Non-amplified Tumors Research Articles

NIMG-34. QUANTITATIVE MR PARAMETERS FOR THE PREDICTION OF EGFR AMPLIFICATION AND THE TERT PROMOTER MUTATION STATUS OF IDH-WILDTYPE LOWER-GRADE GLIOMAS

Abstract OBJECTIVES To assess the predictive role of quantitative MRI parameters from DTI, dynamic susceptibility contrast (DSC), and dynamic contrast-enhanced (DCE) imaging in determining the epidermal growth factor receptor (EGFR) amplification and telomerase reverse transcriptase promoter (TERTp) mutation status of isocitrate dehydrogenase-wildtype (IDHwt) lower-grade gliomas (LGGs). METHODS A total of 49 patients with IDHwt LGGs with either known EGFR amplification (39 EGFR non-amplified, 10 EGFR-amplified) or TERTp mutation (19 TERTp wildtype, 21 TERTp mutant) statuses underwent preoperative MRI. The mean apparent diffusion coefficient (ADC), fractional anisotropy (FA), normalized cerebral blood volume (nCBV), normalized cerebral blood flow (nCBF), volume transfer constant (Ktrans), rate transfer coefficient (Kep), extravascular extracellular volume fraction (Ve), and plasma volume fraction (Vp) values were assessed. Univariate and multivariate logistic regression models were constructed to predict EGFR amplification and TERTp status. RESULTS EGFR-amplified tumors showed lower mean ADC values than EGFR non-amplified tumors (p = 0.019). Mean ADC was an independent predictor of EGFR amplification, with an AUC of 0.75. TERTp mutant tumors showed higher mean nCBV (p = 0.020), higher mean nCBF (p = 0.017), and higher mean Vp (p = 0.002) than TERTp wildtype tumors. With multivariate logistic regression, mean Vp was the only independent predictor of TERTp mutation status, with an AUC of 0.85. CONCLUSION Lower ADC values may be a useful marker for the prediction of EGFR amplification, whereas higher Vp values may be a useful marker for the prediction of the TERTp mutation status of IDHwt LGGs.

The Tumor Microenvironment in Neuroblastoma: New Players, New Mechanisms of Interaction and New Perspectives.

Simple SummaryNeuroblastoma is the second most common solid tumor in children. Our understanding of the contribution of genetic factors (seed) that contribute to neuroblastoma progression has substantially improved in the last 2 decades but the contribution of the tumor microenvironment (TME, soil) is the subject of more recent attention. Here we highlight recent studies pointing to novel mechanisms by which the TME affects neuroblastoma progression. Cancer-associated fibroblasts are present in neuroblastoma tumors and contribute to escape from chemotherapy and immunotherapy. Extracellular vesicles and regulatory micro-RNAs they contain, serve as shuttle mechanisms between neuroblastoma cells and stromal cells. The TME landscape of neuroblastoma differs between MYCN amplified and MYCN-non amplified tumors with the former being “cold” and the latter “hot” and rich in inflammatory cells. These recent observations will have a significant impact on our ability to design precise clinical trials that integrate information on the neuroblastoma cells and on their TME.The contribution of the tumor microenvironment (TME) to cancer progression has been well recognized in recent decades. As cancer therapeutic strategies are increasingly precise and include immunotherapies, knowledge of the nature and function of the TME in a tumor becomes essential. Our understanding of the TME in neuroblastoma (NB), the second most common solid tumor in children, has significantly progressed from an initial focus on its Schwannian component to a better awareness of its complex nature, which includes not only immune but also non-immune cells such as cancer-associated fibroblasts (CAFs), the contribution of which to inflammation and interaction with tumor-associated macrophages (TAMs) is now recognized. Recent studies on the TME landscape of NB tumors also suggest significant differences between MYCN-amplified (MYCN-A) and non-amplified (MYCN-NA) tumors, in their content in stromal and inflammatory cells and their immunosuppressive activity. Extracellular vesicles (EVs) released by cells in the TME and microRNAs (miRs) present in their cargo could play important roles in the communication between NB cells and the TME. This review article discusses these new aspects of the TME in NB and the impact that information on the TME landscape in NB will have in the design of precise, biomarker-integrated clinical trials.

Somatic structural variation targets neurodevelopmental genes and identifies SHANK2 as a tumor suppressor in neuroblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Like many childhood cancers, neuroblastoma shows a relative paucity of somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) compared to adult cancers. Here, we assessed the contribution of somatic structural variation (SV) in neuroblastoma using a combination of whole-genome sequencing (WGS) of tumor-normal pairs (n = 135) and single-nucleotide polymorphism (SNP) genotyping of primary tumors (n = 914). Our study design allowed for orthogonal validation and replication across platforms. SV frequency, type, and localization varied significantly among high-risk tumors. MYCN nonamplified high-risk tumors harbored an increased SV burden overall, including a significant excess of tandem duplication events across the genome. Genes disrupted by SV breakpoints were enriched in neuronal lineages and associated with phenotypes such as autism spectrum disorder (ASD). The postsynaptic adapter protein-coding gene, SHANK2, located on Chromosome 11q13, was disrupted by SVs in 14% of MYCN nonamplified high-risk tumors based on WGS and 10% in the SNP array cohort. Expression of SHANK2 was low across human-derived neuroblastoma cell lines and high-risk neuroblastoma tumors. Forced expression of SHANK2 in neuroblastoma cells resulted in significant growth inhibition (P = 2.6 × 10−2 to 3.4 × 10−5) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P = 3.1 × 10−13 to 2.4 × 10−30). These data further define the complex landscape of somatic structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis in this childhood cancer.

Abstract 5899: Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons

Abstract Introduction: SLIMamp technology allows multiplex-PCR of tiled amplicons in a single tube, which enables targeting of large exons for NGS analysis with a streamlined process. Copy number variants (CNVs) are DNA segments that are present with variation in the number of copies compared to a normal genome. CNVs have a high prevalence in the pathogenesis of cancer and their characterization is important to acquire a more comprehensive picture of the mutations present in a patient sample. Pillar developed a proprietary CNV algorithm and combined it with SLIMamp to develop an integrated multi-cancer plus CNV detection NGS panel which identifies CNVs in the ERBB2, EGFR, MET, and MYC genes. Methods: To assess the ability of the ONCO/Reveal Multi-Cancer with CNV Panel to detect ERBB2 CNV compared to DISH (dual in situ hybridization), libraries were created from 44 well-characterized FFPE breast cancer samples plus 3 Genome in a Bottle (GIAB) samples. 23 tumor samples had known ERBB2 amplification (20 with matched normal tissue), 21 were known non-amplified tumors, and 3 GIAB samples were used as negative controls. Libraries were sequenced on an Illumina MiSeq and data was analyzed by the Pillar Variant Analysis Toolkit (PiVAT). The previous DISH results were not known to Pillar. Results were provided to an independent collaborator who unblinded the study and compared the NGS results to DISH. Results: Overall the assay performed well with a mapping rate of 99.4% ± 2.1%, on-target rate of 99.1% ± 0.4%, and coverage uniformity >0.2x mean coverage of 93.5% ± 4.1%. With respect to CNV calling, the assay and software detected 100% (21/21) of ERBB2 amplification negative samples as confirmed by DISH. For amplification positive samples, the NGS assay detected 100% (23/23) of positive samples. The software correctly identified 91% (21/23) of amplification positive samples. The two samples that were not called by PiVAT had normalized genes counts of 1.2 and 1.4, and also had the lowest DISH scores. All samples with a normalized gene count >1.5 were correctly called by PiVAT. The NGS assay was able to accurately call CNV independent of tumor cellularity with tumor content of 20%-90% not impacting the CNV call. Conclusions: The ONCO/Reveal Multi-Cancer with CNV Panel is a robust assay for the detection of CNVs, SNVs, and Indels of interest across multiple solid tumor cancer types. The workflow is streamlined, with same day loading of finished libraries when starting from as little as 5ng of isolated input DNA. The assay and software demonstrate detection of low CNV with the recommended cutoff set at 1.5. With further optimization of the calling algorithm accurate calling of even lower CNVs may be possible. The detection and identification of CNVs along with other mutations gives a more comprehensive overview of the mutations present in a sample, supporting better clinical management of patients. Citation Format: Nicholas Lodato, Zonghan Liu, Akuah Kontor, Xiaoxi Wu, Lukas Hillmer, Sean Polvino, Yue Ke, Erin Petrilli, Adam Labonte, Geoffrey Richman, Zhaohui Wang. Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5899.

Abstract 1293: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma

Abstract MYCN genomic amplification is one of the risk factors in neuroblastoma. 13-cis retinoic acid (13-cisRA), a differentiating agent, down-regulates MYCN protein and is part of neuroblastoma maintenance therapy. Despite the improvement in clinical outcome with 13-cisRA, anti-GD2 monoclonal antibody plus cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients still die of recurrent disease. Although MYC genomic amplification is rare in neuroblastoma (~1%), 11% of neuroblastoma primary tumors collected at diagnosis (Dx) have high c-MYC protein suggesting that MYC transcriptional activation rather than its gene amplification drives such tumors. Here, we sought to investigate the role of MYC oncogene in progressive disease (PD) and to molecularly characterize mechanisms of MYC expression in neuroblastoma. We report transcriptional activation of MYC medicated by the OCT4 (encoded by POU5F1), functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma. In large panels of neuroblastoma patient-derived cell lines (19 Dx and 16 PD) and patient-derived xenograft PDX models (8 Dx and 9 PD), we confirmed that c-MYC expression levels were higher in PD relative to Dx lines (P = 0.0005). We identified OCT4 and TCF3 as transcription factors highly expressed in neuroblastoma cells with high c-MYC. Subsequently, we confirmed two novel OCT4-binding sites (including OBS1 and OBS2) located in the MYC promoter/enhancer region: -1209 to -1140 and found that OCT4 NH2-terminal domain (NTD) and POU specific domain (POUs) are critical for MYC transcriptional activation. To identify kinases that is associated with OCT4-induced c-MYC activation, we used mass spectrometry and PhosphoMotif Finder® and identified MAPKAPK2 (MK2) as one of the upstream kinases that can bind to and directly regulate the OCT4 biological function by phosphorylation at its amino acid Ser111 residue to transcriptionally activate MYC expression. The data in 175 MYCN non-amplified high-risk primary tumors (TARGET database) showed that MAPKAPK2 positively correlated with MYC expression (P < 0.001) and overall survival was lower (P < 0.001) for patients with high MAPKAPK2. Also, OCT4, MK2, and c-MYC were higher in PD relative to Dx neuroblastomas models. Functional studies by gene knockdown of the POU5F1 or MAPKAPK2 using shRNAs showed decreased c-MYC expression, inhibition of cell proliferation, and restoring neurite outgrowth in response to 13-cisRA. In conclusion, high c-MYC independent of genomic amplification, not MYCN amplification, is associated with disease progression in neuroblastoma. The MK2-mediated OCT4 transcriptional activation is a novel mechanism for MYC activation in PD neuroblastoma and provides a potential novel therapeutic target. Citation Format: Sung Jen Wei, Thinh H. Nguyen, Dustin G. Mook, Monish R. Makena, Dattesh Verlekar, Ashly Hindle, Gloria Martinez, Shengping Yang, Hiroyuki Shimada, C. Patrick Reynolds, Min H. Kang. MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1293.

Amplification of KRAS and its heterogeneity in non-Asian gastric adenocarcinomas

BackgroundGastric cancer is one of the deadliest cancer entities worldwide. While surgery is the only curative treatment option in early tumors, for locally advanced and metastatic patients further therapeutic targets are needed. Several studies not only reported mutations but also amplifications of the KRAS locus in different cancer entities. More recently, KRAS amplification was discussed as a new therapeutic target. Little is known about the (prognostic) relevance and (heterogenic) distribution of KRAS amplification in gastric adenocarcinomas, especially in Non-Asian patients.MethodsAmplification of the KRAS locus and corresponding protein expression was analyzed in 582 gastric adenocarcinomas employing fluorescence in-situ hybridization (FISH) and immunohistochemistry. Amplification status was correlated with clinico-pathological features, clinical outcome and molecular tumor data including a correlation to the TCGA subtypes of gastric carcinoma.ResultsKRAS amplification was detected in 27 out of 470 analysable tumors (5.7%) and correlated with protein expression of KRAS in all amplified tumors. Within the KRAS amplified gastric tumors 14/27 (51.9%) showed a heterogeneous distribution with also KRAS non-amplified tumor parts. According to TCGA 24 tumors (88.8%) were related to chromosomal instable tumors (CIN). The survival analysis of the entire patient cohort did not show any difference in overall survival in dependence on the KRAS status. However, a significant survival difference with a worse outcome for patients with KRAS amplified tumors was identified when analysing patients without neoadjuvant pre-treatment.ConclusionsWe confirm the unfavorable prognosis of KRAS amplified tumors reported by other studies in (Asian) patient groups, at least in patients without neoadjuvant pre-treatment. Within KRAS amplified tumors we revealed intratumoral heterogeneity that may define a (more aggressive) tumor cell population which is more frequently observed in patients with lymph node metastases. Despite the heterogeneous distribution of KRAS amplified tumor clones, KRAS amplified locally advanced or metastasized gastric adenocarcinomas represent a therapeutically highly relevant tumor subgroup.

Phase I Dose-Escalation and -Expansion Study of Telisotuzumab (ABT-700), an Anti–c-Met Antibody, in Patients with Advanced Solid Tumors

This first-in-human phase I study evaluated the pharmacokinetics, safety, and preliminary efficacy of telisotuzumab, formerly called ABT-700, an antagonistic antibody directed against c-Met. For dose escalation (3+3 design), 3 to 6 patients with advanced solid tumors were enrolled into four dose cohorts (5-25 mg/kg). In the dose-expansion phase, a subset of patients was prospectively selected for MET amplification (FISH screening). Patients received telisotuzumab intravenously on day 1 every 21 days. For dose expansion, 15 mg/kg was chosen as the dose on the basis of safety, pharmacokinetics, and other data from the escalation cohorts. Forty-five patients were enrolled and received at least one dose of telisotuzumab (dose escalation, n = 15; dose expansion, n = 30). Telisotuzumab showed a linear pharmacokinetics profile; peak plasma concentration was proportional to dose level. There were no acute infusion reactions and no dose-limiting toxicities were observed. The most common treatment-related adverse events included hypoalbuminemia (n = 9, 20.0%) and fatigue (n = 5, 11.1%). By Response Evaluation Criteria In Solid Tumors (RECIST), 4 of 10 (40.0%) patients with MET-amplified tumors had confirmed partial response in target lesions (one ovarian, two gastric, and one esophageal), two (20.0%) had stable disease, three (30.0%) had progressive disease; one patient was unable to be evaluated. Among patients with nonamplified tumors (n = 35), no objective responses were observed; however, 11 patients had stable disease per RECIST criteria. In conclusion, telisotuzumab has an acceptable safety profile with clinical activity observed in patients with MET-amplified advanced solid tumors.

Impact of MYCN status on response of high-risk neuroblastoma to neoadjuvant chemotherapy

Background/PurposeMYCN-amplification in neuroblastoma is associated with an aggressive clinical phenotype. We evaluated the association of MYCN amplification with tumor response to neoadjuvant chemotherapy. MethodsPrimary tumor response, assessed by percentage volume change on CT scan and degree of tumor resection, assessed by the operating surgeon, were retrospectively compared in 84 high-risk neuroblastoma patients. There were thirty-four (40%) with MYCN-amplified tumors and fifty (60%) with non-amplified tumors treated at our institution from 1999 to 2016. Metastatic disease response was assessed on MIBG scan by change in Curie score. ResultsMYCN-amplification was associated with a greater mean percentage reduction in primary tumor volume after neoadjuvant chemotherapy (72.27% versus 46.83% [non-amplified tumors], p = 0.001). The percentage of patients with a Curie score > 2 at diagnosis who then had a score ≤ 2 after neoadjuvant chemotherapy was not significantly different (8 [61.5%] and 8 [34.8%], respectively, p = 0.37). Twenty-eight (85.7%) patients with MYCN-amplification had ≥90% surgical resection compared to 45 (91.84%) patients with non-amplified tumors (p = 0.303). ConclusionsMYCN-amplification in high-risk neuroblastoma was associated with a better response of the primary tumor to neoadjuvant chemotherapy, but not metastatic sites, than in patients with non-amplified tumors. This did not significantly impact the ability to resect ≥90% of the primary tumor/locoregional disease. Type of StudyTreatment Study Level of EvidenceLevel III

Parathyroid hormone-like hormone plays a dual role in neuroblastoma depending on PTH1R expression.

We have previously reported the expression of parathyroid hormone‐like hormone (PTHLH) in well‐differentiated, Schwannian stroma‐rich neuroblastic tumors. The aim of this study was to functionally assess the role of PTHLH and its receptor, PTH1R, in neuroblastoma. Stable knockdown of PTHLH and PTH1R was conducted in neuroblastoma cell lines to investigate the succeeding phenotype induced both in vitro and in vivo. Downregulation of PTHLH reduced MYCN expression and subsequently induced cell cycle arrest, senescence, and migration and invasion impairment in a MYCN‐amplified, TP53‐mutated neuroblastoma cell line. These phenotypes were associated with reduced tumorigenicity in a murine model. We also show that PTHLH expression is not under the control of the calcium‐sensing receptor in neuroblastoma. Conversely, its production is stimulated by epidermal growth factor receptor (EGFR). Accordingly, irreversible EGFR inhibition with canertinib abolished PTHLH expression. The oncogenic role of PTHLH appeared to be a consequence of its intracrine function, as downregulation of its receptor, PTH1R, increased anchorage‐independent growth and induced a more undifferentiated, invasive phenotype. Respectively, high PTH1R mRNA expression was found in MYCN nonamplified primary tumors and also significantly associated with other prognostic factors of good outcome. This study provides the first evidence of the dual role of PTHLH in the behavior of neuroblastomas. Moreover, the identification of EGFR as a transcriptional regulator of PTHLH in neuroblastoma provides a novel therapeutic opportunity to promote a less aggressive tumor phenotype through irreversible inhibition of EGFR tyrosine kinase activity.

Cyclin D1 differential activation and its prognostic impact in patients with advanced breast cancer treated with trastuzumab

IntroductionWe sought to determine the level of activation of the critical components of the cyclin D1-mediated pathway and to evaluate their prognostic significance across the different molecular subtypes of advanced breast cancer.Patients and methodsThe study population comprised 219 female patients with advanced breast cancer who had been found to have human epidermal growth factor receptor 2 (HER2)-positive disease by local testing and were all treated with trastuzumab-based regimens. For all tumours, central testing for HER2 was performed, and cyclin D1 gene (CCND1) amplification, mRNA and protein expression were assessed by FISH, quantitative real-time-PCR and immunohistochemistry, respectively. Prognostic impact on clinical endpoints was evaluated with Cox regression analyses.ResultsAfter central testing, only 134 (61.2%) of 219 patients were confirmed to have HER2 gene amplification by FISH and/or 3+ HER2 protein expression by immunohistochemistry. After a median follow-up time of 136.0 months (95% CI 123.3 to 148.9), 105 (78.4%) HER2-positive patients and 76 (89.4%) HER2-negative patients had died, while 80% of the former and 87.1% of the latter had experienced a disease relapse. Patients with positive oestrogen receptor/progesterone receptor status presented with higher cyclin D1 mRNA expression. In the HER2-negative subgroup, patients with negative cyclin D1 protein expression were at higher risk of progression (HR= 1.66, 95%CI 1.01 to 2.72, Wald’s p=0.045). Among de novo metastatic patients, the risk of progression was higher for patients with non-amplified CCND1 tumours (HR= 2.00, 95% CI 1.03 to 3.90, p=0.041).ConclusionAberrant activation of the cyclin D1-mediated pathway appears to reduce the risk of progression in HER2-negative tumours, but not in HER2-positive ones.

The long-term prognostic and predictive capacity of cyclin D1 gene amplification in 2305 breast tumours

BackgroundUse of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses.MethodsCCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes.ResultsWhen combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15–2.46), luminal B (1.37; 1.01–1.86) and ER+/LN−/HER2− (1.66; 1.14–2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours.ConclusionsOur results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN−/HER2−, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Abstract OT2-07-06: Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies

Abstract Antibody-Coupled T cell Receptor (ACTR) is an autologous engineered T cell therapy developed to combine with tumor-targeting antibodies to exert potent anti-tumor immune responses and tumor cell killing. The ACTR construct is composed of the extracellular domain of CD16 fused to CD3ζ signaling and T cell co-stimulatory domains. ACTR-expressing T cells are universal in that they can be paired with a therapeutic antibody to target specific antigens on tumors. Unum has two ACTR constructs, ACTR087 and ACTR707, currently in clinical testing. ACTR087 and ACTR707 are being tested in combination with rituximab in subjects with CD20+ B cell lymphoma in two separate trials (NCT02776813 and NCT03189836, respectively). Preliminary data with ACTR087 + rituximab has demonstrated clinical proof-of-concept and a dose-response relationship in subjects with relapsed/refractory B cell lymphoma. ACTR087 is also being tested in combination with a novel BCMA-targeting antibody in subjects with multiple myeloma (NCT03266692). While T cell therapies, such as chimeric antigen receptor (CAR) T cells, have demonstrated clinical activity in hematological cancers, the therapeutic potential of this approach has yet to be established in solid tumors. Challenges associated with targeting solid tumors with CAR-T cells include tumor antigen heterogeneity and antigen expression on normal tissues. HER2 is a well-established therapeutic target that is over-expressed in a number of cancer indications. HER2 is also expressed at low levels on normal epithelial cells, creating a risk for on-target/off-tumor toxicities of HER2-targeted CAR-T cells. Here we present nonclinical studies demonstrating that ACTR T cells in combination with trastuzumab have antigen density-dependent activity on HER2-expressing tumor cell lines, while trastuzumab-based CAR-T cells do not. We observed that ACTR + trastuzumab had robust activity against HER2-amplified tumor cells and more modest activity against non-amplified tumor cells, whereas HER2-targeting CAR-T cells had comparable activity against HER2-amplified and non-amplified tumor cells. On normal human primary cells, ACTR + trastuzumab had minimal activity in comparison to HER2 CAR-T cells, suggesting that ACTR + trastuzumab may exhibit a superior clinical therapeutic index. Furthermore, the activity of ACTR T cells against HER2-amplified tumor cells was titratable with antibody concentration, allowing for control of ACTR activity by modulation of trastuzumab concentration. Together, these data demonstrate the specificity of the ACTR T cell therapeutic approach to target HER2-amplified tumors and support clinical testing in combination with trastuzumab. A phase 1, multicenter, single-arm, open-label dose escalation study, ATTCK-34-01, is proposed to evaluate ACTR T cells in combination with trastuzumab in subjects with advanced HER2-positive malignancies. The primary study objectives are to assess the safety and tolerability of the combination, and to define the recommended phase 2 dose combination for further study. Additional objectives include assessment of anti-tumor activity, ACTR T cell persistence and trastuzumab pharmacokinetics. Enrollment is expected to commence in early 2019. Citation Format: O'Callaghan KM, Shin J, Cheung AS, Cheema T, Judge C, Ranger A, Huet HA, Ettenberg SA, Sachs J, Vasconcelles M, Motz G. Antibody-coupled T cell receptor (ACTR) engineered autologous T cells in combination with trastuzumab for the treatment of HER2-positive malignancies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-07-06.

Frequent ESR1 and CDK Pathway Copy-Number Alterations in Metastatic Breast Cancer.

DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single base-pair mutations in the estrogen receptor (ESR1). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets (>15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER+ tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased ESR1 CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. ESR1-amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No ESR1 CN amplifications and only one gain was identified in ER- tumors. ESR1 copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included ERBB2, GRB7, and cell-cycle pathway members CCND1 and CDK4/6, which showed mutually exclusivity with deletions of CDKN2A, CDKN2B, and CDKN1B. IMPLICATIONS: Copy-number alterations of ESR1 and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.

Differential regulation of human monocytes and NK cells by antibody-opsonized tumors.

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa-, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.

Clinical significance of MYCN amplification in patients with high-risk neuroblastoma.

This study investigated the clinical significance of MYCN amplification within high-risk neuroblastoma (NB). Medical records of 135 patients who were diagnosed with high-risk NB from 2004 to 2016 were reviewed. Fifty-one (38%) patients had MYCN amplified tumors, and the remaining 84 (62%) had nonamplified tumors. MYCN amplification was associated with abdominal primary site, less differentiated pathology, higher levels of lactate dehydrogenase and neuron-specific enolase (NSE), lower vanillylmandelic acid level, and larger primary tumor volume at diagnosis. MYCN amplification was associated with a better early response (faster reduction of primary tumor volume and NSE level). The proportion of patients in complete response or very good partial response after induction treatment was relatively higher in MYCN amplified tumors than in nonamplified tumors; however, all progressions during induction treatment occurred only in MYCN amplified tumors (P=0.007). The time to progression was shorter (median 1.5 years vs. 1.9 years, P=0.037) and survival after relapse/progression was worse in MYCN amplified tumors (3 year overall survival: 7.7 ± 7.4%vs. 20.5 ± 8.8%, P=0.046). There was no difference in event-free survival and overall survival between MYCN amplified and nonamplified tumors. MYCN amplification was associated with more aggressive features at diagnosis and a better early response, but a higher progression rate during induction treatment and lower chance of survival after relapse/progression. There was no difference in survival rates according to MYCN amplification in patients with high-risk NB.

Abstract P5-05-03: Obesity drives breast cancer progression through estrogen dependent and independent mechanisms

Abstract Background: More than 200,000 women are diagnosed with breast cancer each year and 75% develop estrogen receptor positive (ER+) tumors. Obesity is an independent risk factor for the development of ER+ breast cancer, particularly after menopause, and affects 40% of US women. Obese women are more likely to be diagnosed with advanced tumors, lymph node involvement, and less likely to respond to endocrine therapy versus lean women. Mechanisms underlying the increased risk and worse prognosis of obese women are poorly understood. The dogma in the field states that estrogen production is the main contributor to obesity-associated ER+ breast cancer. We show that this is not always the case. Given the epidemic proportions of obesity in the US, we need better pre-clinical models that will inform focused clinical trials and interventions for patients. In this study, we describe a novel mouse model of obesity and ER+ breast cancer patient-derived xenografts (PDX). Our studies highlight the heterogeneity of responses within the ER+ breast cancer subtype to the obese environment and implicate both estrogen-dependent and independent mechanisms of obesity-associated tumor progression. Methods: ER+, FGFR1-amplified or non-amplified human tumors were established in ovariectomized lean and obese mice in the presence of high or low estradiol (E2). To simulate the hormonal environment of women on aromatase inhibitors, E2 was removed from half of the mice in each adiposity group and the study was terminated 3 weeks later. Weight gain, body fat percentage, and adipose tissue as well as tumor characteristics were analyzed. Results: Prior to EWD, obese mice were heavier and had higher body fat percentage than lean mice and also displayed a phenotype of metabolic dysfunction. This trend was accelerated after EWD, with obese mice gaining more weight due to body fat accumulation. Tumors responded in one of two ways: Regardless of FGFR1 amplification, obesity promoted ER+ tumor growth in the presence of low (postmenopausal), but not high (premenopausal) E2. In the presence of low E2, tumor PR levels were higher in obese compared to lean mice, suggesting hyperactive ER signaling. In FGFR1-amplified tumors, obesity promoted tumor growth after EWD. In addition, EWD induced excess fat deposition in visceral depots in both lean and obese mice; however, obese mice also gained fat in mammary adipose depots. Mammary fat pad mass and rate of post-EWD weight gain directly correlated with adipose FGF1 levels. Tumors from obese mice had higher levels of phosphorylated FGFR1, without changes in total FGFR1, compared to lean mice. Conclusions: Utilizing a unique PDX model system, we show that obesity promotes tumor progression in the presence of low E2, and also after EWD, and identify growth factor receptor signaling as a mediator of these phenotypes. The activation of FGFR1 may underlie increased breast cancer risk and recurrence observed in obese, postmenopausal women. Citation Format: Wellberg EA, Johnson SJ, Jacobsen BM, Anderson SM, Sartorius CA, MacLean PS, Kabos P. Obesity drives breast cancer progression through estrogen dependent and independent mechanisms [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-03.

EGFR-amplification plus gene expression profiling predicts response to combined radiotherapy with EGFR-inhibition: A preclinical trial in 10 HNSCC-tumour-xenograft models

Background and purposeImprovement of the results of radiotherapy by EGFR inhibitors is modest, suggesting significant intertumoural heterogeneity of response. To identify potential biomarkers, a preclinical trial was performed on ten different human squamous cell carcinoma xenografts of the head and neck (HNSCC) studying in vivo and ex vivo the effect of fractionated irradiation and EGFR inhibition. Local tumour control and tumour growth delay were correlated with potential biomarkers, e.g. EGFR gene amplification and radioresponse-associated gene expression profiles. Material and methodsLocal tumour control 120days after end of irradiation was determined for fractionated radiotherapy alone (30f, 6weeks) or after simultaneous EGFR-inhibition with cetuximab. The EGFR gene amplification status was determined using FISH. Gene expression analyses were performed using an in-house gene panel. ResultsSix out of 10 investigated tumour models showed a significant increase in local tumour control for the combined treatment of cetuximab and fractionated radiotherapy compared to irradiation alone. For 3 of the 6 responding tumour models, an amplification of the EGFR gene could be demonstrated. Gene expression profiling of untreated tumours revealed significant differences between amplified and non-amplified tumours as well as between responder and non-responder tumours to combined radiotherapy and cetuximab. ConclusionThe EGFR amplification status, in combination with gene expression profiling, may serve as a predictive biomarker for personalized interventional strategies regarding combined treatment of cetuximab and fractionated radiotherapy and should, as a next step, be clinically validated.

Abstract 3945: Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing

Abstract Introduction Chromosome instability (CIN) is a hallmark of cancer, acting by boosting genetic alterations responsible of tumorigenesis, progression and heterogeneity. Whole-genome sequencing (WGS) protocols are established methods for studying copy-number alterations (CNA) in single-cells, following a necessary whole-genome amplification (WGA) step. We previously presented a method for single-cell CNA profiling of CTCs based on shallow WGS of LM-PCR based WGA products. Here for the first time, we show that the same method may be employed even on single FFPE cells, overcoming the challenges of DNA degradation and damage linked to this type of samples. Methods Two 50μm thick FFPE sections from pancreas ductal adenocarcinoma with &amp;lt;20% tumor content were processed following DEPArray™ sample preparation protocol, including cell dissociation and staining with vimentin, keratin and DAPI, and DNA quality was assessed using the DEPArray FFPE QC kit (QC=0.7). Cell pools (n=15, range=6-213 cells) and single-cells (n=11) from pure tumor and stromal populations were recovered in separate tubes and lysed. A pool of 86 tumor cells and a pool of 213 stromal cells were used for low-pass WGS on MiSeq, obtaining ≈2M paired-end reads on average. By contrast, other cell pools and single cells were firstly whole-genome amplified using the Ampli1™ WGA kit and then sequenced at shallow coverage using Ampli1 LowPass kit and IonTorrent PGM, with ≈500k single-end reads as mean throughput per library. The genome integrity (GI) of WGA samples was evaluated by Ampli1 QC, a PCR-assay based on 4 amplicons of different lengths. Results The purity of DEPArray sorting was confirmed by the large number of chromosome alterations in sorted tumor and the parallel lack of gains and losses in sorted stromal pools and single-cells. A titration test was conducted measuring the consistency of CNA profiles of tumor WGA products starting from different number of cells, ranging from 117 down to single cells. For the purpose, we employed a Receiver Operating Characteristic (ROC) curve using the non-amplified tumor population as reference. Results showed an excellent performance level with a mean Area Under Curve (AUC) equals to 0.93. Interestingly, lower AUCs (0.87) were observed for single-cells, due to some level of inter-cell heterogeneity. Moreover, the proposed low-pass WGS method demonstrated a high resiliency to DNA degradation as quality of CNA profiles, measured by Derivative Log Ratio Spread (DLRS), only showed a weak correlation with GI level, with high-quality CNA profiles obtained also with the lowest GI value. Conclusions Presented approach for copy-number profiling of tumor single-cells isolated by DEPArray digital sorter and processed with Ampli1 workflow has proven to be a reliable and valuable application for the molecular characterization of tumor clones in degraded samples as FFPE tissues. Citation Format: Alberto Ferrarini, Genny Buson, Chiara Bolognesi, Claudio Forcato, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2017-3945

MEDU-05. THE ROLE OF GABA METABOLISM IN MEDULLOBLASTOMA

Abstract BACKGROUND: Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (originating in the cerebellum) is the most common malignant pediatric brain tumor. In the microenvironment of the brain, especially the cerebellum, variables related to GABA, the major inhibitory neurotransmitter in the nervous system, are particularly prominent. Abnormal GABAergic Receptor activation has been described in in aggressive MYC-driven Group 3 medulloblastoma. However these studies did not look at the metabolic contribution of GABA for the development of medulloblastomas. In addition to its role in neurotransmission through GABA receptor, GABA can act as a trophic factor during nervous system development to influence cellular events including proliferation, migration, differentiation, synapse maturation, and cell death. Under conditions that inhibit the tricarboxylic acid cycle (TCA), impair respiration, and enhance the accumulation of reactive oxygen intermediates, GABA can be used as an NADH energy source for growth through the GABA-shunt pathway regulators (ABAT, SSADH, GAT-1, GAT-3). Therefore, we hypothesize that blocking GABA-metabolic-shunt will lead to growth suppression and invasiveness of medulloblastoma in the cerebellar GABA-rich microenvironment. RESULTS: Our results show RNA microarray from patient medulloblastoma tissue have high expression of GABA-shunt regulators with ~3-fold increase in the expression of ABAT in MYC amplified versus non-amplified MYC tumors. When medulloblastomas were supplemented with GABA, there was a significant fold change in expression of GABA-shunt mediators and induction of large and stable tumor spheres with Epithelial-Mesenchymal Transition gene expression signature. We next investigated whether a novel perrilyl alcohol-based small molecule NEO216 targeted the GABA-shunt metabolic pathway. NEO216 administration significantly reduced GABA-mediated NADH levels, reversed EMT-profiling, leading to loss of sphere formation. CONCLUSION: Thus, the expression of GABA-metabolic shunt in medulloblastoma could be a malignant microenvironmental adaptation for growth and metastasis that could potentially be exploited through targeted therapy for patients benefit.