R132H Mutation Research Articles

Molecular Characteristics of Response to Olutasidenib (FT-2102) in Patients with Relapsed/Refractory mIDH1 Acute Myeloid Leukemia

Molecular Characteristics of Response to Olutasidenib (FT-2102) in Patients with Relapsed/Refractory mIDH1 Acute Myeloid Leukemia

Selected Articles from This Issue

Medulloblastoma is a common and deadly pediatric malignancy whose lethality is compounded by a lack of targeted therapies, partially owing to the differential biologies of the four major molecular subtypes. The most prevalent subtype, Sonic Hedgehog medulloblastoma (SHH-MB), is driven by hyperactivation of the eponymous SHH pathway within MATH-1-expressing granule cell precursors in the developing brain. Therefore, efforts to develop molecular targeted therapies for SHH-MB have focused largely on downstream targets and effectors of the SHH pathway such as the proto-oncogene SOX9, which has been shown to be specifically overexpressed in SHH-MB but whose functional role in disease progression has yet to be defined. Here, Adolphe and colleagues demonstrate that loss of SOX9 does not significantly impact the course of SHH-MB disease development and progression, indicating a dispensable role for this proto-oncogene in SHH-MB despite its elevated expression in most cases. This finding may have significant implications for ongoing preclinical and translational work aimed at identifying suitable molecular drivers of SHH-MB for therapeutic development.Lymphangioleiomyomatosis is a rare neoplastic disease of unknown origin that is nearly exclusive to women, with therapies targeting mTOR as the recognized standard of care. However, there is substantial variability in disease presentation, progression, and therap. response. Here, Espin, Baiges and colleagues perform multi-omics characterization of patient-derived lymphangioleiomyomatosis cells and tissues to identify the molecular features and potential causes of disease heterogeneity. The data suggest that differential expression of certain transcription factors—such as YB1, RUNX1, IRF1, estrogen receptor, and progesterone receptor—as well as certain immunity factors may account for some of these features. Based on the molecular features identified herein, the authors also nominate a breast mesenchymal cell line and certain pleural mesothelioma cell lines as potential surrogate models of lymphangioleiomyomatosis, expanding on the biological basis of the disease. Taken together, the data provide a high-value resource for further exploration of the molecular drivers of lymphangioleiomyomatosis progression.Glioma patients are frequently treated with temozolomide, a DNA alkylating chemotherapeutic that is known to cross the blood-brain barrier. The isocitrate dehydrogenase (IDH) R132H mutation is a common molecular feature of gliomas and is generally associated with gliomagenesis, but also with a more robust response to temozolomide therapy. However, the molecular underpinnings of this observation are not defined. In this study, Zhan, Ma, and colleagues use a heterozygous knock-in approach to model the effects of IDH R132H/wild-type allele balance on glioma cell biology. They find that the heterozygous IDH genotype in tandem with glioma driver gene alterations in p53, RB, and ATRX predisposes glioma cells to pro-senescent features. This combination of molecular features was associated with increased DNA damage markers and potentiated the effects of temozolomide in this model. However, heterozygous IDH R132H mutation also predisposed astroglial cells toward a senescence-associated secretory phenotype, which is known to exert a pro-tumorigenic effect within a tumor-permissive microenvironment. These data offer a potential mechanistic explanation for how a single mutation within IDH may provide a pro-tumorigenic stimulus while also sensitizing glioma cells to alkylating chemotherapy with temozolomide.Potentiating the effects of genotoxic interventions such as chemo- or radiotherapy requires the suppression of DNA damage response pathways, such as non-homologous end joining (NHEJ). Blocking NHEJ with chemical inhibitors is one strategy, but none have been clinically approved for this application. Building upon prior efforts to develop chemical screening strategies for NHEJ inhibitors, Du and colleagues report a miniaturized screening protocol that can be carried out in 96-well plates. By assessing an FDA-approved drug library in this format, the authors identify several potential candidates that function to inhibit NHEJ at concentrations below their cytotoxic dose. One of these drugs, ompalisib, was shown to suppress the phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in response to chemo- or radiotherapy, a key step in the initiation of NHEJ to mediate their genotoxic effects. Overall, the authors advance a novel screening strategy to examine the effects of pharmacological interventions on DNA damage responses via NHEJ, and further nominate a candidate FDA-approved drug for development as a chemo- and radiosensitizer.

Intracranial myxoid mesenchymal tumour with EWSR1-ATF1 fusion mimicking high grade glioma

Abstract Aims Molecular profiling is increasingly used in the diagnosis of CNS and non-CNS neoplasms. More than a quarter of all soft tissue tumours are characterized by specific recurrent chromosomal translocations which can be used as molecular signatures. With increasing frequency, EWSR1 rearrangements are found on both mesenchymal tumours and primary glial/neuronal tumours. Here we present a case of intracranial myxoid mesenchymal tumour (IMMT), a rare tumour which is becoming more recognised in recent years, affecting mainly children and young adults, and rarely older adults. It can be found in intraaxial and extraaxial location, with frequent dural connection. The tumour is defined by the genetic hallmark of EWSR1-CREB family gene fusion. Including our case, 16 intracranial tumours with this gene fusion have been reported to date. Our goal is to contribute further to the characterisation of the morphological spectrum, fusion partners and biological behaviour of rare EWSR1-CREB (non-ETS)-rearranged tumours of the CNS. Method Case: The patient is a 27 year old woman with a frontal lobe lesion, radiologically described as a tumour with dural attachment. She underwent surgical debulking, and tumour tissue was histologically examined with conventional immunohistochemistry. Additional genetic testing included targeted mutation screening, FISH, EPIC (Illumina BeadChip) methylation array and next generation sequencing. Histology showed a mitotically active neoplasm with relatively uniform cells, round nuclei and oligodendroglioma-like clear cell change, but no myxoid change. Glomeruloid microvascular proliferation and large areas of tumour necrosis were present. Immunohistochemistry was focally positive for GFAP, and negative or normal for synaptophysin, IDH1 R132H mutation, ATRX and p53. The ki-67 index reached ~20%. Sequencing of IDH1 and IDH2 did not reveal rare IDH mutations, and FISH did not show 1p19q codeletion. Testing for BRAF V600 mutation was negative. Results Although the histology initially suggested a diagnosis of oligodendroglioma, the integrated diagnosis was compatible with glioblastoma, IDH wildtype. Methylation array analysis by EPIC array did not result in classification of currently known entities, neither confirming glioblastoma, nor providing a new diagnosis, when analysed on both brain tumour and sarcoma classifier. This suggested a novel tumour entity not yet represented in the classifier algorithm. Additional testing including next generation sequencing revealed EWSR1 gene rearrangement with fusion partner ATF1 (EWSR1-ATF1 fusion). Based on this, the diagnosis was revised to the emerging new entity of ‘intracranial myxoid mesenchymal tumor’ (IMMT) characterised by EWSR1 fusion with members of the cAMP response element binding protein (CREB) family (ATF1, CREB1 and CREM). Subsequent immunohistochemistry demonstrated positive staining for CD99 and EMA but not desmin. The patient underwent various oncological treatments and is recurrence-free 3 years after initial diagnosis. Conclusion Histologically, IMMT demonstrates a spectrum of features that overlaps with other tumours, but often displays circumscribed growth, uniform cellularity, cytoplasmic clearing and variable myxoid change. The clinical behaviour of these tumours is not fully understood, however provisionally considered intermediate grade. EWSR1-CREB family fusion is not specific but shared with a diverse group of extracranial tumours including soft tissue, salivary gland, odontogenic and myoepithelial tumours. Therefore, clinico-radiologico-pathological correlation is essential to achieve the final diagnosis, and ensure the absence of a primary tumour elsewhere. Familiarisation with IMMT, its characteristic genetic profile and its as yet underreported natural course is crucial, as it can clinically mimic other intracranial tumours such as malignant meningioma or glioma but appears to behave less aggressively than high grade glioma. It is also important to further our understanding of its optimal treatment through review of larger case series and global comparison of patient management.

Metachronous oligodendroglial tumours with different IDH mutational profile in a young patient

Abstract Aims Oligodendroglioma is molecularly defined by mutation of isocitrate dehydrogenase (IDH) and 1p19q codeletion. IDH mutation is an early driver of tumorigenesis, via its oncometabolite 2-hydroxyglutarate, regardless of the exact mutational subtype in homologues IDH1 or IDH2. IDH mutant cells then acquire 1p19q codeletion, with haploinsufficiency likely to contribute to oncogenesis by reduced expression of genes on 1p and 19q, as well as mutations in TERT, FUBP1 (on 1p31.1) in ~30% and CIC (on 19q13.2) in ~>60% of 1p19q-codeleted gliomas. We present a case of a young patient with metachronous oligodendroglial tumours, initially thought to represent contralateral recurrence of the same disease. However, IDH mutation analysis in each tumour revealed distinct types of mutations, involving both IDH1 and IDH2, indicating different cellular lineages of tumorigenesis. We aim to present this unusual combination by illustrating the histology and molecular profile, and review the literature with regards to multifocal but molecularly distinct glioma. Method Case: The patient is a 33 year old man initially presenting with seizures, who was found to have a frontal lobe lesion (hence called tumour 1) with focal radiological enhancement, followed by a contralateral lesion in the parietal lobe 6 months later (hence designated as tumour 2). He underwent separate surgical debulking, and each time, tumour tissue was histologically and genetically examined. Testing included targeted mutation screening by immunohistochemistry and PCR based methods, pyrosequencing for MGMT methylation analysis, FISH for chromosomal LOH analysis of 1p and 19q, immunohistochemistry for mismatch repair enzymes and next generation sequencing. Results Histology of tumour 1 revealed a neoplasm with uniform cells, round nuclei and oligodendroglioma-like clear cell change, without mitoses, microvascular proliferation or necrosis. Immunohistochemistry showed absence of IDH1 R132H mutation, retained expression of ATRX and no altered p53 staining. The ki-67 index reached 6%. Sequencing of IDH1/2 mutations revealed a rare IDH2 mutation (non-/R172K). FISH confirmed codeletion of 1p19q, and the integrated diagnosis was oligodendroglioma, IDH mutant and 1p19q codeleted, WHO grade II. Histology of tumour 2 demonstrated oligodendroglioma morphology in areas, but more cellular and nuclear pleomorphism and focally brisk mitotic activity (7 mitoses in 10 hpf; ki67 index 20%), while both microvascular proliferation and necrosis were absent. Immunohistochemistry showed IDH1 R132H mutation and retained ATRX, while p53 was not expressed. FISH studies confirmed codeletion of 1p19q, and the integrated diagnosis was anaplastic oligodendroglioma, IDH mutant and 1p19q codeleted, WHO-2016 grade III. NGS data and MMR results are compared. Conclusion We present a patient with two histologically similar, but molecularly distinct oligodendroglial tumours affecting both cerebral hemispheres. Apart from the grade, the important difference is the presence of different IDH mutations, 1) a rare IDH2 mutation (non-R172K) and 2) the common IDH1 (R132H) mutation. While both types of IDH mutations identified are known to occur in oligodendroglioma, the difference clearly indicates two distinct lineages of tumorigenesis, especially as IDH mutation is considered an early event in gliomagenesis. IDH2 mutations are often associated with oligodendrogliomas, while IDH1 R132H is recognised to be frequent in both diffuse oligodendroglial and astroglial neoplasms. Multifocal divergent gliomas have been described previously but oligodendrogliomas with differing IDH mutations in the same patient have not knowingly been reported yet. Importantly, though therapeutically irrelevant here, multicentric gliomas do not automatically imply relatedness. However, a common origin or predisposition (here, even predating IDH mutation) may not be ruled out.

Mutant IDH1 promotes phagocytic function of microglia/macrophages in gliomas by downregulating ICAM1.

Tumor-associated microglia/macrophages (TAMs) are the main innate immune effector cells in malignant gliomas and have both pro- and anti-tumor functions. The plasticity of TAMs is partially dictated by oncogenic mutations in tumor cells. Heterozygous IDH1 mutation is a cancer driver gene prevalent in grade II/III gliomas, and IDH1 mutant gliomas have relatively favorable clinical outcomes. It is largely unknown how IDH mutation alters TAM phenotypes to influence glioma growth. Here we established clinically relevant isogenic glioma models carrying monoallelic IDH1 R132H mutation (IDH1R132H/WT) and found that IDH1R132H/WT significantly downregulated immune response-related pathways in glioma cells, indicating an immunomodulation role of mutant IDH1. Co-culturing IDH1R132H/WT glioma cells with human macrophages promoted anti-tumor phenotypes of macrophages and increased macrophage migration and phagocytic capacity. In orthotopic xenografts, IDH1R132H/WT decreased tumor growth and prolonged animal survival, accompanied by increased TAM recruitment and upregulated phagocytosis markers, suggesting the induction of anti-tumor TAM functions. Using human cytokine arrays that query 36 proteins, we identified significant downregulation of ICAM-1/CD54 in IDH1R132H/WT gliomas, which was further confirmed by ELISA and immunoblotting analyses. ICAM1 gain-of-function studies revealed that ICAM1 downregulation in IDH1R132H/WT cells played a mechanistic role to mediate the immunomodulation function of IDH1R132H/WT. ICAM-1 silencing in IDH1 wild-type glioma cells decreased tumor growth and increased the anti-tumor function of TAMs. Together, our studies support a new TAM-mediated phagocytic function within IDH1 mutant gliomas, and improved understanding of this process may uncover novel approaches to targeting IDH1 wild type gliomas.

Synchronous supratentorial and infratentorial oligodendrogliomas with incongruous IDH1 mutations, a case report

Infratentorial oligodendrogliomas, a rare pathological entity, are generally considered metastatic lesions from supratentorial primary tumors. Here, we report the case of a 23-year-old man presenting with a histopathologically confirmed right precentral gyrus grade 2 oligodendroglioma and a concurrent pontine grade 3 oligodendroglioma. The pontine lesion was biopsied approximately a year after the biopsy of the precentral lesion due to disease progression despite 4 cycles of procarbazine-CCNU-vincristine (PCV) chemotherapy and stable supratentorial disease. Histology and genetic analysis of the pontine biopsy were consistent with grade 3 oligodendroglioma, and comparison of the two lesions demonstrated common 1p/19q co-deletions and TERT promoter mutations but distinct IDH1 mutations, with a non-canonical IDH1 R132G mutation identified in the infratentorial lesion and a R132H mutation identified in the cortical lesion. Initiation of Temozolomide led to complete response of the supratentorial lesion and durable disease control, while Temozolomide with subsequent radiation therapy of 54 Gy in 30 fractions resulted in partial response of the pontine lesion. This case report supports possible distinct molecular pathogenesis in supratentorial and infratentorial oligodendrogliomas and raises questions about the role of different IDH1 mutant isoforms in explaining treatment resistance to different chemotherapy regimens. Importantly, this case suggests that biopsies of all radiographic lesions, when feasible and safe, should be considered in order to adequately guide management in multicentric oligodendrogliomas.

P04.07 The molecular evolution of oligodendrogliomas

Abstract BACKGROUND Oligodendroglioma (OD) is defined by the presence of both IDH1/2 mutation and 1p/19q codeletion. Although prognosis of OD patients is relatively favorable, tumours usually relapse and often evolve to a higher malignancy grade, with some acquiring the treatment induced hypermutated phenotype. To better understand how these tumours evolve in time, we examined the molecular differences between matched primary and recurrent ODs. MATERIAL AND METHODS We identified 21 patients who underwent surgery at least twice [male: 11, female: 10, median age: 44 years (31–66)]. Clinical data were available for 14/21 patients: 5/14 received a treatment between resections [4 radiotherapy, 1 radiotherapy followed by PCV chemotherapy]; median time from the first to the second surgery was 71.5 months (12–158). Whole genome DNA-methylation analysis was performed using Illumina’s MethylationEPIC ‘850K’ BeadChip. Results were evaluated using the Molecularneuropathology.org platform (version 3.1.5) and in R. RESULTS Most samples were WHO grade 2 ODs [14, 10 and 1 tumours in first, second and third resection group, respectively]; WHO grade 3 was found in 6, 10 and 3 tumours in first, second and third resection, respectively; in 4 patients the tumour showed malignant progression from grade 2 to 3. Most ODs exhibited an IDH1 R132H mutation [17/21 patients]; in no cases was IDH1/2 mutation lost during progression. DNA methylation analysis was successfully performed in 41/45 cases [primary OD: 17, recurrent OD: 24] for a total of 18 matched pairs. 37 samples were assigned to the “IDH mutant glioma, subclass 1p/19q codeleted OD”; the remaining 4 were assigned to various other methylation classes but CNV (copy number variation) analysis confirmed the 1p19q codeletion in all samples. Recurrent tumours exhibited de novo loss of chromosome 4 in 3/24 cases (12.5%) and loss of chromosome 13 in 3/24 cases (12.5%). In unsupervised analysis of the 1000 most variable CpG sites, samples from the same patient clustered together. This indicates that the inter-tumour variability is greater than the intra-, temporal- or grading variability between tumours. There were no overt differences in DNA methylation levels between the primary and matched recurrent OD. However, lower genome wide DNA methylation levels were observed in tumours that dedifferentiated to grade 3 ODs compared to those of grade 2, indicating that DNA demethylation is associated to higher malignancy grade. CONCLUSION DNA methylation analysis in a cohort of primary and recurrent oligodendrogliomas highlights the genomic and epi-genetic changes that are acquired at tumour progression. We are currently expanding the cohort and collecting/integrating the clinical data to better explore the evolution of recurrent ODs.

Monoallelic IDH1 R132H Mutation Mediates Glioma Cell Response to Anticancer Therapies via Induction of Senescence.

Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2-M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non-cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies. IMPLICATIONS: The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant-mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

Abstract 457: A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas

Abstract Introduction In the 2016 World Health Organization Central Nervous System Tumor Classification, diffuse gliomas were reclassified to include astrocytic tumors (grade II and III), oligodendrogliomas (grade II and III), and grade IV glioblastomas with diffuse astrocytoma being distinct from the diffuse gliomas. This reclassification is due to the abundance of IDH1 mutations in many of these tumors. A much better outcome has been observed in these patients than those without this mutation. IDH1 mutation of the R132H type constitutes more than 90% of all IDH1 and IDH2 mutations. Further, IDH1 IHC is now widely applied for the differential diagnosis of these tumors. There are two distinct IDH1 R132H clones on the market, the H09 and IHC132. Comparing these markers for the IDH1 R132H mutations by IHC can help determine if one is superior for diagnostics. Materials and Methods IHC was performed on 3-5 um thick formalin-fixed, paraffin-embedded (FFPE) brain tissue arrays containing glioblastoma, astrocytoma, oligoastrocytoma, oligodendroglioma with adjacent normal tissue (US Biomax, Derwood, USA). These slides were deparaffinized, and peroxidase blocked in the usual manner. Slides were then subjected to HIER in a pressure cooker using a citrate-based buffer at 110°C for 15 minutes before being stained on a semi-automated instrument. The slides were stained with rabbit monoclonal IDH1 R132H antibody H09 (Dianova, Hamburg, Germany) and mouse monoclonal IDH1 R132H antibody IHC132 (GenomeMe, Richmond, Canada) at concentrations and protocols predetermined to match staining intensity. Results The mouse monoclonal anti-IDH1 R132H IHC132 has shown that it is analogous to the current IDH1 R132H [H09]. Upon review of the data, IHC132 marker stains a higher percentage of cases in glioblastoma compared to H09 (11%, n=36) and equal in astrocytoma (73%, n=15), oligoastrocytoma (75%, n=4), oligodendroglioma (100%, n=8). Neither clone stained normal brain tissue per negative controls. Conclusion Mouse monoclonal anti-IDH1 R132H [IHC132] compared with that of the rabbit monoclonal anti-IDH1 R132H [H09] showed equal sensitivity for astrocytoma, oligodendroglioma, and oligoastrocytomas with higher sensitivity for glioblastoma. The results provide evidence to support a higher percentage of positive staining and equal or greater intensity for clone IHC132 versus H09. Citation Format: Sara Figueroa, Joseph Vargas, Jason Ramos. A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 457.

Efficacy of 5-Azacitidine on IDH1/2 Acute Myeloid Leukemia/Myelodysplastic Syndromes and Correlation with 2-Hydroxyglutarate Production

Background. Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations are found in 10 and 15% of acute myeloid leukemia (AML) patients, respectively. IDH1/2 mutants' proteins produce high 2-hydroxyglutarate (2HG) levels that inhibit α-ketoglutarate-dependent dioxygenases including histone and DNA demethylases, as TET2 proteins. Furthermore, serum 2HG can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations at diagnosis (cut-off value >2µM) but also correlate with response after intensive chemotherapy. 5-azacitidine (AZA) is a DNA methyltransferase inhibitor that demonstrated significant clinical benefit not only in high-risk MDS but also in AML. Hypomethylating agents yield higher response rate in the context of CpG island hypermethylation phenotype induced by TET2 mutations in myelodysplastic syndrome (MDS). Thus, we evaluate AZA response in the context of IDH1/2 mutations and the effect of AZA on serum 2HG production.Patients and methods. The present study retrospectively focused on IDH1/2 mutant AML/MDS patients treated in our center with at least 1 cycle of AZA. IDH1/2 mutational status was evaluated in all patients with Sanger or Next-Generation sequencing before therapy. All patients received subcutaneous AZA 75mg/m2/d for 7 days every 28 days. Response to therapy was evaluated according to modified 2003 IWG criteria's for AML. Hematological improvement (HI) was evaluated according to IWG 2006 criteria's for MDS. Overall survival (OS) was censored in case of IDH1/2 inhibitor therapy after AZA. Serum samples were analyzed for total 2HG by reverse-phase liquid chromatography coupled to mass spectrometry.Results. Thirty-six patients (34 AML, 2 MDS) were retrospectively analyzed. Initial characteristics of the patients are shown on Table 1. Median age was 74.3 years (range, 48.4-86). Thirteen patients (36%) had IDH1 mutations (R132C: 10, R132H: 2, R132G: 1) and 22 patients (61%) had IDH2 mutations (R140Q: 19, R172K: 3). One patient had both IDH1 R132H and IDH2 R140Q mutations. Twenty-nine patients had intermediate cytogenetic (including normal karyotype in 20 patients) and 6/36 unfavorable. IDH1/2 mutations were associated with SRSF2 mutations in 13 patients, DNMT3A in 11 patients, NPM1 mutation in 9 patients, ASXL1 in 5 patients and FLT3 -ITD in 5 patients. Ten patients (28%) were treated frontline with AZA while the others received AZA as second line (50%, including 6 patients with primary refractory AML to intensive chemotherapy) or subsequent lines (22%). Five patients received IDH1/2 inhibitors (AG221: 3, AG120: 2) prior to AZA treatment. Patients received a median of 4.5 cycles of AZA (range, 1-23). Four patients (11%) achieved complete remission (CR) and 7 (19%) obtained HI with persistent excess of blasts (overall response rate: 30%). Median OS was 9.5 months (FU range, 0.2-41.6) with a trend toward shorter OS in IDH1 mutant AML patient (6.4 vs 10.1; p≤0.0764) (Figure 1). No difference was observed between patients in 1st or subsequent treatment lines (p≤0.7265).Median 2HG concentration (available in 25/36 patients) before the onset of AZA treatment was 9.7µM. Seventeen patients had available serum during follow-up (median: 2 measures/patient). When 2HG values in responding and non-responding patients were compared at different time-points, 2HG values remained elevated in responders (median 2HG: 6.8µM; range 0.99-13.5) without significant difference compared to baseline or relapse values (Figure 2). All responders to AZA ultimately relapsed or progressed during follow-up. IDH1 or IDH2 mutations persisted at relapse along with elevated 2HG levels (median 2HG: 4.5µM).Discussion. In the present series, IDH1/2 mutations were not associated with an increased sensitivity to AZA, as opposed to TET2 mutations. AZA may decrease production of 2HG in responders compared to diagnosis values. However, unlike intensive chemotherapy, AZA is unable to normalize 2HG values in responding patients. This suggests that 2HG cannot be used as a surrogate marker of AZA response and AZA efficacy might not be related to inhibition of 2HG production in IDH1/2 AML/MDS patients. Thus, combinations of AZA with IDH1/2 mutant proteins inhibitors might lead to an additive or synergistic antitumor effect. [Display omitted] DisclosuresRenneville:CELGENE: Research Funding. Ribrag:Roche: Honoraria, Other: travel, accommodation, expenses; BMS: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Infinity: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Nanostring: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Pharmamar: Consultancy; Esai: Honoraria, Research Funding; ArgenX: Research Funding; Servier: Consultancy, Honoraria. de Botton:Celgene Corporatation: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria; Agios Pharmaceuticals, Inc.: Honoraria, Research Funding.

The Present and Future of Less-invasive Liquid Biopsy for the Diagnosis of Gliomas and Brain Tumors

There is growing interest in liquid biopsy, the less-invasive detection of circulating tumor DNA(ctDNA)or circulating tumor cells(CTCs)from cerebrospinal fluid(CSF)and/or serum of patients, for the diagnosis of brain tumors. We share our experience of detecting hot spot point mutations using droplet digital PCR(ddPCR)in ctDNA obtained from the CSF of patients with brain tumors. The detection of mutations such as IDH1 R132H, BRAF V600E, and TERT promoter mutations in gliomas can be diagnostic. For optimal detection of ctDNA, which is only seen at very low concentrations, proper handling and storage of CSF, high-yield extraction of ctDNA, and usage of sensitive PCR methods for detection are imperative. We discuss which mutations can be assessed when diagnosing brain tumors, with a specific focus on gliomas. Finally, we look at what the near future holds for liquid biopsy of brain tumor patients, including next-generation sequencing panel analysis and accurate assessment of fusion genes.

Improved outcomes associated with maximal extent of resection for butterfly glioblastoma: insights from institutional and national data

Butterfly glioblastomas (bGBMs) are grade IV gliomas that infiltrate the corpus callosum and spread to bilateral cerebral hemispheres. Due to the rarity of cases, there is a dearth of information in existing literature. Herein, we evaluate clinical and genetic characteristics, associated predictors, and survival outcomes in an institutional series and compare them to a national cohort. We identified all adult patients with bGBM treated at Brigham & Women's Hospital (2008-2018). The National Cancer Database (NCDB) was also queried for bGBM patients. Survival was analyzed with Kaplan-Meier methods, and Cox models were built to assess for predictive factors. Of 993 glioblastoma patients, 62 cases (6.2%) of bGBM were identified. Craniotomy for resection was attempted in 26 patients (41.9%), with a median volumetric extent of resection (vEOR) of 72.3% (95% confidence interval [95%CI] 58.3-82.1). The IDH1 R132H mutation was detected in two patients (3.2%), and MGMT promoter was methylated in 55.5% of the assessed cases. In multivariable regression, factors predictive of longer OS were increased vEOR, MGMT promoter methylation, and receipt of adjuvant therapy. Median OS for the resected cases was 11.5months (95%CI 7.7-18.8) vs. 6.3 (95%CI 5.1-8.9) for the biopsied. Of 21,353 GBMs, 719 (3.37%) bGBM patients were identified in the NCDB. Resection was more likely to be pursued in recent years, and GTR was independently associated with prolonged OS (p < 0.01). Surgical resection followed by adjuvant chemoradiation is associated with significant survival gains and should be pursued in carefully selected bGBM patients.

Label-free highly sensitive detection of DNA approximate length and concentration by impedimetric CRISPR-dCas9 based biosensor technology

In this study, we designed a CRISPR-dCas9-based biosensor with potential clinical use in glioblastoma subtype discrimination through detection of isocitrate dehydrogenase R132H (IDH) mutation status. The electrode was modified to detect mutant DNA cysteamine (Cys), PAMAM, dCas9 and sgRNA for R132H mutations, respectively. The biosensor system we proposed was able not only to detect mutant DNA, but also to measure the approximate length of DNA. Therefore, it can be considered that the biosensor technology that we developed is novel in the field of DNA biosensors. Another superior capability of the biosensor system is that it can simultaneously measure DNA concentration by electrochemical impedance spectroscopy and DNA length by capacitive detection, which lowers the concentration-based false-positive signals. The calibration range was obtained between 100 fM and 1000 fM, LOD and LOQ were also calculated as 33.96 fM and 102.91 fM respectively. Moreover, thanks to the sensitivity of the capacitive detection, the biosensor was able to discriminate the same EIS signals of the 200 bp and 250 fM concentration data and 1000 bp and 50 fM concentration data. In conclusion, the biosensor was capable of detect target DNA and DNA length, simultaneously in minutes.

T2/T2-FLAIR mismatch sign as a predictive biomarker for anaplastic astrocytoma genetic profile

T2/T2-FLAIR mismatch sign as a predictive biomarker for anaplastic astrocytoma genetic profile

IDH1 gene mutation activates Smad signaling molecules to regulate the expression levels of cell cycle and biological rhythm genes in human glioma U87‑MG cells.

Isocitrate dehydrogenase1 (IDH1) mutation is the most important genetic change in glioma. The most common IDH1 mutation results in the amino acid substitution of arginine 132 (Arg/R132), which is located at the active site of the enzyme. IDH1 Arg132His (R132H) mutation can reduce the proliferative rate of glioma cells. Numerous diseases follow circadian rhythms, and there is growing evidence that circadian disruption may be a risk factor for cancer in humans. Dysregulation of the circadian clock serves an important role in the development of malignant tumors, including glioma. Brain-Muscle Arnt-Like protein 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) are the main biological rhythm genes. The present study aimed to further study whether there is an association between IDH1 R132H mutation and biological rhythm in glioma, and whether this affects the occurrence of glioma. The Cancer Genome Atlas (TCGA) database was used to detect the expression levels of the biological rhythm genes BMAL1 and CLOCK in various types of tumor. Additionally, U87-MG cells were infected with wild-type and mutant IDH1 lentiviruses. Colony formation experiments were used to detect cell proliferation in each group, cell cycle distribution was detected by flow cytometry and western blotting was used to detect the expression levels of wild-type and mutant IDH1, cyclins, biological rhythm genes and Smad signaling pathway-associated genes in U87-MG cells. TCGA database results suggested that BMAL1 and CLOCK were abnormally expressed in glioma. Cells were successfully infected with wild-type and mutant IDH1 lentiviruses. Colony formation assay revealed decreased cell proliferation in the IDH1 R132H mutant group. The cell cycle distribution detected by flow cytometry indicated that IDH1 gene mutation increased the G1 phase ratio and decreased the S phase ratio in U87-MG cells. The western blotting results demonstrated that IDH1 R132H mutation decreased the expression levels of the S phase-associated proteins Cyclin A and CDK2, and increased the expression levels of the G1 phase-associated proteins Cyclin D3 and CDK4, but did not significantly change the expression levels of the G2/M phase-associated protein Cyclin B1. The expression levels of the positive and negative rhythm regulation genes BMAL1, CLOCK, period (PER s (PER1, 2 and 3) and cryptochrom (CRY)s (CRY1 and 2) were significantly decreased, those of the Smad signaling pathway-associated genes Smad2, Smad3 and Smad2-3 were decreased, and those of phosphorylated (p)-Smad2, p-Smad3 and Smad4 were increased. Therefore, the present results suggested that the IDH1 R132H mutation may alter the cell cycle and biological rhythm genes in U87-MG cells through the TGF-β/Smad signaling pathway.

Concomitant KIAA1549-BRAF fusion and IDH mutation in Pediatric spinal cord astrocytoma: a case report and literature review

Primary tumors of the spinal cord are rare, accounting for 3-6% of tumors in the central nervous system, particularly in children. KIAA1549-BRAF fusion is more common in pilocytic astrocytoma (PA) and IDH1 R132H mutation is rare in infratentorial tumors. Here, we report a 10-year-old male patient who presented with weakness in lower limbs that progressed to difficulty walking. Magnetic resonance imaging (MRI) revealed an intramedullary solid-cystic lesion from the medulla oblongata to the thoracic spin 4 level, with the expansion of the spinal cord. The lesion exhibited patchy enhancement at C4-T1, indicating a tentative diagnosis of astrocytoma. The patient underwent resection of the lesion in the spinal canal from the cervical 6 level to the thoracic 2 level. Histopathology confirmed diagnosis of astrocytoma, WHO grade 2. Genetic analysis showed both IDH1 R132H mutation and KIAA1549-BRAF fusion. Therefore, our integrated diagnosis was astrocytoma, IDH mutation, WHO grade 2. Its molecular analyses include IDH1 R132H mutation and KIAA1549-BRAF fusion. After the operation, the patient did not receive chemo- or radiotherapy, and underwent an aggressive rehabilitation regiment. Follow up 10months later, symptoms improved. To our best knowledge, this is the first case of concomitant IDH mutation and BRAF fusion in pediatric spinal cord astrocytoma.

IDH1 Non-Canonical Mutations and Survival in Patients with Glioma.

Background: Non-canonical mutations of the isocitrate dehydrogenase (IDH) genes have been described in about 20–25% and 5–12% of patients with WHO grade II and III gliomas, respectively. To date, the prognostic value of these rare mutations is still a topic of debate. Methods: We selected patients with WHO grade II and III gliomas and IDH1 mutations with available tissue samples for next-generation sequencing. The clinical outcomes and baseline behaviors of patients with canonical IDH1 R132H and non-canonical IDH1 mutations were compared. Results: We evaluated 433 patients harboring IDH1 mutations. Three hundred and ninety patients (90.1%) had a canonical IDH1 R132H mutation while 43 patients (9.9%) had a non-canonical IDH1 mutation. Compared to those with the IDH1 canonical mutation, patients with non-canonical mutations were younger (p < 0.001) and less frequently presented the 1p19q codeletion (p = 0.017). Multivariate analysis confirmed that the extension of surgery (p = 0.003), the presence of the 1p19q codeletion (p = 0.001), and the presence of a non-canonical mutation (p = 0.041) were variables correlated with improved overall survival. Conclusion: the presence of non-canonical IDH1 mutations could be associated with improved survival among patients with IDH1 mutated grade II–III glioma.

NANOMECHANICAL PROPERTIES OF GLIOMA CELLS WITH IDH1 R132H MUTATION IN PRIMARY CULTURES AS PROSPECTIVE MARKERS FOR ONCODIAGNOSTICS

Phenotyping and genotyping of tumor cells of various genesis provides irreplaceable information for targeted therapy. We have revealed the nanomechanical properties of living cells of cultures of primary cell lines of gliomas with the IDH1 R132H mutation, which make it possible to differentiate them from cells that do not have this mutation, which is necessary for personalized therapy of these diseases.

TROP-2, 5hmC, and IDH1 Expression in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC), a highly aggressive malignancy, has no effective treatment to date. Trophoblast cell-surface antigen 2 (TROP-2), a transmembrane glycoprotein, has been suggested to be a promising novel target for sacituzumab govitecan, an antibody-drug conjugate. 5-Hydroxymethylcytosine (5hmC) has a role in tumor suppression and promoting modification. Additionally, isocitrate dehydrogenase 1 (IDH1) mutations are strongly associated with increased overall survival in gliomas and worse prognosis in leukemias. This study attempts to evaluate the immunoexpression of TROP-2, 5hmC, and IDH1 in ATCs and to determine their potential impact in targeted therapy. Twenty-four ATCs were retrieved, with 9 cases that occurred de novo and 15 cases derived from either papillary thyroid carcinoma (PTC) or follicular thyroid carcinoma (FTC). Sections were immunostained with TROP-2, 5hmC, and IDH1 antibodies, and evaluated using the QuPath program. The t tests were performed using SPSS software. TROP-2 was detected in 12 ATCs with 9 cases demonstrating a high expression and in all PTC components, and absent in all FTC components of secondary ATCs. 5hmC expression was moderately reduced in PTC and FTC components and markedly reduced in ATC. The entire cohort showed a total absence of IDH1. Increased TROP-2 immunoexpression in some ATCs supports that these patients may potentially benefit from an antibody-drug conjugate therapy targeting TROP-2. Markedly reduced 5hmC expression suggests that 5hmC may be used as potential therapeutic targets for ATC. The total lack of IDH1 R132H mutation by immunostain indicates that it has no prognostic and therapeutic value in ATC.

PATH-06. ASSESSMENT OF CIRCULATING TUMOR DNA IN CEREBROSPINAL FLUID BY WHOLE EXOME SEQUENCING TO DETECT GENOMIC ALTERATIONS OF GLIOBLASTOMA

Abstract Cerebrospinal fluid (CSF) has been demonstrated as a better source of circulating tumor DNA (ctDNA) than plasma for brain tumors. However, it is unclear whether whole exome sequencing (WES) is qualified for detection of ctDNA in CSF. The aim of this study was to determine if assessment of ctDNA in CSF by WES is a feasible approach to detect genomic alterations of glioblastoma. CSFs of ten glioblastoma patients were collected pre-operatively at the Department of Neurosurgery, Sun Yat-sen University Cancer Center. ctDNA in CSF and genome DNA in the resected tumor were extracted and subjected to WES. The identified glioblastoma-associated mutations from ctDNA in CSF and genome DNA in the resected tumor were compared. Due to the ctDNA in CSF was unqualified for exome sequencing for one patient, nine patients were included into the final analysis. More glioblastoma-associated mutations tended to be detected in CSF comparing with the corresponding tumor tissue samples (3.56±0.75 vs. 2.22±0.32, P=0.097), while the statistical significance was limited by the small sample size. The average mutation frequencies were similar in CSF and tumor tissue samples (74.12% ± 6.03% vs. 73.83% ± 5.95%, P = 0.924). The R132H mutation of isocitrate dehydrogenase 1 and the G34V mutation of H3F3A which had been reported in the pathological diagnoses were also detected from ctDNA in CSF by WES. Patients who received temozolomide chemotherapy previously or those whose tumor involved subventricular zone tended to harbor more mutations in their CSF. Assessment of ctDNA in CSF by WES is a feasible approach to detect genomic alterations of glioblastoma, which may provide useful information for the decision of treatment strategy.