Amplification Status Research Articles

Abstract P3-18-12: Achieving negative margin after repeated attempts for lumpectomy does not nullify the risk of ipsilateral breast tumor recurrences

Abstract Introduction: Currently, the widely accepted principle in managing lumpectomy margins in early breast cancer patients is to avoid the presence of tumor cell at the margin. The presence of tumor cells on lumpectomy margin can represent two different aspects of local control in breast cancer. One aspect is related to the potential technical failure that may lead to an incomplete resection of the tumor. The other aspect of positive margin is associated with a biologic nature of the tumor. For example, some tumors show features of extensive intraductal component which often result in positive lumpectomy margins and may lead to increased risk of local recurrence after breast conservation surgery. Therefore, the two-fold increase of IBTR risk with positive margin would be the reflection of the combined effect of both technical failure and the biologic features.In this study, we aimed to address this issue by investigating the IBTR rates of patients in whom negative margins were achieved by initial lumpectomy or by re-excision. Our assumption was if the IBTR rates of the two groups are identical, then the two-fold increase of IBTR risk is mostly caused by the technical incomplete resection and therefore the measures to achieve negative margins would be justified. Methods: We retrospectively reviewed the data of 5,633 patients who underwent breast conservation surgery and whole breast irradiation for invasive breast cancer between January 2004 and December 2018 from Seoul National University Hospital. During the period, a total of 828 patients had positive lumpectomy margin after their initial surgery. Among them, thirty-five patients who did not undergo re-operation were excluded due to the small sample size. Results: The median age of the studied patients was 49 years old (19-92), and nearly two third of the patients had T1 tumors (64.3%). 4,293 patients (76.7%) were node negative and 4,332 patients (77.4%) had hormone receptor positive tumors. During the median follow-up period of 76.6 ( ± 44.6) months, a total of 121 patients (2.2%) experienced IBTR. Patients who underwent re-excision to achieve negative margins experienced significantly higher rate of IBTR compared to patients in whom the margins were clear at the first lumpectomy (p=0.031, HR: 1.61; 95% C.I., 1.04-2.48). The survival curves began to separate around 4-5 years after surgery (98.5% vs 98.0% at 5 year and 97.4% vs 94.7% at 10 years of follow-up). The survival difference was more clearly observed for younger patients (p=0.033, HR 1.72; 95% C.I., 1.04-2.85 for age less than 50). When the patients were divided according to their hormone receptor and HER2 amplification status, we observed significant difference in HR+/HER2- and HR-/HER2- subtypes while the HER2-amplified tumors showed no significant differences.By using Cox regression analysis, we adjusted for other significant predictors of IBTR such as age, histologic grade, lymphovascular invasion, hormone receptor status, HER2 amplification status, and Ki-67 levels. The results of the Cox regression analysis showed that re-excision to achieve negative margin is significantly associated with the risk of IBTR after adjusting for these variables (p=0.023, HR: 1.72; 95% C.I., 1.08-2.74). Conclusion: Patients who underwent re-excision after lumpectomy for initial positive margin carry increased risk of developing IBTR even they achieve final negative margin when compared to patients with initially negative lumpectomy margin. Our observation indicate that a substantial proportion of the increasing risk of IBTR associated with positive lumpectomy margin can be attributed to the biologic characteristics of the tumor rather than technical incomplete resection. Citation Format: Jong Ho Cheun, Hong-Kyu Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Achieving negative margin after repeated attempts for lumpectomy does not nullify the risk of ipsilateral breast tumor recurrences [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-18-12.

CO2 pneumoperitoneum effects on proliferation and apoptosis in two different neuroblastoma cell lines.

The proto-oncogene MYCN is considered a transcription factor involved in the regulation of neuroblastoma (NB) cell biology. Since minimally invasive-surgery represents a debated treatment of NB, we investigated CO2 effects on proliferative activity and apoptotic pathway in two NB cell lines, SH-SY5Y (MYCN-non-amplified) and IMR-32 (MYCN-amplified). SH-SY5Y and IMR-32 were exposed to CO2 (100%) at a pressure of 15mmHg for 4h and then moved to normal condition for 24h. Cell proliferation, caspase 3 activity and transcript levels of BAX, BCL-2, cyclin B, cyclin D and MMP-2 were evaluated. CO2 exposure caused a decrease in cell proliferation associated to increases in BAX/BCL-2 ratio and caspase 3 activity in SH-SY5Y, while opposite effects have been found in IMR-32. CO2 exposure induced a decrease of cyclin B1 in SH-SY5Y, while an increase in cyclin B1 and D1 was observed in IMR-32. A slight up-regulation of MMP-2 expression in SH-SY5Y and a significant increase of 2.2 folds in IMR-32 was observed (p < 0.05). Our results suggest that CO2 exposure may cause different effects on various NB cell lines, likely due toMYCN amplification status. Further in vitro and in vivo studies are needed to highlight the role of laparoscopy on NB behaviour.

Alpha-actinin-4 (ACTN4) gene amplification is a predictive biomarker for adjuvant chemotherapy with tegafur/uracil in stage I lung adenocarcinomas.

Although adjuvant tegafur/uracil (UFT) is recommended for patients with completely resected stage I non‐small‐cell lung cancer (NSCLC) in Japan, only one‐third of cases has received adjuvant chemotherapy (ADJ) according to real‐world data. Therefore, robust predictive biomarkers for selecting ADJ or observation (OBS) without ADJ are needed. Patients who underwent complete resection of stage I lung adenocarcinoma with or without adjuvant UFT were enrolled. The status of ACTN4 gene amplification was analyzed by FISH. Statistical analyses to determine whether the status of ACTN4 gene amplification affected recurrence‐free survival (RFS) were carried out. Formalin‐fixed, paraffin‐embedded samples from 1136 lung adenocarcinomas were submitted for analysis of ACTN4 gene amplification. Ninety‐nine (8.9%) of 1114 cases were positive for ACTN4 gene amplification. In the subgroup analysis of patients aged 65 years or older, the ADJ group had better RFS than the OBS group in the ACTN4‐positive cohort (hazard ratio [HR], 0.084, 95% confidence interval [CI], 0.009‐0.806; P = .032). The difference in RFS between the ADJ group and the OBS group was not significant in ACTN4‐negative cases (all ages: HR, 1.214; 95% CI, 0.848‐1.738; P = .289). Analyses of ACTN4 gene amplification contributed to the decision regarding postoperative ADJ for stage I lung adenocarcinomas, preventing recurrence, improving the quality of medical care, preventing the unnecessary side‐effects of ADJ, and saving medical costs.

Computer-aided scoring of erb-b2 receptor tyrosine kinase 2 (HER2) gene amplification status in breast cancer

BackgroundIdentification of HER2 protein overexpression and/or amplification of the HER2 gene are required to qualify breast cancer patients for HER2 targeted therapies. In situ hybridization (ISH) assays that identify HER2 gene amplification function as a stand-alone test for determination of HER2 status and rely on the manual quantification of the number of HER2 genes and copies of chromosome 17 to determine HER2 amplification. MethodsTo assist pathologists, we have developed the uPath HER2 Dual ISH Image Analysis for Breast (uPath HER2 DISH IA) algorithm, as an adjunctive aid in the determination of HER2 gene status in breast cancer specimens. The objective of this study was to compare uPath HER2 DISH image analysis vs manual read scoring of VENTANA HER2 DISH-stained breast carcinoma specimens with ground truth (GT) gene status as the reference. Three reader pathologists reviewed 220, formalin-fixed, paraffin-embedded (FFPE) breast cancer cases by both manual and uPath HER2 DISH IA methods. Scoring results from manual read (MR) and computer-assisted scores (image analysis, IA) were compared against the GT gene status generated by consensus of a panel of pathologists. The differences in agreement rates of HER2 gene status between manual, computer-assisted, and GT gene status were determined. ResultsThe positive percent agreement (PPA) and negative percent agreement (NPA) rates for image analysis (IA) vs GT were 97.2% (95% confidence interval [CI]: 95.0, 99.3) and 94.3% (95% CI: 90.8, 97.3) respectively. Comparison of agreement rates showed that the lower bounds of the 95% CIs for the difference of PPA and NPA for IA vs MR were –0.9% and –6.2%, respectively. Further, inter- and intra-reader agreement rates in the IA method were observed with point estimates of at least 96.7%. ConclusionsOverall, our data show that the uPath HER2 DISH IA is non-inferior to manual scoring and supports its use as an aid for pathologists in routine diagnosis of breast cancer.

IDH-mutant astrocytoma with EGFR amplification-Genomic profiling in four cases and review of literature.

Abstract EGFR amplification is associated with aggressive glioma behavior and regarded as a molecular feature of glioblastoma. Although rare, IDH-mutant astrocytomas with EGFR amplification exist but remain poorly understood. We report the clinical and molecular profile of four grade 4 IDH-mutant astrocytomas with EGFR amplification, evaluated using histology, DNA sequencing, cytogenetics, and DNA methylation profiling. Other alterations included ATRX, TP53, and PIK3CA mutations; CDKN2A/B loss; PDGFRA+KIT amplifications; and MGMT methylation in two cases. None disclosed microsatellite instability. DNA methylation confidently classified all tumors as “IDH-mutant High-Grade Astrocytoma” (0.99 0.997, 0.98, and 0.99 scores), despite their EGFR-amplified status. Literature review indicated EGFR amplification occurs within 8-19% of IDH-mutant gliomas, with significantly lower overall survival only in the co-presence of CDKN2A/B loss and MET amplification. Our report suggests IDH-mutant astrocytomas with EGFR amplification are under-recognized and that EGFR amplification status alone does not carry diagnostic or prognostic significance in these tumors.

Liquid Biopsy and Tissue Biopsy Comparison with Digital PCR and IHC/FISH for HER2 Amplification Detection in Breast Cancer Patients.

Two hundred twenty-four breast cancer patients with paired tissue and plasma samples were enrolled from 3 clinical centers to evaluate sensitivity and specificity of a digital PCR HER2 amplification assay. All patients were histologically confirmed diagnosis of locally advanced and recurrent or metastatic breast cancer with stage III/IV and had tissue HER2 status determinations using IHC/FISH. For the whole 224 advanced breast cancer patients, the sensitivity between dPCR in plasma and IHC/FISH in tissue samples is 43.75% (42/96), the specificity is 84.38% (108/128) and the overall concordance is 66.96% (150/224). Interestingly, when we looked at stage III, stage IV and recurrent or metastatic breast cancer separately, compared with IHC/FISH in tissue samples, the sensitivity of dPCR in plasma increases from 37.93% (11/29) for stage III to 41.67% (15/36) for stage IV cancer. Recurrent breast cancer patient had an increased sensitivity of 51.61% (16/31). This is consistent with our expectation sensitivity would increase concordantly as tumor burden goes up. On the other hand, specificity decreased from 92.68% (38/41) for stage III to 86.44% (51/59) for stage IV cancer. Recurrent breast cancer patient had a specificity of only 67.86% (19/28). This is, in part, due to inter- and intra-tumor heterogeneity. Many patients determined to be negative for HER2 amplification in tissue biopsy could have HER2 positive tumors at other sites, which was detected by the liquid biopsy. This study suggested the necessity of liquid biopsy for HER2 amplification detection and demonstrated digital PCR can be used as a companion diagnostic tool to determine HER2 amplification status. It also suggested that a liquid biopsy should follow a negative result from tissue biopsy to avoid false negative results especially for late-stage breast cancer patients and ones who experienced relapse or became resistant to current therapy. Future studies should focus on therapeutic effects on patients determined to be HER2 positive through liquid biopsy and collecting additional tissue biopsies to identify HER2 positive tumor when the original tissue biopsy and liquid biopsy don't agree.

Establishment of Rapid and Accurate Screening System for Molecular Target Therapy of Osteosarcoma.

Introduction Comprehensive analyses using clinical sequences subcategorized osteosarcoma (OS) into several groups according to the activated signaling pathways. Mutually exclusive co-occurrences of gene amplification (PDGFRA/KIT/KDR, VEGFA/CCND3, and MDM2/CDK4) have been identified in approximately 40% of OS, representing candidate subsets for clinical evaluation of additional therapeutic options. Thus, it would be desirable to evaluate the specific gene amplification before starting therapy in patients with OS. Materials and Methods This is a retrospective study. We examined 13 cases of clinical OS samples using NanoString-based copy number variation (CNV) analysis. Decalcification and chemotherapeutic effects on this analysis were also assessed. Results First, the accuracy of this system was validated by showing that amplification/deletion data obtained from this system using various types of cancer cell lines almost perfectly matched to that from the Cancer Cell Line Encyclopedia (CCLE). We identified potentially actionable alterations in CDK4/MDM2 amplification in 10% of samples and potential additional therapeutic targets (PDGFRA/KIT/KDR and VEGFA/CCND3) in 20% of samples, which is consistent with the reported frequencies. Furthermore, this assay could identify these potential therapeutic targets regardless of the sample status (frozen vs formalin-fixed paraffin-embedded [FFPE] tissues). Conclusion We established a NanoString-based rapid and cost-effective method with a short turnaround time (TAT) to examine gene amplification status in OS. This CNV analysis using FFPE samples is recommended where the histological evaluation of viable tumor cells is possible, especially for tumors after chemotherapy with higher chemotherapeutic effects.

Iron-Chelation Treatment by Novel Thiosemicarbazone Targets Major Signaling Pathways in Neuroblastoma.

Despite constant advances in the field of pediatric oncology, the survival rate of high-risk neuroblastoma patients remains poor. The molecular and genetic features of neuroblastoma, such as MYCN amplification and stemness status, have established themselves not only as potent prognostic and predictive factors but also as intriguing targets for personalized therapy. Novel thiosemicarbazones target both total level and activity of a number of proteins involved in some of the most important signaling pathways in neuroblastoma. In this study, we found that di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) potently decreases N-MYC in MYCN-amplified and c-MYC in MYCN-nonamplified neuroblastoma cell lines. Furthermore, DpC succeeded in downregulating total EGFR and phosphorylation of its most prominent tyrosine residues through the involvement of NDRG1, a positive prognostic marker in neuroblastoma, which was markedly upregulated after thiosemicarbazone treatment. These findings could provide useful knowledge for the treatment of MYC-driven neuroblastomas that are unresponsive to conventional therapies.

Identification of a Five-Gene Signature Derived From MYCN Amplification and Establishment of a Nomogram for Predicting the Prognosis of Neuroblastoma.

Background: Neuroblastoma (NB), the most common solid tumor in children, exhibits vastly different genomic abnormalities and clinical behaviors. While significant progress has been made on the research of relations between clinical manifestations and genetic abnormalities, it remains a major challenge to predict the prognosis of patients to facilitate personalized treatments. Materials and Methods: Six data sets of gene expression and related clinical data were downloaded from the Gene Expression Omnibus (GEO) database, ArrayExpress database, and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. According to the presence or absence of MYCN amplification, patients were divided into two groups. Differentially expressed genes (DEGs) were identified between the two groups. Enrichment analyses of these DEGs were performed to dig further into the molecular mechanism of NB. Stepwise Cox regression analyses were used to establish a five-gene prognostic signature whose predictive performance was further evaluated by external validation. Multivariate Cox regression analyses were used to explore independent prognostic factors for NB. The relevance of immunity was evaluated by using algorithms, and a nomogram was constructed. Results: A five-gene signature comprising CPLX3, GDPD5, SPAG6, NXPH1, and AHI1 was established. The five-gene signature had good performance in predicting survival and was demonstrated to be superior to International Neuroblastoma Staging System (INSS) staging and the MYCN amplification status. Finally, a nomogram based on the five-gene signature was established, and its clinical efficacy was demonstrated. Conclusion: Collectively, our study developed a novel five-gene signature and successfully built a prognostic nomogram that accurately predicted survival in NB. The findings presented here could help to stratify patients into subgroups and determine the optimal individualized therapy.

FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 Oncogene

(Cell 129, 1275–1286; June 29, 2007) We, the editors of Cell, were contacted by the authors of this article regarding a duplication in Figure 7 that was not identified in the previous 2008 correction. Dr. Yang Liu, the corresponding author, has determined that the error was introduced during revision of the manuscript. In preparing the final figure, three images from the experiment showing that Foxp3 expression inhibits the growth of drug-resistant clones in breast cancer cell lines shown in Figure 7A—SKBR3 cells transfected with Otc cDNA, SKBR3 cells with vector, and MCF-7 cells with Foxp3—were mistakenly inserted into Figure 7D showing that ectopic expression of ErbB2 abrogated Foxp3-mediated repression. These images were incorrectly presented as TSA cells transfected with ErbB2 + pEF1 vector, ErbB2 + Foxp3, and pcDNA6 vector + Foxp3, respectively. The authors no longer have access to the original data, and consequently, a correction is not possible. Given the age of the paper and that the duplication does not compromise the conclusions of the paper, based on the information available to us at this time, we believe that no further action is warranted. FOXP3 Is an X-Linked Breast Cancer Suppressor Gene and an Important Repressor of the HER-2/ErbB2 OncogeneZuo et al.CellJune 29, 2007In BriefThe X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germline mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that female mice heterozygous for the “scurfin” mutation of the Foxp3 gene (Foxp3sf/+) developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and HER-2/ErbB2 was overexpressed. Foxp3 bound and repressed the HER-2/ErbB2 promoter. Deletion, functionally significant somatic mutations, and downregulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2/ErbB2 overexpression, regardless of the status of HER-2 amplification. Full-Text PDF Open Archive

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors

BackgroundDysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies.MethodsWe performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(−) HNSCC cell lines.ResultsWe found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3–selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1.ConclusionsOur results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

DNA methylation profiling identifies two distinct subgroups in breast cancers with low hormone receptor expression, mainly associated with HER2 amplification status

BackgroundCurrent clinical guidelines suggest that breast cancers with low hormone receptor expression (LowHR) in 1–10% of tumor cells should be regarded as hormone receptor positive. However, clinical data show that these patients have worse outcome compared to patients with hormone receptor expression above 10%. We performed DNA methylation profiling on 23 LowHR breast cancer specimens, including 13 samples with HER2 amplification and compared our results with a reference breast cancer cohort from The Cancer Genome Atlas to clarify the status for this infrequent but important patient subgroup.ResultsIn unsupervised clustering and dimensionality reduction, breast cancers with low hormone receptor expression that lacked HER2 amplification usually clustered with triple negative breast cancer (TNBC) reference samples (8/10; “LowHR TNBC-like”). In contrast, most specimens with low hormone receptor expression and HER2 amplification grouped with hormone receptor positive cancers (11/13; “LowHR HRpos-like”). We observed highly similar DNA methylation patterns of LowHR TNBC-like samples and true TNBCs. Furthermore, the Ki67 proliferation index of LowHR TNBC-like samples and clinical outcome parameters were more similar to TNBCs and differed from LowHR HRpos-like cases.ConclusionsWe here demonstrate that LowHR breast cancer comprises two epigenetically distinct groups. Our data strongly suggest that LowHR TNBC-like samples are molecularly, histologically and clinically closely related to TNBC, while LowHR HRpos-like specimens are closely related to hormone receptor positive tumors.

P59.01 Clinical Characteristics of Patients With MET Amplification-Positive NSCLC After EGFR-TKI Therapy

Patients with epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) ultimately acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) during treatment. In 5–22% of these patients, resistance is mediated by aberrant mesenchymal epithelial transition factor (MET) gene amplification. Here, we evaluated the emergence of MET amplification after EGFR-TKI treatment failure based on clinical parameters. We retrospectively analyzed 186 patients with advanced EGFR-mutant NSCLC for MET amplification status by in situ hybridization (ISH) assay after EGFR-TKI failure. We collected information including baseline patient characteristics, metastatic locations and generation, line, and progression free survival (PFS) of EGFR-TKI used before MET evaluation. Multivariate logistic regression analysis was conducted to evaluate associations between MET amplification status and clinical variables. View Large Image Figure ViewerDownload Hi-res image Download (PPT) Regarding baseline EGFR mutations, exon 19 deletion was predominant (57.5%), followed by exon 21 mutation (39.2%). The proportions of MET ISH assays performed after first/second-generation and third-generation TKI failure were 66.7% and 33.1%, respectively. The median PFS for the most recent EGFR-TKI treatment was shorter in MET amplification-positive patients than in MET amplification-negative patients (mPFS of 1st/2nd generation TKI cohort, 7.0 vs. 10.4 months, p = 0.003, PFS of 3rd generation TKI cohort, 7.2 vs. 11.0 months, p=0.163). Multivariate logistic regression demonstrated that a history of smoking, short PFS on the most recent TKI, and less intracranial progression were associated with a high probability of MET amplification (all p < 0.05). Our results demonstrated the distinct clinical characteristics of patients with MET amplification-positive NSCLC after EGFR-TKI therapy. Our clinical prediction can aid physicians in selecting patients eligible for MET amplification screening and therapeutic targeting.

Magnetic Resonance Imaging Parameters for Noninvasive Prediction of Epidermal Growth Factor Receptor Amplification in Isocitrate Dehydrogenase-Wild-Type Lower-Grade Gliomas: A Multicenter Study.

The epidermal growth factor receptor (EGFR) amplification status of isocitrate dehydrogenase-wild-type (IDHwt) lower-grade gliomas (LGGs; grade II/III) is one of the key markers for diagnosing molecular glioblastoma. However, the association between EGFR status and imaging parameters is unclear. To identify noninvasive imaging parameters from diffusion-weighted and dynamic susceptibility contrast imaging for predicting the EGFR amplification status of IDHwt LGGs. A total of 86 IDHwt LGG patients with known EGFR amplification status (62 nonamplified and 24 amplified) from 3 tertiary institutions were included. Qualitative and quantitative imaging features, including histogram parameters from apparent diffusion coefficient (ADC), normalized cerebral blood volume (nCBV), and normalized cerebral blood flow (nCBF), were assessed. Univariable and multivariable logistic regression models were constructed. On multivariable analysis, multifocal/multicentric distribution (odds ratio [OR] = 11.77, P=.006), mean ADC (OR=0.01, P=.044), 5th percentile of ADC (OR=0.01, P=.046), and 95th percentile of nCBF (OR=1.24, P=.031) were independent predictors of EGFR amplification. The diagnostic performance of the model with qualitative imaging parameters increased significantly when quantitative imaging parameters were added, with areas under the curves of 0.81 and 0.93, respectively (P=.004). The presence of multifocal/multicentric distribution patterns, lower mean ADC, lower 5th percentile of ADC, and higher 95th percentile of nCBF may be useful imaging biomarkers for EGFR amplification in IDHwt LGGs. Moreover, quantitative imaging biomarkers may add value to qualitative imaging parameters.

A case report of response to crizotinib in chemotherapy-refractory metastatic gallbladder cancer with met amplification and acquired resistance resulting from the loss of MET amplification.

Gallbladder cancer (GBC) is a highly invasive disease and the most prevalent malignancy of the biliary system. Patients with GBC are commonly diagnosed at a late stage and have an unfavorable prognosis. Palliative chemotherapy has been the standard care for recurrent or metastatic disease in the past decades. Recently, several targeted therapies have been investigated in advanced biliary tract cancer (BTC) including inhibitors of genes or pathways such as FGFR2 fusions or rearrangements, IDH1 mutations, and NTRK gene fusions. Also, several clinical studies involving molecular stratification have been performed in defined patient groups, for example, BRAF V600E and HER2. Mesenchymal epithelial transition(MET)encodes a tyrosine kinase receptor and its ligand hepatocyte growth factor is a proto-oncogene. Targeting the MET signaling pathway is an effective strategy in numerous cancer types. However, the poor efficacy of MET inhibitors has been demonstrated in several phase II studies, but currently no reports have explained the potential mechanisms of resistance to MET inhibitors in BTC. In this article, we report a case of metastatic GBC with MET amplification that exhibited a rapid response to crizotinib after the failure of two lines of chemotherapy. After the patient had progressed and discontinued crizotinib, cabozantinib was introduced. Analysis of circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) indicated a loss of MET amplification status. To our knowledge, this is the first case study demonstrating the use of NGS in ctDNA to monitor the development of acquired resistance during anti-MET treatment in GBC.

Dual targeting of MEK and PI3K effectively controls the proliferation of human EGFR-TKI resistant non-small cell lung carcinoma cell lines with different genetic backgrounds

BackgroundMolecular targeted therapy for non-small cell lung carcinoma (NSCLC) is restricted due to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This study evaluated the effects of dual targeting of MEK and PI3K in human EGFR-TKI resistant NSCLC cell lines.MethodsEGFR-TKI resistant NSCLC cell lines H1975, H460, and A549, with different mutation and amplification status in EGFR, K-RAS, PIK3CA, and MET genes, were treated with a MEK162 (MEK inhibitor) and BKM120 (PI3K inhibitor) combination or a BIBW2992 (EGFR inhibitor) and ARQ197 (MET inhibitor) combination and assayed for cell proliferation, apoptosis, and cell cycle distribution.ResultsDual targeting of MEK and PI3K efficiently inhibited the cell proliferation, induced apoptosis and the G0/G1 cell cycle, and decreased the phosphorylation of ERK1/2, AKT, S6, and 4E-BP1. H460 cells with K-RAS and PIK3CA mutation were most sensitive to MEK162 and BKM120 combinations. H1975 cells with EGFR and PIK3CA mutation and MET amplification were sensitive to BIBW2992 and ARQ197 combinations.ConclusionDual targeting regulated the proliferation of EGFR-TKI-resistant NSCLC cells, especially mutants in K-RAS and PIK3CA that are promising for EGFR-TKI-resistant NSCLC therapeutics.

Immunogenomic determinants of tumor microenvironment correlate with superior survival in high-risk neuroblastoma

BackgroundTumor-infiltrating CD8+ T cells and neoantigens are predictors of a favorable prognosis and response to immunotherapy with checkpoint inhibitors in many types of adult cancer, but little is known about...

Abstract 2163: Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients

Abstract Background: Cyclin-dependent kinase 4 (CDK4) and mouse double minute 2 homolog (MDM2) amplification occur at high frequency in well-differentiated liposarcoma and dedifferentiated liposarcoma, which are generally considered to be diagnostic criteria for these tumors. Studies have also reported that CDK4 and/or MDM2 amplification can emerge in STS subtypes, such as Ewing's sarcoma (ES) and fibrosarcoma (FS). However, the clinical pathological and molecular characteristics of CDK4 and/or MDM2 amplification in STS are still unclear. We systematically depicted the clinical characteristics of MDM2 and CDK4 gene amplification in Chinese STS patients (pts), aiming to assist the precise diagnosis and treatment of STS. Methods: Totally 184 Chinese pts with different STS subtypes were included in this study. In terms of subtypes distribution, the cohort contained multiple STS subtypes including myofibroblastic sarcoma (MFS, n = 23), liposarcoma (LPS, n = 21), FS (n = 20), rhabdomyosarcoma (RMS, n = 19), liomyosarcoma (LMS, n = 12), undifferentiated pleomorphic sarcoma (n = 11), clear cell sarcoma (n = 10), synovial sarcoma (n = 8), ES (n = 7), hemangiosarcoma (n=7), other rare subtypes (n = 37) and unknown types (n = 9). The amplification status of CDK4 and MDM2 in the tumor tissues of these pts was reviewed by the DNA and RNA next generation sequencing method. Results: Totally 16.8% (31/184) of the Chinese STS pts harbored CDK4 and/or MDM2 amplification. Among the 31 pts, 17 were with co-amplification of CDK4 and MDM2, including 12 LPSs, two RMSs, one MFS, one LMS and one FS. Fourteen pts were with single amplification of MDM2 or CDK4, including LPS (n = 4), RMS (n = 3), MFS (n = 3), FS (n = 2), ES (n = 1), and malignant peripheral nerve sheath tumor (n = 1). In LPS, the ratios of co-amplification and single amplification of CDK4/MDM2 were 57.1% (12/21) and 19.0% (4/21) respectively. Other STS subtypes with high frequency of CDK4 and/or MDM2 amplification included RMS (26.3%, 5/19) and MFS (19.4%, 4/23). Meanwhile, our study revealed that co-amplification of CDK4 and MDM2 appeared to be a strong driver event in STS, which mutated exclusively with other driver genes, such as TP53, MYOD1, ATRX and PTEN. Pts with co-amplification of CDK4 and MDM2 were likely to benefit from the treatment of CDK4 and/or MDM2 inhibitors. However, more than half of the pts with single amplification of CDK4 or MDM2 were accompanied by mutations in other driven genes, such as FLI1-EWSR1 fusion, PAX3-FOXO1 fusion, MYOD1 p.L122R, TP53 p.R273S, and NRAS p.G13C. In these pts, single amplification of CDK4 or MDM2 may not be the main driver event, who may be less likely to benefit from CDK4 or MDM2 inhibitor monotherapy. Conclusion: Our study revealed the clinicopathologic and molecular features of CDK4 and/or MDM2 amplification in Chinese STS pts, which can guide the selection of personalized treatment strategies in STS pts. Citation Format: Gu Jin, Chunyang Wang, Xiaojuan Wang, Qifan He, Tonghui Ma. Molecular characteristics of CDK4 and/or MDM2 amplification in Chinese soft tissue sarcoma (STS) patients [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 2163.

Abstract 3030: Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences

Abstract Background: Neuroblastoma is a childhood cancer that arises from the sympathetic nervous system. Approximately 1% of patients have a family history of disease with causal germline mutations in neuroblastoma predisposition genes, ALK or PHOX2B. Recent genome-wide association studies (GWAS) of sporadic cases have identified over two dozen susceptibility loci, and next-generation sequencing studies estimate that 8-10% of children with cancer harbor a rare pathogenic (P) or likely pathogenic (LP) germline variant in a cancer predisposition gene (CPG). To date, the heritability and clinical significance of germline P/LP variants remains unknown due to a lack of parental germline DNA. Methods: Through the Gabriella Miller Kids First program, we performed whole genome sequencing (WGS) at 30x depth on patient-parent triads (n=457) and dyads (n=99), along with WGS and exome sequencing on matched tumor DNA (n=336) and RNA-sequencing on matched tumor RNA (n=207). Rare variants (allele frequency &amp;lt;0.1% in ExAC, 1000 Genomes, and gnomAD) in a predefined set of CPGs (n=197) were annotated, and pathogenicity was assessed using ClinVar, InterVar, and manual review. Gene-based enrichment was performed by comparison to gnomAD WGS data (n=143,068), excluding TCGA samples. Heritability was evaluated using family-based variant calling. Tumor DNA sequencing was analyzed for second hits or somatic enrichment of the germline P/LP variant. Tumor RNA sequencing was interrogated to identify expressed P/LP variants and preferential allelic expression. Ongoing analyses are utilizing polygenic risk scores from our GWAS study to inform tumor penetrance. Results: We observed 78 P/LP germline variants in CPGs in 75 probands (13.5%). Of the 72 probands with trio data, 95% of the P/LP variants were inherited (54% maternal, 46% paternal). Several CPGs showed enrichment of P/LP variants, including BARD1 (p=0.001; OR 16.16; 95% CI 3.21-50.45). We observed one canonical ALK mutation (R1275Q), but no PHOX2B mutations. For patients with corresponding tumor samples, all germline P/LP variants were observed in the tumor DNA. Germline P/LP variants were associated with high-risk disease as defined by risk group (high vs. low/intermediate, p=0.005), INRG stage (stage M vs stage L1/L2/MS, p=0.024), and MYCN amplification status (amplified vs not amplified, p=0.016). Conclusions: Here we show that approximately 13.5% of patients with neuroblastoma harbor a rare P/LP germline variant in a CPG, and the vast majority of these are inherited. Neuroblastoma patients with P/LP germline variants are more likely to have high-risk disease and several variants suggest potential therapeutic opportunities. Work is ongoing to understand the genetic factors that explain why parents harboring the same P/LP variant did not develop neuroblastoma, and to determine the genetic counseling implications of these data. Citation Format: Emily Blauel, Zalman Vaksman, Alex Lee, Rebecca Kaufman, Laura Egolf, Andrew Olshan, John Maris, Diskin Sharon. Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences [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 3030.

Distinct Characteristics and Clinical Outcomes to Predict the Emergence of MET Amplification in Patients with Non-Small Cell Lung Cancer Who Developed Resistance after Treatment with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

Simple SummaryMET amplification is one of the resistance determinants after EGFR-TKI therapy in EGFR mutant NSCLC. In this study, we evaluated the emergence of MET amplification after EGFR-TKI treatment failure. The median progression-free survival associated with the most recent EGFR-TKI treatment was shorter in MET amplification-positive patients than in negative patients. Smoking history and less intracranial progression are associated with MET amplification. Suboptimal responses with previous EGFR-TKI are associated with MET amplification. Proper MET amplification screening for therapeutic targeting is needed.Objectives: Patients with epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) ultimately acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) during treatment. In 5–22% of these patients, resistance is mediated by aberrant mesenchymal epithelial transition factor (MET) gene amplification. Here, we evaluated the emergence of MET amplification after EGFR-TKI treatment failure based on clinical parameters. Materials and Methods: We retrospectively analyzed 186 patients with advanced EGFR-mutant NSCLC for MET amplification status by in situ hybridization (ISH) assay after EGFR-TKI failure. We collected information including baseline patient characteristics, metastatic locations and generation, line, and progression-free survival (PFS) of EGFR-TKI used before MET evaluation. Multivariate logistic regression analysis was conducted to evaluate associations between MET amplification status and clinical variables. Results: Regarding baseline EGFR mutations, exon 19 deletion was predominant (57.5%), followed by L858R mutation (37.1%). The proportions of MET ISH assays performed after first/second-generation and third-generation TKI failure were 66.7% and 33.1%, respectively. The median PFS for the most recent EGFR-TKI treatment was shorter in MET amplification-positive patients than in MET amplification-negative patients (median PFS 7.0 vs. 10.4 months, p = 0.004). Multivariate logistic regression demonstrated that a history of smoking, short PFS on the most recent TKI, and less intracranial progression were associated with a high probability of MET amplification (all p < 0.05). Conclusions: Our results demonstrated the distinct clinical characteristics of patients with MET amplification-positive NSCLC after EGFR-TKI therapy. Our clinical prediction can aid physicians in selecting patients eligible for MET amplification screening and therapeutic targeting.