR-MDS) and the mechanism underlying dyserythropoiesis is multifactorial, leading to apoptosis of erythroid progenitors and precursors. LR-MDS anemia is mainly treated with ESAs (e.g., Erythropoietin or EPO), but response is not optimal and eventually patients will suffer from chronic anemia and transfusion dependence. Moreover, the only strong predictor of ESAs treatment is the endogenous concentration of serum EPO (sEPO). The primary aim of this study was to detect quantitative and qualitative flow cytometry (FC) alterations of erythroid populations in the bone marrow (BM) of LR-MDS patients associated with resistance or response to ESAs and compared them with age-matched non cytopenic BM controls (CTRLs, 6). Secondly, we wanted to investigate if different WHO diagnostic MDS subgroups displayed specific pattern of erythroid maturation and/or FC abnormalities. We selected 97 LR-MDS anemic patients treated with ESAs at MDS Unit, Hematology dpt. AOU Careggi (Florence) and divided them according to type of ESAs response (e.g., primary refractory, PR; long responders, LR, with DOR longer than 24 months and secondary refractory, SR, with DOR shorter than 24 months). Baseline BM FC analysis was performed using an erythroid panel containing antibodies for CD33, CD34, CD36, HLA-DR, CD45, CD71, CD105, CD117. We also measured the coefficient of variance (CV) of CD71 and CD36 to assess qualitative FC abnormalities within the erythroid populations. We then identified all stages of erythroid maturation from A) CD34+ committed erythroid cells (CD34+, CD45dim, CD117+, CD71+, CD33+, HLA-DR +), or erythroid progenitors (COM-E), expressed both as fraction of all CD34+ cells and all 117+ progenitor cells, to B) CD34- erythroid precursors (from proerythroblasts, PROE, to orthochromatic erythroblasts). CV CD71 was increased in PR patients compared to CTRLs (p=0,009) and to responders (p=0,008). COM-E/tot.CD34+ cells were increased in all response categories, but there was a significant trend of higher values in LR vs. PR (p=0,055). Finally, (COM-E + PROE)/tot.117+ progenitors were only enriched in LR compared to CTRLs (p=0,009). Regarding WHO subgroups, MDS 5q- displayed a decrease in COM-E/CD34+ cells and in (COM-E + PROE)/tot.117+ cells compared to MDS-RS (p=0,00094 and p=0,009 respectively) and to CTRLs for the latter population (p=0,03). Moreover, total erythroid precursors were significantly reduced compared to MDS-SLD, -MLD and -RS. MDS-RS showed a marked erythroid hyperplasia and elevation of CV CD71 compared to CTRLs (p=0,0079 and p=0,024 respectively). Taken together, these results show that in LR-MDS, the increase of CV CD71 in erythroid cells is associated with resistance to ESAs, whereas response to ESAs is present in cases with enriched EPO sensitive COM-E and PROE populations compared to CTRLs, especially for LR vs PR (p=0,055). Finally, MDS-5q- and MDS-RS were the WHO subgroups with the highest rate of PR in our cohort (46% and 59% respectively), irrespective of sEPO. These 2 subgroups of MDS exhibited different repartition of erythroid subpopulations with characteristics associated with ESAs resistance. In 5q- COM-E and PROE were depauperate and, consequently, erythroid precursors reduced, while MDS-RS displayed an expansion of erythroid precursors and higher CV of CD71 compared to CTRLs. Finally, these FC parameters, easily and rapidly assessed at baseline BM evaluation in MDS patients, correlate with response to ESAs and with different WHO subgroups. FC erythroid abnormalities could partly explain the complex pathobiology underlying dyserythropoiesis and its treatment. The different repartition of erythroid progenitors observed could lead to molecular and transcriptome analyses focused on these altered subpopulations in order to investigate the exact mechanism of anemia in LR-MDS(s). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-502 Supportive Care in Low-Risk Myelodysplastic Syndromes: Role of Erythropoiesis Stimulating Agents (ESAs)

Erythropoiesis stimulating agents (ESAs) are the frontline treatment in low-risk anemic MDS patients, and an employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS. An analysis of the erythropoietic response to ESAs therapy in a cohort of anemic non transfusion-dependent MDS patients, enrolled in a retrospective register, RECAMDS, subgroup of Italian register, was performed. 183 patients, treated with standard-dose ESAs, have been retrospectively analyzed. Data analysis was performed, according to IWG 2006 criteria, at the baseline, after 3 and 6 months of continuous treatment, with a subanalysis of the patients according to WHO and R-IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl, mean serum erythropoietin (EPO) concentration: 51 mU/L, after a mean time from diagnosis of 6 months (r.1-118). ORR was 83.6% (153/183); no difference among WHO and IPSS subgroups was found. 132/183 (72.1%) achieved response after 3 months of treatment, while other 21/183 (11.2%) after 6 months. 19 patients with stable disease (non-responders, according to IWG criteria), in which treatment was continued, achieved response after 9 months. In the macrocytic-responders group, 83.2% exhibited again macrocytosis after 3 months, while 16.8% become normocytic. In the normocytic-responders group, 89.8% exhibited again normocytosis, while 10.2% become macrocytic. In these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia, with transfusion-dependence, regarded as first signs of progression of disease. Non-responders were 30/183 (16.3%): in the macrocytic non-responders group 89% exhibited again macrocytosis after 3 months, while 11% become normocytic; in the normocytic group 76% exhibited again macrocytosis, while 24% become normocytic. These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs mainly due to stimulating erythroid production in MDS clones; in the minority of patients probably it happens due to recruiting residual polyclonal erythropoiesis. It is interesting to note that stimulating effects of ESAs last even when the expression of dysplasia progresses.

Evaluating Complete Remission with Incomplete Hematologic Recovery (CRh) As a Response Criterion in Myelodysplastic Syndromes (MDS)

Evaluating Complete Remission with Incomplete Hematologic Recovery (CRh) As a Response Criterion in Myelodysplastic Syndromes (MDS)

Erythropoiesis Stimulating Agents (ESAS) in Supportive Care of Low-Risk Myelodysplastic Syndromes: Recamds Registry FINAL Results

Erythropoiesis stimulating agents (ESAs) are the frontline treatment in low-risk anemic MDS patients and an employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. It's matter of debate whether the clinical response is a result of proliferation and maturation of the dysplastic clone or stimulation of residual normal erythropoiesis by ESAs.Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS: an analysis of the erythropoietic response to ESAs therapy in a cohort of anemic non trasfusion-dependent MDS patients, enrolled in a retrospective register, RECAMDS, subgroup of Italian register, was performed.183 patients, treated with standard-dose ESAs, have been retrospectively analyzed. Data analysis was performed, according to IWG 2006 criteria, at the baseline, after 3 and 6 months of continuous treatment, with a subanalysis of the patients according to WHO and R-IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl, mean serum EPO concentration: 51 mU/L, after a mean time from diagnosis of 6 months (r.1-118).ORR was 83.6% (153/183), no difference among WHO and IPSS subgroups was found: 132/183 (72.1%) achieved response after 3 months of treatment, while other 21/183 (11.2%) after 6 months. 19 patients with stable disease(non-responders, according to IWG criteria), in which treatment was continued, achieved response after 9 months. In the macrocytic-responders group 83.2% exhibits again macrocytosis after 3 months, while 16.8% become normocytic. In the normocytic-responders group 89.8% exhibits again normocytosis, while 10.2% become macrocytic: in these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia, with transfusion-dependence, regarded as first signs of progression of disease. Non-responders were 30/183 (16.3%): in the macrocytic non-responders group 89% exhibit again macrocytosis after 3 months, while 11% become normocytic; in the normocytic group 76% exhibits again macrocytosis, while 24% become normocytic.These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs mainly stimulating erythroid production in MDS clones; in the minority of patients probably it happens recruiting residual polyclonal erythropoiesis.It is interesting to note that stimulating effects of ESAs last even when the expression of dysplasia progresses. [Display omitted] DisclosuresMartinelli: Roche: Consultancy; Stemline Therapeutics: Consultancy; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Daichii Sankyo: Consultancy; Astellas: Consultancy, Speakers Bureau.

Plasma markers in pulmonary hypertension subgroups correlate with patient survival

BackgroundRecent studies have provided evidence for an important contribution of the immune system in the pathophysiology of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we investigated whether the inflammatory profile of pulmonary hypertension patients changes over time and correlates with patient WHO subgroups or survival.Methods50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients and 18 healthy controls (HCs) were included in the study. Plasma inflammatory markers at baseline and after 1-year follow-up were measured using ELISAs. Subsequently, correlations with hemodynamic parameters and survival were explored and data sets were subjected to unbiased multivariate analyses.ResultsAt diagnosis, we found that plasma levels of interleukin-6 (IL-6) and the chemokines (C-X3-C) motif legend CXCL9 and CXCL13 in CTD-PAH patients were significantly increased, compared with HCs. In idiopathic PAH patients the levels of tumor growth factor-β (TGFβ), IL-10 and CXCL9 were elevated, compared with HCs. The increased CXCL9 and IL-8 concentrations in CETPH patients correlated significantly with decreased survival, suggesting that CXCL9 and IL-8 may be prognostic markers. After one year of treatment, IL-10, CXCL13 and TGFβ levels changed significantly in the PAH subgroups and CTEPH patients. Unbiased multivariate analysis revealed clustering of PH patients based on inflammatory mediators and clinical parameters, but did not separate the WHO subgroups. Importantly, these multivariate analyses separated patients with < 3 years and > 3 years survival, in particular when inflammatory mediators were combined with clinical parameters.DiscussionOur study revealed elevated plasma levels of inflammatory mediators in different PAH subgroups and CTEPH at baseline and at 1-year follow-up, whereby CXCL9 and IL-8 may prove to be prognostic markers for CTEPH patients. While this study is exploratory and hypothesis generating, our data indicate an important role for IL-8 and CXCL9 in CHD and CTEPH patients considering the increased plasma levels and the observed correlation with survival.ConclusionIn conclusion, our studies identified an inflammatory signature that clustered PH patients into WHO classification-independent subgroups that correlated with patient survival.

MBRS-29. PROSPECTIVE MOLECULAR PROFILING IN PEDIATRIC MEDULLOBLASTOMA PATIENTS ENROLLED ON THE “HEAD START 4” PROTOCOL

Medulloblastoma is the most common malignant embryonal brain tumor in children with only modest improvements in outcomes achieved over the last 20 years. The implementation of irradiation-avoiding strategies, including trials by the “Head Start” consortium, have demonstrated improved cure rates along with enhanced quality of life. Simultaneously, the classification of medulloblastomas has undergone a dramatic shift as molecular testing has made it possible to divide these tumors into distinctive subtypes. Currently, the WHO recognizes four medulloblastoma molecular subgroups; however it remains unclear how patients within these subgroups respond to modern irradiation-avoiding therapies. This study aims to demonstrate the feasibility of prospective molecular profiling in medulloblastoma patients enrolled on the “Head Start 4” trial. Whole-exome sequencing (SureSelect Human All Exon V6+COSMIC) and DNA methylation (Illumina EPIC Array) profiling were performed on 10 paired tumor/blood samples and 4 tumor samples, respectively. High-quality mutational and copy number data were produced for each of the 10 subjects demonstrating well-described gene mutations (SUFU) and chromosomal losses (9q and 10q). Four subjects had methylation profiling which successfully separated them into the WHO subgroups (two SHH and two Group 3). These data showed the feasibility of prospective high-dimensional mutational and DNA methylation analysis using “Head Start 4” patients. Future work will focus on finalizing these profiling efforts, enabling the development of models that predict response to irradiation-avoiding treatment and, in general, a better understanding of the molecular mechanisms underlying treatment resistance and tumor progression, leading to more personalized approaches to treating children with medulloblastoma.

Extent and prognostic value of MGMT promotor methylation in glioma WHO grade II

MGMT promotor methylation is associated with favourable outcome in high-grade glioma. In glioma WHO grade II, it is unclear whether the extent of MGMT promotor methylation and its prognostic role is independent from other molecular markers. We performed a retrospective analysis of 155 patients with glioma WHO grade II. First, all 155 patients were assigned to three molecular groups according to the 2016 WHO classification system: (1) oligodendroglioma, IDH-mutant and 1p19q co-deleted (n = 81); (2) astrocytoma, IDH-mutant and 1p19q non-codeleted (n = 54); (3) astrocytoma, IDH-wildtype (n = 20). MGMT promotor methylation was quantified using Sanger sequencing of the CpG sites 74–98 within the MGMT promotor region. Highest numbers of methylated CpG sites were found for oligodendroglioma, IDH-mutant and 1p19q co-deleted. When 1p19q co-deletion was absent, numbers of methylated CpG sites were higher in the presence of IDH-mutation. Accordingly, lowest numbers were seen in the IDH-wildtype subpopulation. In the entire cohort, larger numbers of methylated CpG sites were associated with favourable outcome. When analysed separately for the three WHO subgroups, a similar association was only retained in astrocytoma, IDH-wildtype. Collectively, extent of MGMT promotor methylation was strongly associated with other molecular markers and added prognostic information in astrocytoma, IDH-wildtype. Evaluation in prospective cohorts is warranted.

PATH-28. EXTENT AND PROGNOSTIC VALUE OF MGMT PROMOTOR METHYLATION DEPEND ON IDH MUTATION AND 1p19q CO-DELETION IN GLIOMA WHO GRADE II

Abstract INTRODUCTION Methylation of the promotor region of the O6-methylguanin-DNA-methyltransferase (MGMT) gene is associated with increased survival in low- and high-grade glioma. It is unknown whether this association also applies to the 2016 WHO categories of glioma WHO grade II. MATERIAL AND METHODS We retrospectively searched the institutional database of the Center for Neuro-Oncology for patients with glioma WHO grade II. Patients were assigned to one of three groups according to the 2016 WHO classification system: 1. 1p19q co-deleted oligodendroglioma, IDH mutant; 2. 1p19q non-codeleted astrocytoma, IDH mutant; 3. 1p19q non-codeleted astrocytoma, IDH wild-type. MGMT methylation status was analysed using Sanger sequencing of the CpG sites 74-98 within the MGMT promotor region. The total number of methylated CpG sites was calculated for each patient. RESULTS 155 patients with glioma WHO grade II were encountered, including 81 1p19q co-deleted, IDH mutant oligodendrogliomas; 54 IDH mutant astrocytomas; and 20 IDH wild-type astrocytomas. The mean number of methylated CpG sites among oligodendrogliomas was significantly higher when compared to IDH mutant astrocytomas (18.9 ± 0.4 vs. 16.3 ± 0.6; p = 0.001). In turn, the number of methylated CpG sites among IDH mutant astrocytomas was higher when compared to IDH wild-type astrocytomas (16.3 ± 0.6 vs. 12.3 ± 1.9; p = 0.007). Median follow-up was estimated at 35 months. Median time to malignant progression was 87 months for all patients, and median overall survival was not reached. In the entire cohort, a larger number of methylated CpG sites was prognostic of overall survival and time to malignant progression. When analysed separately for the three WHO subgroups, a similar association was only retained in IDH wild-type astrocytoma. CONCLUSION In our series of WHO II gliomas, MGMT promotor methylation appeared strongly associated with 1p19q codeletion and IDH mutations. MGMT promotor methylation was only prognostic in IDH wild-type astrocytoma.

Prediction of Relapse after Allogeneic Stem Cell Transplantation Using Individualized Minimal Residual Markers; The Prospective Nordic Study NMDSG14B

Introduction One third of patients with myelodysplastic syndrome (MDS) will relapse after allogeneic stem cell transplantation (SCT), with a dismal prognosis. Early detection of relapse enables pre-emptive treatment and may potentially reduce relapse risk, but is limited by the lack of sensitive markers for minimal residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using sensitive digital droplet PCR (ddPCR). Method We designed a prospective Nordic study (NMDSG14B; NCT02872662) enrolling all patients with MDS, mixed MDS / MPN or AML with myelodysplasia related disease and &amp;lt; 30% marrow blasts undergoing SCT in the Nordic region. We hypothesized that personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Patients were included before SCT and serial bone marrow samples were collected before, and 1 and 3 months post SCT, and thereafter every third month for 2 years or until relapse or death. Blood samples were collected monthly. The MRD results were not available for the treating physicians. MRD positivity was defined based on the background noise of the specific ddPCR-assays and varied between 0.05-0.1% VAF. Results Three-hundred and sixteen patients were screened between 2016 and 2020, of which 19 were excluded due to lack of mutation or disease progression preventing SCT. We here present data of 254 patients followed ≥ 6 months after SCT. Median age was 64 (18-78) years and 59% were male. Most WHO subgroups of MDS (n=166), MDS/MPN (n=39), AML (n=8) and therapy-related disease (n=41), were represented. Risk profile according to IPSS-R was very low (n=13), low (n=32), intermediate (n=46), high (n=60) and very high (n=32). The majority of patients received pre-SCT treatment consisting of HMA (n=159) and / or intensive chemotherapy (n=59) while 60 patients did not receive disease-modifying treatment prior to SCT. The most common mutations were ASXL1 (n=69), TET2 (n=58), SRSF2 (n=57) and TP53 (n=44). No mutation was identified in 10 pts, and NGS data is still pending for 11 patients. After a median follow-up of 436 days, estimated 2 years overall survival and relapse free survival were 72% and 63%, respectively. Cumulative incidence of NRM and relapse at 2 years was 16% and 20%, respectively. Forty-six patients relapsed after a median of 170 (53-733) days, and the estimated median survival following relapse was 197 days. The most common pre-SCT mutations in the relapsed cohort were TP53 (n=19), DNMT3A (n=11) and RUNX1 (n=9). Thirty-seven patients died due to non-relapse mortality (NRM) after a median of 83 (4-754) days. To date, MRD results are available for 64 patients. Relapse was preceded by positive MRD in 14 out of 15 patients a median of 79 (21-173) days before clinical relapse. The 15th patient had an extra-medullary relapse only. Borderline positive MRD samples &amp;lt; 0.2% VAF within 100 days after SCT followed by negative samples were seen in 11 non-relapse patients. Twenty-four of 37 patients in continuous complete remission (CCR) were consistently MRD neg. Six CCR patients had positive MRD after 100 days; two with transient borderline peaks (&amp;lt;0.1%) at 6 months; two with transient peaks &amp;gt; 0.1%, which turned negative when the patients developed GVHD; one patient with slowly decreasing MRD which turned negative first after 1 y, and finally one patient with prevailing KIT mutation (&amp;gt;700 days post-SCT) despite negative BCOR and STAG2. Two MRD+ patients died from NRM without showing signs of clinical relapse. Discussion In summary, we show that our pipeline of personalized MRD-assessment, based on patient-specific mutations is feasible with a high sensitivity to predict relapse. An update of study progression will be presented at the meeting. Figure 1 Disclosures Illman: Sanofi-Genzyme: Other: Travel Support; Celgene: Other: Travel Support. Mielke:DNA Prime: Honoraria, Other: received via my institution , Speakers Bureau; KIADIS Pharma: Honoraria, Other: received via my institution , Speakers Bureau; Miltenyi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: received via my institution , Speakers Bureau; Kite/Gilead: Honoraria, Other: received via my institution , Speakers Bureau; Bellicum: Honoraria, Other: received via my institution, Speakers Bureau; Novartis: Honoraria, Other: received via my institution, Speakers Bureau; Celgene/BMS: Honoraria, Other: received via my institution , Speakers Bureau. Ebeling:Accord Healthcare: Other: Travel Support; Amgen: Other: Travel Support; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Otsuka Pharma Scandanavia AB: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MDS-391: Role of Erythropoiesis-Stimulating Agents (ESAs) in Supportive Care of Low-Risk Myelodysplastic Syndromes

Context: Erythropoiesis-stimulating agents (ESAs) are the frontline treatment in low-risk anemic MDS patients, and employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. It is a matter of debate whether the clinical response is a result of proliferation and maturation of the dysplastic clone or stimulation of residual normal erythropoiesis by ESAs. Objective: Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS. An analysis of the erythropoietic response to ESA therapy in a cohort of anemic, non-transfusion-dependent MDS patient, enrolled in a retrospective registry, RECAMDS, a subgroup of Italian register, was performed. Design: Real-world analysis. Setting: Low-risk MDS. Patients: One hundred eighty-three patients treated with standard-dose ESAs have been retrospectively analyzed. Data analysis was performed according to IWG 2006 criteria at baseline and after 3 and 6 months of continuous treatment, with a sub-analysis of the patients according to WHO and R-IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl and mean serum EPO concentration of 51 mU/L after a mean time from diagnosis of 6 months (r.1–118). Interventions: ESAs to manage low-risk MDS. Main outcome measures: Results were evaluated according to IMWG 2006 criteria. Results: ORR was 83.6% (153/183); no difference among WHO and IPSS subgroups was found: 132/183 (72.1%) achieved response after 3 months of treatment, while the other 21/183 (11.2%) achieved response after 6 months. Nineteen patients with stable disease (non-responders, according to IWG criteria), in whom treatment was continued, achieved response after 9 months. In the macrocytic-responders group, 83.2% exhibited repeat macrocytosis after 3 months, while 16.8% became normocytic. In the normocytic-responders group, 89.8% exhibited repeat normocytosis, while 10.2% become macrocytic: in these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia with transfusion-dependence, which was regarded as first signs of progression of disease. Non-responders were 30/183 (16.3%): in the macrocytic non-responders group, 89% exhibit macrocytosis again after 3 months, while 11% became normocytic; in the normocytic group, 76% exhibited macrocytosis again, while 24% became normocytic. Conclusions: These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs, mainly stimulation of erythroid production in MDS clones. In the minority of patients, it likely happens due to recruiting residual polyclonal erythropoiesis. It is interesting to note that stimulating effects of ESAs last, even when the expression of dysplasia progresses.

Results from a Genome-Wide Association Study (GWAS) in Mastocytosis Reveal New Gene Polymorphisms Associated with WHO Subgroups.

Mastocytosis is rare disease in which genetic predisposition is not fully understood. The aim of this study was to analyze associations between mastocytosis and single nucleotide polymorphisms (SNPs) by a genome-wide association study (GWAS) approach. A total of 234 patients were enrolled in our study, including 141 with cutaneous mastocytosis (CM; 78 children and 63 adults) and 93 with systemic mastocytosis (SM, all adults). The control group consisted of 5606 healthy individuals. DNA samples from saliva or blood were genotyped for 551 945 variants using DNA microarrays. The prevalence of certain SNPs was found to vary substantially when comparing patients and healthy controls: rs10838094 of 5OR51Q1 was less frequently detected in CM and SM patients (OR = 0.2071, p = 2.21 × 10−29), rs80138802 in ABCA2 (OR = 5.739, p = 1.98 × 10−28), and rs11845537 in OTX2-AS1 (rs11845537, OR = 6.587, p = 6.16 × 10−17) were more frequently detected in CM in children and adults. Additionally, we found that rs2279343 in CYP2B6 and rs7601511 in RPTN are less prevalent in CM compared to controls. We identified a number of hitherto unknown associations between certain SNPs and CM and/or SM. Whether these associations are clinically relevant concerning diagnosis, prognosis, or prevention remains to be determined in future studies.

Comprehensive Genomic Analysis in NRG Oncology/RTOG 9802: A Phase III Trial of Radiation Versus Radiation Plus Procarbazine, Lomustine (CCNU), and Vincristine in High-Risk Low-Grade Glioma

NRG Oncology/RTOG 9802 (ClinicalTrials.gov Identifier: NCT00003375) is a practice-changing study for patients with WHO low-grade glioma (LGG, grade II), as it was the first to demonstrate a survival benefit of adjuvant chemoradiotherapy over radiotherapy. This post hoc study sought to determine the prognostic and predictive impact of the WHO-defined molecular subgroups and corresponding molecular alterations within NRG Oncology/RTOG 9802. IDH1/2 mutations were determined by immunohistochemistry and/or deep sequencing. A custom Ion AmpliSeq panel was used for mutation analysis. 1p/19q codeletion and MGMT promoter methylation were determined by copy-number arrays and/or Illumina 450K array, respectively. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were calculated using the Cox proportional hazard model and tested using the log-rank test. Multivariable analyses (MVAs) were performed incorporating treatment and common prognostic factors as covariates. Of the eligible patients successfully profiled for the WHO-defined molecular groups (n = 106/251), 26 (24%) were IDH-wild type, 43 (41%) were IDH-mutant/non-codeleted, and 37(35%) were IDH-mutant/codeleted. MVAs demonstrated that WHO subgroup was a significant predictor of PFS after adjustment for clinical variables and treatment. Notably, treatment with postradiation chemotherapy (PCV; procarbazine, lomustine (CCNU), and vincristine) was associated with longer PFS (HR, 0.32; P = .003; HR, 0.13; P < .001) and OS (HR, 0.38; P = .013; HR, 0.21; P = .029) in the IDH-mutant/non-codeleted and IDH-mutant/codeleted subgroups, respectively. In contrast, no significant difference in either PFS or OS was observed with the addition of PCV in the IDH-wild-type subgroup. This study is the first to report the predictive value of the WHO-defined diagnostic classification in a set of uniformly treated patients with LGG in a clinical trial. Importantly, this post hoc analysis supports the notion that patients with IDH-mutant high-risk LGG regardless of codeletion status receive benefit from the addition of PCV.

Long-term analysis of the WHO-defined molecular subgroups of high-risk grade II gliomas treated with radiation and temozolomide on NRG Oncology/RTOG 0424.

2518 Background: This study sought to evaluate the prognostic significance of the three WHO-defined molecular glioma subgroups ( IDHwt, IDHmt/non-codel, and IDHmt/codel) in NRG Oncology/RTOG 0424, a phase II trial of high-risk low-grade gliomas treated with radiation (RT) and concurrent and adjuvant temozolomide (TMZ) after biopsy/surgical resection. Notably, this is the first clinical study to evaluate the prognostic value of the WHO subgroups in RT + TMZ-treated high-risk grade II (G2) gliomas using prospectively-collected long-term survival data. Methods: IDH1/2 mutation status was determined by next-generation sequencing. 1p/19q co-deletion status was determined by Oncoscan and/or 450K methylation data. Overall survival (OS) and progression-free survival (PFS) by marker status were determined by the Cox proportional hazard model and tested using the log-rank test in a post-hoc analysis. Patient pre-treatment characteristics were included as covariates in multivariate analyses. Results: Of all the eligible patients (N=129), 80 (62%) had sufficient quality DNA for both IDH and 1p/19q analyses. Of these 80, 54 (67.5%) were IDHmt, and 26 (32.5%) were IDHwt. Of the 54 IDHmt patients, 26 (32.5% of total, 48% of IDHmt) were IDHmt/codel, and 28 (35% of total, 52% of IDHmt) were IDHmt/non-codel. Both IDHmt subgroups were significantly correlated with longer PFS ( IDHmt/co-del = 8.1yrs (5.2-not reached (NR)); IDHmt/non-codel = 7.5yrs (3.9-11.8); IDHwt = 1.0yr (0.6-1.7), p&lt;0.001) and OS ( IDHmt/co-del = 9.4yrs (8.2-NR); IDHmt/non-codel = 8.8yrs (5.9-NR); IDHwt = 2.3yrs (1.4-3.4), p&lt;0.001) relative to the IDHwt subgroup. Upon univariate and multivariate analyses, both molecular IDHmt subgroup comparisons relative to IDHwt remained significant (p&lt;0.001) even after incorporation of known clinical variables. Conclusions: These analyses suggest that G2 glioma patients harboring IDH1/2 mutations, regardless of co-deletion status, demonstrated longer survival with RT + TMZ relative to IDHwt tumors, although sample size is limited and analyses were post-hoc. These results also support the notion that outcomes for IDHwt high-risk G2 gliomas remain dismal (median = 2.3yrs, similar to G3 anaplastic astrocytoma); these patients should be separated from IDHmt patients in future G2 glioma trials, and warrant novel treatment strategies. Funding: U10CA180868, U10CA180822, U24CA196067, CURE, PA Dept. of Health, and Merck. Also, R01CA108633, R01CA169368, RC2CA148190, U10CA180850, BTFC, OSUCCC (all to AC). Clinical trial information: NCT00114140 .

Nuclear expression of β-catenin in endometrial hyperplasia as marker of premalignancy.

We aimedto assess (1)-whether nuclear β-catenin is a marker of endometrial precancer, and (2)-the diagnostic accuracy of β-catenin immunohistochemistry in the differential diagnosis between benign and premalignant endometrial hyperplasia (EH), defining criteria for its use. Electronic databases were searched for studies evaluating β-catenin immunohistochemistry in normal endometrium (NE), benign and/or premalignant EH, and endometrioid carcinoma (EC). Odds ratio (OR; p<0.05), sensitivity, specificity, diagnostic OR (DOR), positive and negative likelihood ratios (LR+, LR-) were calculated. Subgroup analyses were based on the classification system used (WHO or EIN) and criteria to define aberrant β-catenin expression (only nuclear or cytoplasmic/nuclear). Twelve studies with 1510 specimens were included. Nuclear β-catenin rate significantly increased from NE tobenign EH (OR=26.01; p=0.0002, only in WHO subgroup), and from benign EH topremalignant EH (OR=3.89; p=0.0002; more markedly in EIN subgroup), but not from premalignant EH to EC (OR=0.78; p=0.29). Nuclear β-catenin accuracy was very low in WHO subgroup (sensitivity=0.40, specificity=0.76, LR+=1.85, LR-=0.72; DOR=2.89) and moderate in EIN subgroup (sensitivity=0.19, specificity=1.00, LR+=14.80, LR-=0.83; DOR=18.14). Cytoplasmic/nuclear β-catenin accuracy was absent in WHO subgroup (sensitivity=0.45, specificity=0.54, LR+=1.01, LR-=1.01; DOR=0.99) and low in EIN subgroup (sensitivity=0.57, specificity=0.86, LR+=3.63, LR-=0.51; DOR=8.30). Considering nuclear expression and using EIN system, β-catenin immunohistochemistry might be reliable as rule-in test for diagnosis of endometrial precancer, with perfect specificity and moderate overall accuracy.

Tumor Heterogeneity in Gliomas – a Histopathology-Targeted Proteomic Pilot Study

Intra- and inter-tumor heterogeneity underlies both tumor evolution and response to treatment. Our aim was to explore the protein expression of infiltrative gliomas (grades II to IV) with high throughput protein-based mass spectrometry (MS), comparing different histopathologic areas of glioblastomas (GBM) and lower grade gliomas (LGG), to further characterize gliomas pathways, and identify druggable targets and protein signatures to be used as diagnostic and prognostic biomarkers. We studied 12 patients with diffuse gliomas (1 diffuse astrocytoma, 1 anaplastic astrocytoma, 3 anaplastic oligodendrogliomas, and 7 glioblastomas). Each tumor was reclassified according to the 2016 WHO classification of tumors of CNS (IDHmutant-1p19q Codeleted; IDHmutant-1p19q intact; IDH wild type glioma), and mapped using H&E stained slides to identify 2-3 areas with different histopathologic phenotypes, different cellularity, and perinecrotic areas. The selected areas with at least 80% of tumor cells were cored with 1.0mm needle using a semi-automated tissue microarray platform, in a total of 35 samples – hemorrhagic and necrotic portions of the tumors were avoided in all cases. Total protein was extracted from the FFPE tissue and analyzed with 4 hour liquid chromatography tandem MS (LC-MS-MS) for label-free expression proteomics. We compared the average of protein expression in different tumor areas from each patient to identify associations with Wilcoxon rank-sum test. The results were further interpreted following a top-bottom approach and compared in silico with TCGA expression data (mRNA). The statistical analysis was performed using R software. Overall, 9,222 peptides were mapped to 1758 non-redundant proteins in the samples, 320 of which had a significant differential expression in GBM versus LGG. The PCA analysis showed that according to the molecular signatures, the samples clustered well by disease and by patient. Samples also clustered by IDH1 status and WHO subgroup. One patient had non-neoplastic brain adjacent to a GBM available for analysis and this sample clustered with the LGGs. Among the top 35 proteins most differentially expressed in the 2 groups (raw p-value= 0.00252; FDR=0.12684), 14 had higher expression in GBM, and 21 in LGG. This included one protein previously explored by our group in previous studies (TAGLN2), validating our findings with different approach. Our results showed that LC-MS-MS analysis of morphologically different glioma areas is feasible and was able to identify clusters with different patterns of protein expression. Further correlation of proteome profiles with clinical data, histological features, pathway analysis, and druggable target analysis are ongoing. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and the Ohio State University CCC (all to AC).

PF535 CLINICAL AND MOLECULAR CHARACTERISTICS OF SRSF2‐MUTATED NEOPLASMS

Background:Splicing factors are the most frequently mutated genes in myeloid neoplasms. SF3B1‐mutated myelodysplastic syndromes (MDS) have been recently recognized as a distinct subtype strongly associated with ring sideroblasts. Conversely, SRSF2 has been found to be recurrently mutated in chronic myelomonocytic leukemia (CMML), and predicting worse prognosis in MDS, myelofibrosis (MF) and acute myeloid leukemia (AML).Aims:Hence, SRSF2‐mutated neoplasms constitute an heterogenous molecular‐defined subgroup, whose clinical characteristics have not been systematically characterized. We aimed at a comprehensive characterization of clinical features and co‐mutational determinants of SRSF2‐mutated neoplasms across WHO classification boundaries.Methods:We analyzed clinical and molecular characteristics of SRSF2‐mutated myeloid neoplasms diagnosed at 4 referral centers. All clinical information was prospectively annotated in clinical chart records and a common panel of 23 recurrently mutated genes in myeloid neoplasms were analysed using Illumina HiSeq sequencing of peripheral blood cell DNA. Multivariate analyses (MVA) accounted for left censoring at mutation assessment time and right‐censoring in case of disease‐modifying treatments.Results:Overall, 279 patients carrying SRSF2‐mutation were identified, including 84 MDS (49 blast excess, 21 multi‐lineage dysplasia, 7 ring sideroblasts, 7 unclassified), 76 MPN (64 primary‐MF [PMF], 5 secondary‐MF, 3 polycytemia vera, 2 essential thrombocytemia, 2 chronic neutrophilic leukemia), 71 MDS/MPN (59 CMML, 12 MDS/MPN unclassified), 47 AML (38 with MDS‐related changes, 9 NOS), 1 blastic plasmacytoid dendritic cell neoplasm. No difference in age was found between WHO subgroups. Male/female ratio was 3.3. Median overall survival (mOS) was 27.3 months (95%CI: 19.9–34.7). The median number of co‐mutated genes was 3 (range 0–7). SRSF2 mutation was the unique driver mutation detected in 13 cases. The most frequently co‐mutated genes were ASXL1 (38%), TET2 (35%), JAK2 (23%), RUNX1 (17%), IDH2 (15%), CBL (10%), STAG2 (10%), NRAS (8%). We used sequencing data to identify the order of acquisition of SRSF2 mutations in double‐mutant patients and found that SRSF2 mutation was acquired first in 80 (28.7%) cases, sub‐clonal in 111 (39.8%). Mutational order could not be assessed in 88 (31.5%) cases. In MVA, AML phenotype was independently predicted by RUNX1 (OR 4.2, p &lt; .001), IDH2 (OR 4.3, p = .001), STAG2 mutation (OR 3.2, p = .02). Similarly, co‐occurring mutation in JAK2 (OR 77.5, p &lt; .001), MPL (OR 194, p &lt; .001) and ASXL1 (OR 3.1, p = .01) predicted MPN phenotype. MDS/MPN was predicted by TET2 (OR 7.2, p &lt; .001), SETBP1 (OR 4.3, p = .02) mutation and SRSF2‐first mutation (OR 3.1, P = .004). Factors predicting OS were blast count (p &lt; .001), white blood cells (p = .009), TET2 (HR. 54, p &lt; .001), IDH2 (HR. 5, p = .021), NRAS (HR 2.23, P = .017) mutation. Notably, fibrosis, cytogenetics, WHO categories were not associated with different survival in MVA.Summary/Conclusion:In conclusion, SRSF2‐mutated myeloid neoplasms are a subset of myeloid neoplasms characterized by bone marrow dysplasia and overall dismal prognosis, whose fibrotic or proliferative phenotype results from a restricted co‐mutation pattern. Among MPN, SRSF2 mutation is almost invariably associated with MF, further supporting the concept that PMF is an MDS/MPN disorder. Co‐mutational pattern is the best predictor of clinical phenotype, suggesting that recognition of a distinct entity with a unique molecular basis may significantly improve classification and treatment of patients with these disorders.

PB2258 ERYTHROPOIESIS STIMULATING AGENTS (ESAS) IN MANAGEMENT OF MYELODYSPLASTIC SYNDROMES: RECAMDS TRIAL

Background:Erythropoiesis stimulating agents (ESAs) are the frontline treatment in low‐risk anemic MDS patients and an employment of this therapy in the earlier stage of the disease can delay the need for RBC transfusion, hypothetically by slowing the disease course. It's matter of debate whether the clinical response is a result of proliferation and maturation of the dysplastic clone or stimulation of residual normal erythropoiesis by ESAs.Aims:Macrocytosis is one of the cytological hallmarks of dyserithropoiesis in MDS: an analysis of the erythropoietic response to ESAs therapy in a cohort of anemic non trasfusion‐dependent MDS patients, enrolled in a retrospective register, RECAMDS, subgroup of Italian register, was performed.Methods:183 patients, treated with standard‐dose ESAs, have been retrospectively analyzed (Table 1). Data analysis was performed, according to IWG 2006 criteria, at the baseline, after 3 and 6 months of continuous treatment, with a subanalysis of the patients according to WHO and R‐IPSS risk stratification. ESAs were started at mean Hb concentration of 9.31 g/dl, mean serum EPO concentration: 51 mU/L, after a mean time from diagnosis of 6 months (r.1‐118).Results:Overall response rate (ORR) was 83.6% (153/183), no difference among WHO and IPSS subgroups was found: 132/183 (72.1%) achieved response after 3 months of treatment, while other 21/183 (11.2%) after 6 months. 19 patients with stable disease (non‐responders, according to IWG criteria), in which treatment was continued, achieved response after 9 months. In the macrocytic‐responders group 83.2% exhibits again macrocytosis after 3 months, while 16.8% become normocytic. In the normocytic‐responders group 89.8% exhibits again normocytosis, while 10.2% become macrocytic: in these patients, after 3 months, there was a contemporary worsening in neutropenia and thrombocytopenia, with transfusion‐dependence, regarded as first signs of progression of disease. Non‐responders were 30/183 (16.3%): in the macrocytic non‐responders group 89% exhibit again macrocytosis after 3 months, while 11% become normocytic; in the normocytic group 76% exhibits again macrocytosis, while 24% become normocytic.Summary/Conclusion:These preliminary data can suggest that, in the majority of MDS patients responsive to ESAs, the increase of Hb concentration occurs mainly stimulating erythroid production in MDS clones; in the minority of patients probably it happens recruiting residual polyclonal erythropoiesis.It is interesting to note that stimulating effects of ESAs last even when the expression of dysplasia progresses.

Updated predictive analysis of the WHO-defined molecular subgroups of low-grade gliomas within the high-risk treatment arms of NRG Oncology/RTOG 9802.

2002 Background: This study sought to update the predictive significance of the three WHO-defined molecular glioma subgroups ( IDHwt, IDHmt/noncodel, and IDHmt/codel) in the subset of specimens available for analysis in NRG Oncology/RTOG 9802, a phase III trial of high-risk low-grade gliomas (LGGs) treated with radiation (RT) with and without PCV after biopsy/surgical resection. Notably, this is the first phase III study to evaluate the predictive value of the WHO subgroups in LGGs using prospectively-collected, well-annotated long-term overall survival data, in a post-hoc analysis. Methods: IDH1/2 mutation status was determined by immunohistochemistry and/or next-generation sequencing. 1p/19q status was determined by Oncoscan and/or 450K methylation data. Treatment effects on overall survival (OS) and progression-free survival (PFS) by marker status were determined by the Cox proportional hazard model and tested using the log-rank test in a secondary and exploratory analysis. Results: Of all the randomized eligible high-risk G2 patients (N = 251) in NRG Oncology/RTOG 9802, 106(42%) patients had tissue available with sufficient quality DNA for profiling. Of these, 80(75%) were IDHmut; 43(41%) were IDHmut/non-co-deleted, 37(35%) were IDHmut/co-deleted, and 26(24%) were IDHwt. Upon univariate analyses, no significant difference in either PFS or OS was observed with the addition of PCV in the IDHwt subgroup. Both the IDHmut/non-co-deleted and IDHmut/co-deleted subgroups were significantly correlated with longer PFS (HR = 0.32; p = 0.003; HR = 0.13; p &lt; 0.001) and OS (HR = 0.38; p = 0.013; HR = 0.21; p = 0.029) in the RT plus PCV arm, respectively. Conclusions: Our analyses suggest that both IDHmut/non-co-deleted and IDHmut/co-deleted subgroups received benefit from treatment with PCV although sample size is limited and analyses are post-hoc. Our results also support the notion that IDHwt high-risk LGG patients do not benefit from the addition of PCV to RT. Funding: U10CA180868, U10CA180822, and U24CA196067. Also, R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01, BTFC, OSU-CCC (all to AC). Clinical trial information: NCT00003375.