TMOD-08. DEVELOPING DERIVATIVE GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

HGG-44. DEFECTS OF MISMATCH REPAIR PROTEINS IN PEDIATRIC HIGH GRADE GLIOMAS

Hetero- and homozygous germline mutations of the mismatch repair genes MLH1, PMS2, MSH2 and MSH6 cause Lynch and constitutional mismatch repair (CMMRD) cancer predisposition syndrome, respectively. Affected CMMRD individuals are at risk to develop a variety of neoplasms including CNS tumors, particularly high grade gliomas (HGG), during childhood. Currently, few data exist on the prevalence of CMMRD in children with pediatric HGG. We screened a consecutive series of 79 supratentorial HGGs. Tumor tissue was available in 42 patients, 5 were reclassified as non-HGGs. Immunohistochemistry with antibodies against MLH1, PMS2, MSH2 and MSH6 was performed in 37 tumors. Four patients (3 families) with known CMMRD were included. The evaluation of the slides was performed blinded to the CMMRD status. All four patients with known CMMRD (3 patients with PMS2, one with MSH6 mutation) were identified, showing loss of PMS2 and MSH2/MSH6, respectively. Additionally, we identified 6 patients with loss of MSH2/MSH6 staining in tumor cells, but retained staining in preexisting cells, indicating a pattern like in Lynch syndrome. NGS sequencing of these tumor tissues revealed in 2 patients MSH2 mutations and in one patient a hypermutator phenotype with MSH2 and MSH6 mutations. In 3/6 patients no mutations in the MMR genes were detectable. In summary, we found a low prevalence of CMMRD among HGGs, but identified also 2 patients with probable Lynch syndrome. Immunhistochemistry is an effective tool to screen for patients with MMR defects and should be performed in HGGs to optimize treatment and offer affected families genetic counseling.

TMOD-35. DEVELOPING RECURRENT GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Tumor mutational load, microsatellite instability and actionable mutations in metastatic colorectal cancer: Results from the TRIBE2 study.

4077 Background: In the TRIBE2 study molecularly unselected and untreated mCRC patients were randomized to receive FOLFOXIRI/bevacizumab (bev) followed by the same agents after disease progression (PD) or FOLFOX/bev followed by FOLFIRI/bev after PD. We performed a comprehensive NGS analysis of samples from randomized patients in order to investigate the prognostic impact of tumor mutational load (TML), its additional value with respect to the assessment of microsatellite instability (MSI), and the overall prevalence of potentially actionable alterations. Methods: Tumor DNA was obtained from formalin-fixed, paraffin-embedded blocks from primary tumors of 296 (44%) out of 679 randomized patients and underwent NGS analysis using the Caris MI TumorSeek panel, assessing 592 genes. TML was defined low, intermediate or high if < 7, 7-16 or > 16 mutations/Mb were found. MSI status was determined both by NGS and by IHC. Results: TML and MSI were successfully determined by NGS in 224 (76%) cases. NGS and IHC results were concordant in 221 (99%) cases. TML was low, intermediate or high in 56 (25%), 157 (70%) and 11 (5%) cases, respectively. When compared with TML low and intermediate tumors, TML high were more frequently right-sided (p = 0.013), mucinous (p < 0.001) and MSI-high (p < 0.001). TML high tumors were MSI-high or MSS in 8 (73%) and 3 (27%) cases, respectively. Two out of 3 TML high and MSS tumours showed a pathogenic POLE mutation (p.S459F and p.P286R). The other TML high, MSS and POLE wt tumor was dMMR at IHC (loss of MSH6 expression) and showed a pathogenic MSH6 mutation (p.F1040fs). As compared with low and intermediate TML, high TML was associated with longer PFS (median PFS: 17.3 vs 10.6; HR: 0.54 [95%CI: 0.35-1.09], p = 0.098) and OS (median OS: not reached vs 23.7: HR: 0.45 [95%CI:0-28-1.13], p = 0.106). No interaction effect between TML and treatment arm was observed, and no difference between TML low and intermediate tumors was reported in terms of baseline characteristics and prognosis. Actionable alterations ( HER2 mutations [N = 2] and amplifications [N = 4], KRAS G12C [N = 10] and BRAF V600E mutation [N = 39]) were found in 55 (19%) out of 296 cases. No NTRK/ROS/ALK or MET amplification was found. Conclusions: TML high tumors are not limited to MSI-high ones but showed POLE or MSH6 somatic mutation and shower longer PFS and OS. No differences are reported between TML low and intermediate tumors. Molecular alterations predictive of benefit from targeted strategies currently available are detectable only in a small percentage of mCRCs.

PATH-45. DIFFERENT TYPES OF HYPERMUTATOR PHENOTYPE OF GLIOBLASTOMA ACCORDING TO MUTATION PATTERNS OF MISMATCH REPAIR GENES

BACKGROUND Although precision medicine has been widely applied to routine care, only few molecular targeted drugs have been developed for glioma. Immune checkpoint inhibitors hold a potential promise to improve outcome of malignant glioma especially with the hypermutator phenotype. Mismatch repair deficiency could induce hypermutation during temozolomide (TMZ) treatment, but the mechanism of mutation acquisition is not well understood. METHODS We present the results of target sequencing of 57 longitudinal specimens from 27 individuals with glioblastoma (GBM). We used Ion AmpliSeq Cancer Hotspot Panel v2 (CHP) and/or Ion AmpliSeq Comprehensive Cancer Panel (CCP) for target sequencing. RESULTS Acquired mutations with G:C >A:T transition at non-CpG sites were found in 70% of recurrent MGMT methylated samples, whereas only 8% in MGMT unmethylated cases (p< 0.001). Two cases of hypermutator phenotype were identified in MGMT methylated, IDH wild type recurrent GBMs after TMZ chemotherapy. One case gained mutations in mismatch repair genes of MLH1, MSH2, MSH6, and PMS1. Most of the acquired mutations were G:C >A:T mutations typical to TMZ-induced hypermutation. The recurrent tumor was highly aggressive with overall survival after recurrence of 3.7 months. The other case gained mutations in mismatch repair genes of MLH1 and PMS1 only. Most of the acquired mutations were insertion mutations. The prognosis after recurrence was much longer. CONCLUSIONS We identified two types of hypermutator phenotype according to mutation pattern of mismatch repair genes. It has been suggested that MSH2-MSH6 complex deficient tumor cannot repair TMZ-induced mismatch mutation, thereby causing hypermutation. Conversely, MLH1-PMS1 complex deficient tumor with intact MSH2-MSH6 complex can repair mismatch mutation but is susceptible to insertion mutation. Taken together, MSH6 mutation plays a key role in TMZ-induced hypermutation, while MLH1 and PMS1 mutations might cause insertion-based hypermutation. Larger and prospective studies are warranted to clarify the mechanism, outcome, and effectiveness of checkpoint inhibitors.

Ultra-Mutation in IDH Wild-Type Glioblastomas of Patients Younger than 55 Years is Associated with Defective Mismatch Repair, Microsatellite Instability, and Giant Cell Enrichment

Background: Glioblastomas (GBMs) are classified into isocitrate dehydrogenase (IDH) mutants and IDH wild-types (IDH-wt). This study aimed at identifying the mutational assets of IDH-wt GBMs in patients aged 18–54 years for which limited data are available. Methods: Sixteen IDH-wt GBMs from adults < 55 years old were explored for mutations, copy number variations, tumour mutational load (TML), and mutational spectrum by a 409 genes TML panel. Results: Eight (50%) IDH-wt GBMs were hypermutated (TML > 9 mutations/Mb) and two (12.5%) were ultra-mutated (TML > 100 mutations/Mb). One ultra-mutated GBM had microsatellite instability (MSI), a somatic MSH6 mutation, and a germline POLE mutation. The other ultra-mutated GBMs had MSI and two somatic mutations in MSH2. Both ultra-mutated GBMs featured at least 25% giant cells. The overall survival of eight patients with hypermutated GBMs was significantly longer than that of patients with non-hypermutated GBMs (p = 0.04). Conclusions: We identified a hyper-mutated subgroup among IDH-wt GBMs in adults < 55 years that had improved prognosis. Two cases were ultra-mutated and characterized by the presence of at least 25% giant cells, MMR mutations, and MSI. Since high TML has been associated with response to immune checkpoint inhibition in paediatric gliomas, the identification of a subtype of ultra-mutated IDH-wt GBM may have implications for immunotherapy.

Landscape of multilocus inherited neoplasia alleles syndrome in China.

e13027 Background: Comprehensive gene panels are able to identify patients with more than one pathogenic germline mutations. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS). The frequency and phenotype of double heterozygous carriers in China are still unknown. Methods: A cohort of 12514 unrelated patients with solid tumor were screened using a validated panel. DNA damage repair-associated genes ( BRCA1, BRCA2, PALB2, ATM) and DNA mismatch repair genes ( MLH1, MSH2, MSH6, PMS2, MLH3, MSH3, PMS1) are taken focused on this study. Results: 10 (2.16%) cases harboring two pathogenic/likely pathogenic mutations were identified in 464 patients with germline variant. All patients had at least one high-risk gene mutation except for G10(LC, 73y), which carried 2 ATM variants in the same allele. All patients developed tumors associated with only one of the mutation identified until now. Among 3 patients had PMS2/MSH6 mutation, G6 & G7 did not translated into recognized Lynch Syndrome due to abnormal BRCA1/2(H-risk for BC), while G8 harboring ATM (M-risk for BC) mutation developed to CRC. All of the five patients developed to breast cancer were TNBC molecular subtype. The median ages of diagnosis for BC were similar to single variation patients (44.3 vs 42, p=0.6190). Conclusions: Two double heterozygotes did not appear to have severe or different manifestations than single heterozygote. With the growing implementation of NGS based panel testing, relevant variants in two (or more) genes will be found more frequently. [Table: see text]