Recurrent Tumor Samples Research Articles

Abstract 2220: Next-generation sequencing of the primary and recurrent tumors from patients with extranodal natural killer/T cell lymphoma

Abstract Introduction: Nasal-type extranodal natural killer/T cell lymphoma (ENKTL) is an aggressive, EBV-associated tumor commonly found in the nasal cavity. It is more prevalent in patients in Asia and Latin America mainly affecting the extranodal sites. Upfront radiotherapy (RT) with or without chemotherapy is the mainstay of treatment but the patient response is variable. ENKTL is often resistant to conventional chemotherapy and early relapse is common. This study evaluated the mutational profiles of recurrent ENKTL for the mechanism of resistance and attempted to look for potential therapeutic targets. Materials and Methods: This pilot study involved paired samples obtained from three patients with recurrent ENKTL. Next-generation sequencing was performed to investigate the gene alteration profiles of paired pre- and post-treatment recurrent tumor samples in these patients following chemoradiotherapy (CRT) or RT alone. Results: A total of 55 variants was identified, with an average of 20 ± 5 variants per patient. Four genes were frequently mutated in the ENKTL samples, including MUC16 (100%), ARID1A (83%), ADAMTSL1 (67%), and KMT2C (50%). After CRT, case 1 developed local recurrence at nasal cavity within 46 months. The number of mutations in case 1 was nearly identical regardless of recurrent status, with only one acquired amplification (CTLA4) in the recurrent tumor. After RT alone, case 2 relapsed at nasal cavity and skin within 16 months. This patient harbored six mutations (ABCC2, FBXW7, FLT1, FOXL2, JAK1, and JAK3) and three deletions (BMPR1A, FAS, and PTEN) in the recurrent tumor, but not in the primary tumor. After RT alone, case 3 relapsed at nasal cavity and stomach within 42 months. Two mutations (FAT1 and NBN) and three amplifications (CDKN1B, HIST1H1C, and HIST1H1E) were only detected in the primary tumor of case 3, whereas one mutation (ARID2) and two amplifications (EZH2 and PIK3CG) were only identified in the recurrent tumor. On the other hand, two clinically relevant targets (TSC2 and RAD54L) were identified in case 2 and case 3, respectively. TSC2 is an FDA-recognized predictive marker for everolimus in central nervous system cancer and RAD54L is an FDA-recognized predictive marker for olaparib in prostate cancer. Conclusion: Our data showed that MUC16, ARID1A and ADAMTSL1 were observed in more than half of nasal-type ENKTL. Additional mutations can be found in recurrent tumors but the relationship between the acquisition of new mutations and the treatment received was still poorly understood. TSC2 and RAD54L mutations were identified in some recurrent tumors which may serve as potential therapeutic targets for ENKTL. Despite the small sample size, NGS seems to be a powerful tool to shed light on the mutational landscape and biology of recurrent NKTCL. Citation Format: William C. Cho, Kien Thiam Tan, Roger K. Ngan, Wah Cheuk, Yi-Ting Yang, Shu-Jen Chen. Next-generation sequencing of the primary and recurrent tumors from patients with extranodal natural killer/T cell lymphoma [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 2220.

Abstract 1412: Whole exome sequencing reveals PI3K-ATK pathway alterations are frequent in relapsed small cell-lung cancer after chemoradiation

Abstract Background: Although small-cell lung cancer (SCLC) is sensitive to chemotherapy and radiotherapy initially, nearly all patients recur often with treatment-resistant disease. Comparing genomic profiles of paired treatment-naïve and recurrent tumors to understand the clonal architecture and molecular evolution of SCLC under treatment may provide novel insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Paired tumor samples procured at diagnosis and relapse were collected from 11 patients with limited-stage SCLC treated with concurrent chemoradiation (CCRT). All tissues underwent whole exome sequencing (WES). Genomic landscape including somatic mutations, somatic copy number alterations (SCNAs), and clonal architecture were compared between treatment-naïve and paired recurrent tumor samples. Baseline and paired recurrent plasma samples from another 9 patients with SCLC treated with CCRT were performed deep sequencing using a targeted panel containing 1021 cancer related genes. Results: In both pre- and post-treatment tumors, TP53 (73% vs 73%), FAM135B (55% vs 64%), RB1 (45% vs 55%), and CTNND2 (45% vs 55%) were the top four most frequently mutated genes. Tobacco exposure related mutational signature was predominant in all samples. Compared with primary tumors, relapsed tumors showed significantly increased number of mutations (220 in recurrent SCLC vs 210 in primary tumor, Wilcoxon paired test, p-value = 0.016). Six of the 11 patients had increased chromosomal instability (CIS) in recurrent tumors. Relapsed tumors had more genes with copy number amplification compared to pre-treatment primary tumors although the difference did not reach statistical significance (18 vs 10, p-value = 0.066). In all of the patients, an average of 94% mutations at baseline were present in relapsed tumors. The number of clones in recurrent samples was higher than that in pre-treatment samples (13 vs 12, p-value = 0.004), indicating that new clones emerged under the treatment and tumor heterogeneity increased. A total of 648 acquired mutations in 600 genes and 140 acquired SCNAs in 123 genes were identified, in which 126 mutations were clonal (CCF>0.6) in relapsed tumors. These genes were enriched in PI3K-ATK signaling pathway and covered 91% (10/11) patients, implying the potential mechanism of chemoradiation resistance. Based on the specific mutations and mutations with increased CCF in relapsed plasma samples from the other 9 SCLCs, acquired alterations were also enriched in PI3K-ATK signaling pathway. Conclusions: Acquired mutations and chromosomal copy number gains may play a role in relapse of limited stage SCLC post CCRT. PI3K-ATK pathway alterations are frequent in recurrent SCLCs, which may be a candidate resistant mechanism after chemoradiation. Citation Format: Ying Jin, Yamei Chen, Xiao Hu, Huarong Tang, Qian Li, Pansong Li, Xinze Lv, Xuefeng Xia, Jianjun Zhang, Ming Chen. Whole exome sequencing reveals PI3K-ATK pathway alterations are frequent in relapsed small cell-lung cancer after chemoradiation [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 1412.

PI3K-AKT pathway alterations are revealed as a potential mechanism of chemoradiation resistance in small cell lung cancer by whole-exome sequencing.

e20573 Background: Although small-cell lung cancer (SCLC) is sensitive to chemotherapy and radiotherapy initially, nearly all patients recur often with treatment-resistant disease. Comparing genomic profiles and clonal architecture of paired treatment-naïve and recurrent tumors may provide novel insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Paired tumor samples procured at diagnosis and relapse were collected from 11 patients (pts) with limited-stage SCLC treated with concurrent chemoradiation (CCRT). All tissues underwent whole exome sequencing (WES). Genomic landscape including somatic mutations, somatic copy number alterations (SCNAs), and clonal architecture were compared between treatment-naïve and paired recurrent tumor samples. Baseline and paired recurrent plasma samples from another 9 pts with SCLC treated with CCRT were performed deep sequencing using a targeted panel containing 1021 cancer related genes. Results: We observed TP53 and RB1 alterations including mutations and SCNAs in the majority of treatment-naïve ( TP53 in 10 of 11, RB1 in 7 of 11 pts) and relapse samples ( TP53 in 8 of 11, RB1 in 8 of 11 pts). Tobacco exposure related mutational signature was most or second predominant in 11 pre-treatment and 9 recurrent samples, respectively. Compared with primary tumors, relapsed tumors showed significantly increased number of mutations (220 vs 210, p = 0.016). A high proportion of shared mutations between baseline and PD samples ranging from 39% to 94% was observed. More than half (n = 6) of the 11 pts had increased chromosomal instability in recurrent tumors. A total of 687 relapse-specific also called acquired mutations in 633 genes were identified, in which 158 mutations with cancer cell fraction (CCF) > 0.6. These genes were enriched in PI3K-ATK signaling pathway and covered 73% (8/11) pts. Of the 9 paired plasma samples performed by targeted sequencing, genes with specific mutations and mutations with increased CCF in relapsed plasma samples were also enriched in PI3K-ATK pathway. In vitro proliferation assay indicated that PI3K inhibitor gave rise to a marked decrease in proliferation after combined with cisplatin, implying the potential mechanism of chemoradiation resistance. The number of clones in recurrent samples was higher than that in pre-treatment samples (13 vs 12, p = 0.004), indicating that new clones emerged under the treatment and tumor heterogeneity increased. Patients characterized by weakening of the major treatment-naïve clone tended to exhibit relatively longer PFS than those with sustaining major clone in both b baseline and relapsed tumors (p = 0.034). Conclusions: PI3K-ATK pathway alterations are frequent in recurrent SCLCs, which may be a candidate resistant mechanism after chemoradiation. PFS is probably associated with clonal evolutionary pattern.

PSMA Expression in Glioblastoma as a Basis for Theranostic Approaches: A Retrospective, Correlational Panel Study Including Immunohistochemistry, Clinical Parameters and PET Imaging

AimThe aim of the current study was to enlighten the evolution of prostate-specific membrane antigen (PSMA) expression in glioblastoma between initial diagnosis and recurrence in order to provide preliminary insight for further clinical investigations into innovative PSMA-directed treatment concepts in neuro-oncology.MethodsPatients who underwent resection for de-novo glioblastoma (GBM) and had a re-resection in case of a recurrent tumor following radiochemotherapy and subsequent chemotherapy were included (n = 16). Histological and immunohistochemical stainings were performed at initial diagnosis and at recurrence (n = 96 tissue specimens). Levels of PSMA expression both in endothelial and non-endothelial cells as well as vascular density (CD34) were quantified via immunohistochemistry and changes between initial diagnosis and recurrence were determined. Immunohistochemical findings were correlated with survival and established clinical parameters.ResultsPSMA expression was found to be present in all GBM tissue samples at initial diagnosis as well as in all but one case of recurrent tumor samples. The level of PSMA expression in glioblastoma varied inter-individually both in endothelial and non-endothelial cells. Likewise, the temporal evolution of PSMA expression highly varied in between patients. The level of vascular PSMA expression at recurrence and its change between initial diagnosis and recurrence was associated with post recurrence survival time: Patients with high vascular PSMA expression at recurrence as well as patients with increasing PSMA expression throughout the disease course survived shorter than patients with low vascular PSMA expression or decreasing vascular PSMA expression. There was no significant correlation of PSMA expression with MGMT promoter methylation status or Ki-67 labelling index.ConclusionPSMA is expressed in glioblastoma both at initial diagnosis and at recurrence. High vascular PSMA expression at recurrence seems to be a negative prognostic marker. Thus, PSMA expression in GBM might present a promising target for theranostic approaches in recurrent glioblastoma. Especially PSMA PET imaging and PSMA-directed radioligand therapy warrant further studies in brain tumor patients.

Full-coverage TP53 deep sequencing of recurrent head and neck squamous cell carcinoma facilitates prognostic assessment after recurrence.

This study aims to evaluate whether the accumulation of TP53 mutations is associated with clinical outcome by comparing full-coverage TP53 deep sequencing of the initial and recurrent head and neck squamous cell carcinoma (HNSCC). Medical records and surgical specimens of 400 patients with HNSCC surgically treated with curative intent, of which 95 patients developed local or locoregional recurrence, were reviewed. Of these patients, 63 were eligible for genomic analysis. Full-coverage TP53 deep sequencing of 126 paired initial and recurrent tumor samples was examined using next-generation sequencing (NGS). Temporal changes in the mutation status, molecular characterization, and clinical outcome were compared. Fisher's exact test, Kaplan-Meier method, log-rank test, and Cox regression models were used for statistical analysis. Of the recurrent tumors, 22% harbored accumulation of TP53 mutations, and 16% lost the original mutation. The accumulation of TP53 mutations was significantly more frequent in oral cancer than in pharyngeal or laryngeal cancer (33% vs. 7%, p=0.016). Two-year post-recurrence survival (PRS) was associated with TP53 status for recurrent tumors, but not for initial tumors. The TP53 status for recurrent tumors was an independent risk factor in multivariate analysis (hazard ratio, 5.76; 95% confidence interval, 1.86-17.8; p=0.0023). Approximately one-third of the recurrent HNSCC cases showed a different TP53 status from the initial tumor. Temporal changes in the mutation status differed by primary site. Full-coverage TP53 deep sequencing of recurrent tumors was useful in predicting post-recurrence prognosis.

Identifying the mRNAs associated with Bladder cancer recurrence.

To identify the mRNAs associated with bladder cancer (BC) recurrence. The transcription profile of GSE31684 including 39 recurrent BC tumor samples and 54 non-recurrent BC tumor samples as well as transcription profile of GSE13507 including 36 recurrent BC tumor samples and 67 non-recurrent BC tumor samples were downlaoded from the Gene Expression Omnibus. Then, the differentially expressed genes (DEGs) were identified using linear models for microarray data (limma) and the intersections of DEGs from the two datasets were further screened. The weighed gene co-expression network analysis (WGCNA) was used to screen the modules related to BC recurrence. Protein-protein interaction (PPI) network analysis was used to analyze the genes interaction. Their functions were predicted by Gene Ontology and KEGG pathway enrichment. Moreover, The Comparative Toxicogenomics Database 2017 update (CTD) was used to search the BC related pathway. The univariate cox regression analysis was used to identify DEGs associated to the recurrence. Kaplan-Meier plots were used to illustrate recurrence free survival time (RFS). A total of 692 intersections DEGs were screened. WGCNA showed that 7 modules (2279 genes) were stable in both the datasets. A total of 169 intersection DEGs were mapped to the 7 modules. There existed 149 interaction relationships among 81 proteins (18 down-regulated and 63 up-regulated DEGs) in the PPI network. Two KEGG pathways including Focal adhesion and ECM-receptor interaction were enriched which were also found in the CTD. The univariate cox regression analysis showed that 3 DEGs (COL4A1, COL1A2 and COL5A1) were significant related to the prognosis. Multivariate cox regression analysis revealed that pathologic_N (N0-N1 vs N2-N3, p= 0.033) were independent prognostic factors for overall survival in patients with BC. COL4A1, COL1A2 and COL5A1 could be associated with BC recurrence.

Sparsity of dendritic cells and cytotoxic T cells in tumor microenvironment may lead to recurrence in basal cell carcinoma.

Antitumor immune response affects tumor growth. The effect of antitumor immune response on recurrence has been poorly studied in basal cell carcinoma (BCC). To investigate the effects of the peritumoral immune infiltrate on BCC recurrence. A total of 30 BCC patients without recurrence and 29 BCC patients with recurrence were included in this retrospective study. Non-recurrent tumor samples as well as primary and recurrent tumor samples from the recurrent group were stained immunohistochemically with anti-CD4, CD8, CD25, FOXP3, CD68, CD163, and CD1a antibodies. Immune infiltrates were semiquantitatively evaluated. BCC tumor microenvironment was rich in CD4+ cells. CD163 expression was higher than CD68. In primary tumors of the recurrent group, CD8 expression was significantly lower than CD4 expression. CD1a expression was lower in primary tumors of the recurrent group than in nonrecurrent tumors. Our results suggest the existence of an immunosuppressive microenvironment in BCC. Lower CD8+ T-cell numbers and sparsity of dendritic cells in primary tumors of recurrent patients suggest further immunosuppression in the tumor microenvironment and an increase in recurrence risk. This is the first study that evaluates and compares tumor immune microenvironments of primary and recurrent BCC lesions with several markers and investigates the role of antitumor immunity on BCC recurrence.

Multiomics profiling of paired primary and recurrent glioblastoma patient tissues.

BackgroundDespite maximal therapy with surgery, chemotherapy, and radiotherapy, glioblastoma (GBM) patients have a median survival of only 15 months. Almost all patients inevitably experience symptomatic tumor recurrence. A hallmark of this tumor type is the large heterogeneity between patients and within tumors itself which relates to the failure of standardized tumor treatment. In this study, tissue samples of paired primary and recurrent GBM tumors were investigated to identify individual factors related to tumor progression.MethodsPaired primary and recurrent GBM tumor tissues from 8 patients were investigated with a multiomics approach using transcriptomics, proteomics, and phosphoproteomics.ResultsIn the studied patient cohort, large variations between and within patients are observed for all omics analyses. A few pathways affected at the different omics levels partly overlapped if patients are analyzed at the individual level, such as synaptogenesis (containing the SNARE complex) and cholesterol metabolism. Phosphoproteomics revealed increased STMN1(S38) phosphorylation as part of ERBB4 signaling. A pathway tool has been developed to visualize and compare different omics datasets per patient and showed potential therapeutic drugs, such as abobotulinumtoxinA (synaptogenesis) and afatinib (ERBB4 signaling). Afatinib is currently in clinical trials for GBM.ConclusionsA large variation on all omics levels exists between and within GBM patients. Therefore, it will be rather unlikely to find a drug treatment that would fit all patients. Instead, a multiomics approach offers the potential to identify affected pathways on the individual patient level and select treatment options.

Temporospatial genomic profiling in glioblastoma identifies commonly altered core pathways underlying tumor progression.

BackgroundTumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions.MethodsWhole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens.ResultsPrivate mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions.ConclusionsSubclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Pathological feature change and prognostic role of primary and local recurrent chordoma in skull base

Objective To investigate the pathological characteristics and changing trend in primary and local recurrent skull base chordoma, and to analyze their correlation with the outcomes. Methods A total of 89 samples of primary and local recurrent tumors pathologically confirmed from 38 patients who were treated in Neurosurgery Department of Beijing Tiantan Hospital, Capital Medical University between February 2005 and December 2014 were retrospectively enrolled into this self-control study. After HE staining, the pathological features such as nuclear atypia, mitosis, matrix ratio, necrosis, bone invasion and pathological subtypes were analyzed. The expressions of p53, Ki-67, EGFR, VEGFR and TRAF6 were detected by immunohistochemical staining. The changes of pathological indexes as above in primary and recurrence stages were compared. Cox analyses were used to analyze the effects of above pathological indexes on the progression-free survival (PFS) of patients with primary tumor, and the effects of the changes regarding those factors on the overall survival (OS). Results Ten cases (35.7%) of classical subtype changed to myeloid subtype, while none of chondroid chordoma changed to myeloid subtype after recurrence. Twelve cases (31.6%) demonstrated necrosis companied with tumor recurrence. Twenty-five cases (73.5%) of primary tumor showed high-expression of TRAF6, and 19 cases (50.0%) had decreased expression intensity of TRAF6 after tumor recurrence. There were 16 cases (42.1%) and 15 cases (39.5%) with upregulation of P53 and EGFR respectively. Necrosis (HR=7.1, 95% CI: 1.4-37.1, P=0.006), pathological subtype (HR=3.9, 95% CI: 1.2-7.2, P=0.040) and TRAF6≥3 grade (HR=0.2, 95% CI: 0.1-0.5, P<0.001) in primary tumors were directly related to PFS, and TRAF6≥3 grade (HR=0.2, 95% CI: 0.1-0.5, P<0.010) was an independent protective factor for PFS. There was no significant difference in the OS rate among the above pathological changes. Conclusions Classical chordoma shows malignant transformation to the myeloid subtype, which is not found in chondroid subtype. The expression of TRAF6 decreases gradually with tumor recurrence, and its high expression in primary tumor is an independent protective factor for PFS. Key words: Chordoma; Skull base neoplasms; Pathology; TNF receptor-associated factor 6; Prognosis

GENE-02. ESTABLISHING PERSONALIZED TREATMENT OPTIONS FOR RECURRENT HIGH-GRADE GLIOMAS

Abstract BACKGROUND High grade glioma (HGG) patients develop resistance to standard treatment leading to disease progression and limited life expectancy. Recent advances in the molecular characterisation of treatment-naïve HGGs based on next generation sequencing and DNA methylation analyses have led to a better delineation of HGG-subtypes and identification of distinct genomic abnormalities opening opportunities for personalized treatment strategies. METHODS We collected 300 fresh glioma specimen with approximately 100 longitudinal samples of initial and recurrent tumors from 43 matched patients. We succeeded in generating a live-biobank of HGG patient-derived orthotopic xenografts (PDOX) and 3D tumor organoids that neatly recapitulates the mutational spectrum including structural DNA variations and methylation-based subtypes of gliomas. A highlight is the generation of 19 PDOXs of paired initial and relapse HGGs from 9 glioma patients, enabling high-throughput drug screens. We performed comprehensive molecular profiling using arrayCGH, DNA-methylation and targeted DNA sequencing on patient specimen and their derivatives, 3D tumor organoids and PDOXs. RESULTS Detailed analysis of the paired longitudinal samples indicated that PDOXs closely recapitulate the evolutionary trajectory of the parental tumors. Furthermore, targeted genomic sequencing of paired HGGs suggests that relapse tumors also accumulate somatic mutations in epigenetic effectors. Based on patient-derived material we carried out drug response screening on 3D tumor organoids using a compound library matching the majority of genes that were assessed with targeted sequencing. Differential drug responses between initial and recurrent tumors were observed and the prevailing primary drug response profiles were essentially recapitulad in the relapse setting. CONCLUSIONS Response assessment of treatment-naïve gliomas and their recurrences provides crucial information on the differential sensitivity between initial and relapsed HGGs and offers novel personalized therapeutic options for the relapse setting. Furthermore, in depth correlation of the profiled somatic molecular landscape with drug response will enable pharmacogenomic predictions of potential inhibitors in the clinical setting.

INNV-41. PROSPECTIVE SELECTION OF RECURRENT GLIOBLASTOMA PATIENTS FOR TAILORED THERAPIES. RESULTS OF AN INSTITUTIONAL EXPERIENCE

Abstract INTRODUCTION Failure of clinical trials with targeted therapies in glioblastoma (GBM) is probably due to the enrollment of molecularly unselected patients. In preliminary studies, we prospectively selected recurrent GBM patients on the basis of molecular pattern and administered targeted therapy accordingly. This tailored approach gave encouraging results in term of low recurrence rate (RR) and high 6-month progression free survival (PFS-6). Here, we present the long-term results of our work. METHODS On recurrent tumor samples of 34 adult patients, we assessed the expression of VEGF and PTEN through immunohistochemistry and of EGFRvIII through RT-PCR. Patients with VEGF overexpression were treated with bevacizumab (10 mg/Kg i.v. every 2 weeks in 6-week cycles). Patients with EGFRvIII expression and normal PTEN expression added erlotinib (150 mg/day orally). Patients with loss of PTEN expression, irrespective of EGFRvIII status, added sirolimus (1–10 mg/day orally). RESULTS Sixteen patients received bevacizumab alone (bev), 14 bevacizumab plus erlotinib (bev+erl) and 4 bevacizumab plus sirolimus (bev+sir). RR was 50% in the whole cohort and 37.5%, 57.1% and 75% in bev, bev+erl and bev+sir groups, respectively. PFS-6 was 55.9% in the whole cohort and 50%, 64.3% and 50% in bev, bev+erl and bev+sir groups, respectively. Our data compare favorably with those from the large EORTC 26101 trial, which showed a RR of 41.5% and a PFS-6 lower than 30%. When considering sustained response (defined as PFS ≥ 12 months), we overall observed a 20.6% rate, with the highest value in bev+erl subgroup (28.6%). Interestingly, in EORTC 26101 trial, less than 10% of patients achieved PFS ≥ 12 months. CONCLUSIONS Our results confirm that the tailored approach in recurrent GBM provides an advantage in terms of RR, PFS and, above all, of long-term responses, compared with trials without molecular selection.

OS12.2 Targeting epigenetic pathways in the treatment of recurrent high-grade glioma

Abstract BACKGROUND High grade glioma (HGG) patients develop resistance to standard treatment leading to disease progression and limited life expectancy. Advances in the molecular characterisation of treatment-naïve HGGs based on next-generation sequencing and DNA methylation analyses have led to a better delineation of HGG subtypes and the identification of distinct genomic abnormalities. Furthermore, using large patient cohorts of longitudinal tumor samples, comprehensive genomic profiling studies emerged to investigate therapy-associated evolution of gliomas. All together, those studies point out the need for personalised treatment strategies, where applied drugs will be adapted to the unique patient-specific genetic abnormalities. MATERIAL AND METHODS We collected fresh samples of more than 800 brain tumors containing almost 300 glioma specimen with approximately 100 longitudinal samples of initial and recurrent tumors from 43 matched patients. By now, we have successfully established 34 patient-derived orthotopic xenografts (PDOXs) in mice. We performed comprehensive molecular profiling using array comparative genomic hybridisation, DNA methylation analysis and targeted DNA sequencing on patient specimen and their derivatives such as 3D tumor organoids and PDOXs. The custom-design sequencing panel comprises 234 genes that reflect both established genetic identifiers for individual glioma subtype classification and novel genes encoding mainly epigenetic effector genes. Based on patient-derived material we carried out drug response screening on 3D tumor organoids using a compound library matching the majority of genes that were assessed by targeted sequencing. RESULTS We succeeded in generating a live biobank of HGG patient-derived xenografts and 3D organoids that neatly recapitulates the mutational spectrum including structural DNA variation and methylation-based subtypes of gliomas. A highlight is the generation of 19 PDOXs of paired initial and relapse HGGs from a total of 9 glioma patients. A detailed analysis of the paired longitudinal samples indicated that PDOX models closely recapitulate the evolutionary trajectory of the parental tumors. Targeted sequencing of longitudinal HGG PDOXs suggests that relapse tumors accumulate somatic mutations in epigenetic effectors compared with the Initial. Differential drug responses between initial and relapse tumors were observed after screening of in vitro 3D tumor organoids. CONCLUSION Response assessment of naïve initial gliomas and recurrences provides crucial information on the differential sensitivity between initial and relapsed HGGs and offers novel personalised therapeutic options in the relapse setting. Furthermore, in depth correlation of the profiled somatic molecular landscape with drug response will enable pharmacogenomic predictions of potential inhibitors in the clinical setting.

Reduced Neoantigen Expression Revealed by Longitudinal Multiomics as a Possible Immune Evasion Mechanism in Glioma.

Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II-IV glioma patients (glioblastoma, IDH-wild-type, n = 8; grade II-III astrocytoma, IDH-mutant, n = 9; and grade II-III oligodendroglioma, IDH-mutant, 1p/19q-codeleted, n = 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the "neoantigen expression ratio," decreased significantly at recurrence (P = 0.003). This phenomenon was particularly pronounced for "high-affinity," "clonal," and "passenger gene-derived" neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumor-infiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.

MNGI-11. LONGITUDINAL GENOMIC ANALYSIS OF SPORADIC MENINGIOMAS WITH MULTIPLE RECURRENCES

Recently, genomic patterns and methylation profiles of meningiomas were described as prognostic factors for aggressiveness and recurrence (e.g. TERT, SMO and AKT1 mutations). However, the particular molecular profiles of recurrence and malignant progression of specific meningioma subtypes remain undefined. In this regard, longitudinal molecular studies are very interesting. In this study, we characterized somatic mutations in a cohort of sporadic meningioma patients. We included 9 patients with multiple recurrences and up to 11 subsequent resections of sporadic meningiomas. We performed a comparative whole-exome sequencing (WES). Moreover, we used the whole-exome data to predict the HLA MHC class I peptides of the subsequent tumor samples in silico. Our study included patients with follow-up times accounted for up to 26 years (average: 12.6 years, range 3–26). We detected a TRAF7 mutation (c.1189G>C) in only 2 of 6 recurrent tumor samples of a female patient with a malignant progression from a WHO I to a WHO grade II meningioma. In a male patient with 9 analyzed meningioma tumor samples, recurrent somatic PLEKHG5, AGBL1, ALK, RICTOR, DEPDC5 mutations were detected. This individual pattern remained constantly evident under different treatments, even after systemic treatment with the mTOR inhibitor everolimus. Moreover, unique mutations in HLA-DQA1, OR14J1, PRH1 genes occurred after mTOR inhibition in this male patient. Furthermore, we identified somatic mutations in the tumor suppressor candidate NBPF1 in 7 of 9 patients. HLA allele types were predicted in silico, with A*02:01, B*57:01, C*03:03, C07:01 and B51:01 being most frequent. Neither ubiquitously shared somatic mutations nor shared MHC class I peptides were identified among this cohort. Taken together, our study outlines a heterogeneous molecular profile in this analysis. Since longitudinal samples in sporadic meningioma are rather rare, the ICOM consortium would be an ideal frame for studying the molecular profile of recurrence in meningioma in larger cohorts.

L1CAM Immunopositivity in Anaplastic Supratentorial Ependymomas: Correlation With Clinical and Histological Parameters.

Supratentorial ependymomas (ST EPNs) are molecularly characterized, of which the RELA fusion positive tumors are the most common and aggressive subgroup. Moreover, histologically, anaplastic ST EPN (ST-AE) often mimic other central nervous system primary high-grade tumors resulting in a diagnostic dilemma. We aimed to study a cohort of ST-AE; evaluate the expression of two RELA fusion-associated markers-L1CAM and p65 (NF-κB); and correlate their expression with clinical and histological parameters. Cases of ST-AE diagnosed in our department from January 2011 to June 2016 (n = 72) were reviewed. A battery of immunohistochemical markers was employed. A total of 65 confirmed ST-AE were included in the study. Age ranged from 9 months to 60 years. There was a slight predominance in the pediatric population (57%). Male-to-female ratio was 1:1.16. Histomorphological features were varied and mimicked other high-grade tumors in several cases. L1CAM immunopositive tumors constituted 26% of cases and were predominantly seen in young children, in the frontoparietal location, and exhibited clear cell morphology with calcification. A consistent pattern of L1CAM immunopositivity was noted in paired primary and recurrent tumor samples. Our study portrays the varied clinical and histomorphological spectrum of ST-AE. The study emphasizes the association of L1CAM immunopositivity with a wide spectrum of histological parameters, literature on which is scant till date. Since ST EPN-RELA are tumors with aggressive behavior, such a correlation would be clinically relevant, particularly when there is limited access to molecular testing.

A novel enhancer regulates MGMT expression and promotes temozolomide resistance in glioblastoma

Temozolomide (TMZ) was used for the treatment of glioblastoma (GBM) for over a decade, but its treatment benefits are limited by acquired resistance, a process that remains incompletely understood. Here we report that an enhancer, located between the promoters of marker of proliferation Ki67 (MKI67) and O6-methylguanine-DNA-methyltransferase (MGMT) genes, is activated in TMZ-resistant patient-derived xenograft (PDX) lines and recurrent tumor samples. Activation of the enhancer correlates with increased MGMT expression, a major known mechanism for TMZ resistance. We show that forced activation of the enhancer in cell lines with low MGMT expression results in elevated MGMT expression. Deletion of this enhancer in cell lines with high MGMT expression leads to a dramatic reduction of MGMT and a lesser extent of Ki67 expression, increased TMZ sensitivity, and impaired proliferation. Together, these studies uncover a mechanism that regulates MGMT expression, confers TMZ resistance, and potentially regulates tumor proliferation.

Abstract 3695: Mapping of molecular landscape underlying drug resistance and recurrence in glioblastoma: Paired analysis of primary and recurrent tumors

Abstract Background: Glioblastoma (GBM) is the most frequent and the most malignant brain tumor in adults. Despite aggressive therapy, overall survival of GBM patients remains poor due to intrinsic or acquired resistance to combined radiation and chemotherapy. Underlying biology behind GBM progression after first line therapy is mostly unknown and thus, there are only limited targeted therapeutic options for the 2nd line treatment. The aim of this study was to investigate genetic landscape in paired samples of primary versus recurrent tumors and to validate prognostic and predictive value of the most significant differences in independent cohort of GBMs. Patients and methods: 43 paired tumor samples were obtained during the 1st surgery followed by radiochemotherapy and after tumor recurrence during the 2nd palliative surgery. 24/43 paired samples were analyzed for whole genome copy number variations (CNV), 23/43 by fluorescent in situ hybridization for determination of EGFR, p53, RB1, MDM2, CDKN2A, 1p, 19q and 10p statuses. Methylation-specific PCR was used for analysis of MGMT promoter methylation and competitive amplification of differentially melting amplicons PCR for IDH1/2 mutations detection. The impact of most relevant alterations identified in paired GBMs was then validated in independent cohort of unpaired 104 tumors. Results: The most significant differences induced by chemoradiotherapy and/or GBM progression in primary versus recurrent tumors were EGFR amplification (19/24 primary tumors, 16/24 recurrent tumors), CDKN2A loss (18/24 primary tumors, 16/24 recurrent tumors), MDM2 gain (4/24 primary tumors), PDGFRA gain (3/24 recurrent tumors) and GSTT1 loss in primary tumors (7/24) and GSTM1 gain in recurrent tumors (7/24). Univariate analysis of overall survival for significant copy number alterations shown significantly worse prognosis for patients with GSTT1 deletion (HR = 5.356, CI = 1.161-24.701, p = 0.031) in primary tumor and trend for better prognosis in patients with CDKN2A deletion (HR = 0.391, CI = 0.124-1.229, p = 0.108) and GSTM1 amplification (HR = 0.341, CI = 0.090-1.288, p = 0.112) in recurrent tumors. Significantly worse prognosis for GBM patients with aberrations of GSTT1 gene was also confirmed in independent validation cohort of unpaired GBMs in multivariate analysis adjusted for age and Karnofsky score (gain: HR = 1.95, CI = 1.116-3.260, p = 0.011; loss: HR = 2.15, CI = 1.185-3.900, p = 0.012). Conclusion: Molecular genetic profiling of 24 recurrent GBM showed association between recurrence and status of glutathione S-transferases genes. Significance of GSTT1 gene aberration was validated in independent group of glioblastomas and indicates involvement of redox enzymes in GBM chemoradiation response and tumor progression. Acknowledgment: The financial support of grants TE02000058, IGA UP LF_2017_013 is gratefully acknowledged. Citation Format: Magdalena Houdova Megova, Zuzana Sporikova, Radek Trojanec, Ondrej Kalita, Jana Vrbkova, Jiri Drabek, Vladimira Koudelakova, Marian Hajduch. Mapping of molecular landscape underlying drug resistance and recurrence in glioblastoma: Paired analysis of primary and recurrent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3695.

Abstract 2562: Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4

Abstract Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922). Citation Format: Joana Senra, Pamela Villalobos, Barbara Mino, Luisa Solis, Carmen Behrens, Joseph P. Sanderson, Manoj Saini, Andrew B. Gerry, Nicholas J. Pumphrey, Miguel Maroto, Boris Sepesi, Patrick Hwu, David S. Hong, Elizabeht A. Mittendorf, George S. Blumenschein, Ignacio I. Wistuba, Gwendolyn Binder-Scholl, Rafael Amado. Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2562.

Abstract 213: Exome sequencing of paired primary and relapsed small cell lung cancers reveals increased copy number aberration complexity to be associated with disease relapse

Abstract Introduction: Although small-cell lung cancer (SCLC) is sensitive to first-line radiation and chemotherapy, nearly all patients recur often with treatment-resistant disease. Therefore, delineating subclonal architecture and molecular evolution of SCLC after treatment by comparing genomic profiles of recurrent and paired treatment-naïve tumors may provide new insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Treatment-naïve and paired recurrent tumor samples from 9 patients with limited-stage SCLC treated with concurrent chemoradiation (CCRT) underwent whole exome sequencing (WES) and data is currently available for 6 pairs. All 6 patients had disease recurrence more than 3 months after finishing treatment. Genomic landscape including somatic mutations, somatic copy number aberrations (SCNA), subclonal architecture and transversion rate (percentage of base changes from pyrimidine to purine and vice-versa) were compared between treatment-naïve and paired recurrent tumor samples. Circulating cell-free DNA (cfDNA) collected at the time of recurrence was also subject to deep sequencing of 461 cancer genes. Results: SCNA analyses revealed significantly more complex chromosomal gains in relapsed SCLCs compared to pre-treatment primary tumors (7.7% vs 13.2% genes with copy number gain in primary and relapsed SCLC respectively, p=0.034). As expected, TP53 and RB1 were the most commonly mutated genes (83% and 67% respectively) and tobacco exposure related signatures were predominant in all samples. Primary and relapsed tumor pairs showed very similar genomic landscape including tumor mutational burden (11.8/MB in primary SCLC vs 9.7/MB in relapsed tumors), intra-tumor heterogeneity complexity (MATH score 38.2 in primary vs 39.2 in relapse) and transversion rates, that have been reported to be associated with alkylating-agent treatment (62% in primary vs 65% in relapse). An average of 72.25% (range: 53-86%) somatic mutations, including all canonical cancer gene mutations, were shared between primary SCLC and relapsed tumors. In addition, the subclonal architecture was also very similar between treatment-naïve primary SCLC and paired relapsed tumors. Of note, 5 out of 6 patients achieved disease control on retreatment with chemotherapy. Conclusion: Copy number gains may play a role in post-CCRT relapse of SCLC. Paired pre-treatment and relapse samples show genomic similarities which could account for benefit to subsequent treatment. Additional sample analysis and ctDNA assessment is ongoing to better address these findings, especially in patients with chemoresistant relapse. These findings also suggest that other molecular changes involving gene expression (eg. methylation) may play a role in SCLC relapse and resistance. Citation Format: Marcelo V. Negrao, Ming Tang, Ying Jin, Yamei Chen, Xiao Hu, Huarong Tang, Haimiao Xu, Kelly Quek, Jianhua Zhang, Xizeng Mao, Xingzhi Song, John V. Heymach, Ignacio Wistuba, Lauren A. Byers, Bonnie S. Glisson, Andrew Futreal, Ming Chen, Jianjun Zhang. Exome sequencing of paired primary and relapsed small cell lung cancers reveals increased copy number aberration complexity to be associated with disease relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 213.