Trunk Mutations Research Articles

Comprehensive genomic profiling of infiltrative follicular variant of papillary thyroid carcinoma.

Infiltrative follicular variant of papillary thyroid carcinoma (IFVPTC) exhibits nuclear characteristics typical of papillary thyroid carcinoma (PTC) but demonstrates a follicular growth pattern. The diagnosis of IFVPTC presenting with atypical nuclear features of PTC poses challenges for both preoperative cytopathology and postoperative histopathology. In such cases, molecular markers are needed to serve as diagnostic aids. Given the limited knowledge of IFVPTC's genomic features, this study aimed to characterize its genetic alterations and identify clinically relevant molecular markers. Whole-exome sequencing of 50 IFVPTC tumor-normal pairs identified single-nucleotide variants, somatic copy number alterations (sCNAs), and subclonal architecture. Key mutations were verified via polymerase chain reaction and Sanger sequencing, whereas valuable biomarkers were validated via immunohistochemistry (IHC). This study found that endogenous processes rather than exogenous mutagens dominated the shaping of the genome of IFVPTC during tumorigenesis. BRAF V600E was the only common trunk mutation and significantly mutated gene in IFVPTC. Subcloning analysis found that most IFVPTC samples harbored two or more coexisting clones. sCNA analysis revealed that human leukocyte antigen C (HLA-C) and HLA-A were significantly amplified. Subsequent IHC investigations indicated that HLA-C shows promise in averting the misclassification of challenging-to-interpret IFVPTC and invasive encapsulated follicular variant of PTC (I-EFVPTC) as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Although there were several similarities between classic PTC and IFVPTC, they differed significantly in their sCNA patterns. This study provides valuable insights into IFVPTC's genetic alterations and highlights the potential of HLA-C IHC to distinguish challenging-to-interpret IFVPTC and I-EFVPTC from NIFTP, which will enhance the understanding of its molecular features for improved diagnosis and management.

Using molecular characteristics to distinguish multiple primary lung cancers and intrapulmonary metastases.

Multiple lung cancers may present as multiple primary lung cancers (MPLC) or intrapulmonary metastasis (IPM) with variations in clinical stage, treatment, and prognosis. However, the existing differentiation criteria based on histology do not fully meet the clinical needs. Next-generation sequencing (NGS) may play an important role in assisting the identification of different pathologies. Here, we extended the relevant data by combining histology and NGS to develop detailed identification criteria for MPLC and IPM. Patients with lung cancer (each patient had ≥2 tumors) were enrolled in the training (n = 22) and validation (n = 13) cohorts. Genomic profiles obtained from 450-gene-targeted NGS were analyzed, and the new criteria were developed based on our findings and pre-existing Martini & Melamed criteria and molecular benchmarks. The analysis of the training cohort indicated that patients identified with MPLC had no (or <2) trunk or shared mutations. However, 98.02% of mutations were branch mutations, and 69.23% of MPLC had no common mutations. In contrast, a higher percentage of trunk (33.08%) or shared (9.02%) mutations were identified in IPM, suggesting significant differences among mutated components. Subsequently, eight MPLC and five IPM cases were identified in the validation cohort, aligning with the independent imaging and pathologic distinction. Overall, the percentage of trunk and shared mutations was higher in patients with IPM than in patients with MPLC. Based on these results and the establishment of new determination criteria for MPLC and IPM, we emphasize that the type and number of shared variants based on histologic consistency assist in identification. Determining genetic alterations may be an effective method for differentiating MPLC and IPM, and NGS can be used as a valuable assisting tool.

Abstract 2094: A user-friendly R Shiny app for predicting drug response of cancer using deep learning

Abstract Realizing the full potential of precision oncology requires accurate prediction of treatment response. We previously developed a deep learning model, namely DeepDR, to predict drug sensitivity by integrating baseline profiles of mutations and gene expression of a cancer sample (Chiu et al.; DOI: 10.1186/s12920-018-0460-9). The model features a transfer learning design that captures two types of features: i) tumor relevant representations of mutation and expression data learned from tumor samples, and ii) predictive model for drug response (measured by IC50 values) learned from high-throughput drug screens of pan-cancer cell lines. DeepDR was applicable to both cell lines and tumors and achieved superior performance over conventional methods. To make the DeepDR model more accessible to biomedical researchers with limited programming skills, here we present a user-friendly platform implemented by using an R Shiny framework. The R Shiny app is a web interface that allows users to upload mutation and/or gene expression profiles of a cancer sample (cell or tumor model) and predict the sample’s response to 265 FDA-approved and investigational compounds covered by the screening library of the Genomics of Drug Sensitivity in Cancer (GDSC) project. The app provides an intuitive user interface to interactively visualize, search, and filter prediction results among 265 compounds. It also enables downstream analyses including statistical tests and provides links to external compound databases, such as PubChem. In the original publication of DeepDR, we have validated its performance across cell lines and tumors using well-known pharmacogenomic associations, such as estrogen receptor antagonist (tamoxifen) and EGFR-targeting drugs (afatinib and gefitinib), as well as a novel agent, CX-5461, in treating hematopoietic malignancies. Here we utilized the proposed Shiny app to further predict drug sensitivity of patients with hepatocellular carcinoma (HCC; n=356) of The Cancer Genome Atlas (TCGA). We investigated CTNNB1 as a demonstrating example for it is one of the most prevalent, yet undruggable, trunk mutations of HCC. A systematic search yielded nine compounds that were predicted to be significantly more effective in HCC tumors harboring CTNNB1 mutations (n=92) compared to others (n=264) (with one-tailed t-test P &amp;lt; 0.0001). These top compounds target critical cancer pathways, such as cell apoptosis (p53 activator and Bcl-2 inhibitor), the PI3K/AKT and MAPK signaling pathways, and DNA-dependent protein kinases, suggesting candidates for further in vitro and in vivo investigations. In summary, the present R Shiny app is a user-friendly platform that enables in silico drug screening using deep learning with no requirement of programming experience. We expect the tool to foster accessibility of our deep learning prediction machine and facilitate the process of drug development in cancer. Citation Format: Li-Ju Wang, Michael Ning, Tapsya Nayak, Satdarshan P. Monga, Yufei Huang, Yidong Chen, Yu-Chiao Chiu. A user-friendly R Shiny app for predicting drug response of cancer using deep learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2094.

Safety and tolerability of stereotactic radiotherapy combined with durvalumab with or without tremelimumab in advanced non-small cell lung cancer, the phase I SICI trial

IntroductionThis phase I study primarily addresses the safety and tolerability of Stereotactic radiotherapy on the primary tumor combined with double Immune Checkpoint Inhibition (SICI) in patients with non-small cell lung cancer (NSCLC). Increasing the release of neoantigens by radiotherapy might enhance response to immunotherapy. Especially, by targeting trunk mutations in the primary tumor. Materials and MethodsIn three sequential cohorts, immunotherapy regimes combined with stereotactic body radiotherapy (SBRT) on the primary tumor (1x20 Gy on 9 cc) were studied in stage IIIB/IV NSCLC patients progressing on chemotherapy. The first cohort (n = 3) received durvalumab. The second (n = 6) received a combination of tremelimumab and durvalumab followed by durvalumab monotherapy. The third cohort (n = 6) was similar except that the combination was reversed. Descriptive statistics were used to assess safety parameters and the exploratory outcomes of efficacy. Adverse events were reported using NCI CTCAE version 4.03. Exhaled breath was analyzed at baseline. ResultsFifteen patients were included. Median irradiated volume was 9.13 cc, on a median primary tumor volume of 79 cc. There were seven patients with grade 1–2, and two patients with grade 3 treatment related adverse events. There was 1 dose limiting toxicity (colitis) with double immunotherapy. ConclusionThe combination of SBRT to the primary tumor and double immunotherapy in advanced NSCLC patients is safe and feasible.

Elucidation of genomic origin of synchronous endometrial and ovarian cancer (SEO) by genomic and microsatellite analysis.

Elucidation of clonal origin of synchronous endometrial and ovarian cancers (SEOs). We reviewed 852 patients who diagnosed endometrial and/or ovarian cancer. Forty-five (5.3%) patients were diagnosed as SEOs. We evaluated blood and tissue samples from 17 patients. We analyzed the clonal origins of 41 samples from 17 patients by gene sequencing, mismatch microsatellite instability (MSI) polymerase chain reaction assay and immunohistochemical (IHC) staining of 4 repair genes. Sixteen of 17 patients had at least 2 or more trunk mutations shared between endometrial and ovarian cancer suggesting the identical clonal origins. The shared trunk mutation are frequently found in endometrial cancer of the uterus, suggesting the uterine primary. Four out of 17 (24%) SEOs had mismatch repair (MMR) protein deficiency and MSI-high (MSI-H) states. One case was an endometrial carcinoma with local loss of MSH6 protein expression by IHC staining, and the result of MSI analysis using the whole formalin-fixed, paraffin-embedded specimen was microsatellite stable. In contrast, ovarian tissue was deficient MMR and MSI-H in the whole specimen. This indicated that MMR protein deficiency could occur during the progression of disease. Most SEOs are likely to be a single tumor with metastasis instead of double primaries, and their origin could be endometrium. In addition, SEOs have a high frequency of MMR gene abnormalities. These findings not only can support the notion of uterine primary, but also can help to expect the benefit for patients with SEOs by immuno-oncology treatment.

Liquid Biopsy Detects Early Molecular Response and Predicts Benefit to First-Line Chemotherapy plus Cetuximab in Metastatic Colorectal Cancer: PLATFORM-B Study.

Chemotherapy plus anti-EGFR is standard first-line therapy in RAS wild-type (wt) metastatic colorectal cancer (mCRC), but biomarkers of early response are clinically needed. We aimed to define the utility of ctDNA to assess early response in patients with mCRC receiving first-line anti-EGFR therapy. Prospective multicentric study of tissue patients with RAS wt mCRC treated with first-line chemotherapy plus cetuximab undergoing sequential liquid biopsies. Baseline and early (C3) ctDNA were analyzed by NGS. Trunk mutations were assessed as surrogate marker of total tumor burden. RAS/BRAF/MEK/EGFR-ECD were considered mutations of resistance. ctDNA results were correlated with clinical outcome. One hundred patients were included. ctDNA was detected in 72% of patients at baseline and 34% at C3. Decrease in ctDNA trunk mutations correlated with progression-free survival (PFS; HR, 0.23; P = 0.001). RAS/BRAF were the only resistant mutations detected at C3. An increase in the relative fraction of RAS/BRAF at C3 was followed by an expansion of the RAS clone until PD, and was associated with shorter PFS (HR, 10.5; P < 0.001). The best predictor of response was the combined analysis of trunk and resistant mutations at C3. Accordingly, patients with "early molecular response" (decrease in trunk and decrease in resistant mutations) had better response (77.5% vs. 25%, P = 0.008) and longer PFS (HR, 0.18; P < 0.001) compared with patients with "early molecular progression" (increase in trunk and/or increase in resistant mutations). ctDNA detects early molecular response and predicts benefit to chemotherapy plus cetuximab. A comprehensive NGS-based approach is recommended to integrate information on total disease burden and resistant mutations. See related commentary by Eluri et al., p. 302.

Abstract 666: A phase 1 study to detect adverse events after SBRT and immunotherapy by electronic nose in advanced NSCLC

Abstract It is increasingly understood that cancers can be recognized by the immune system and that inflammation relates to response. Combining stereotactic radiotherapy (SBRT) to increase release of cancer cell antigens with an anti-CTLA-4 and PD-L1 inhibitor may lead to increased response rates. Due to tumor heterogeneity it may be important to irradiate the primary tumor (addressing trunk mutations), instead of its metastasis (branch mutations). No solid predictive biomarker for response and/or immunotherapy related adverse events (TRAEs) is available. Electronic nose (eNose) technology measures the complete mixture of volatile organic compounds in exhaled breath and can detect lung cancer based on pattern recognition of the breath profile. We hypothesize that eNose is able to predict TRAEs and response in patients with NSCLC. Methods: In 3 sequential cohorts, immunotherapy regimes combined with SBRT were studied in stage IIIB/IV NSCLC patients progressing on chemotherapy. All patients were irradiated on the primary tumor (1x20 Gy on 9cc) 1 week after the 1st dose of immunotherapy. The 1st cohort (n=3) received durvalumab. The 2nd and 3rd cohort (both n=6) received a combination of durvalumab and tremelimumab followed by durvalumab monotherapy. Duplicate eNose measurements were performed by using the SpiroNose that contains 7 metal oxide semiconductor sensors. TRAEs were categorized using NCI CTCAE version 4.3. Descriptive statistics were used to summarize baseline characteristics and TRAEs. The relationship between breath profiles and response and TRAEs was analyzed with advanced signal processing, ambient correction and Mann-Whitney U test. Linear discriminant and receiver operating characteristics analysis followed. Pearson correlation and regression assessed duration of response. Findings: Fifteen patients were included as described above. Baseline characteristics of the groups were comparable. Median progression free survival was 2 months, overall survival 10 months (immature). No statistical difference was found in (duration of) response. There was 1 low grade TRAE (CTC 1-2) in cohort 1 and 10 in cohort 2/3. High grade TRAEs (CTC 3) were only present in cohort 2/3 (n=3) and 1 patient discontinued treatment. There was one dose limiting toxicity. At baseline, 12/15 patients performed eNose measurements; 7 with TRAEs and 5 without. The TRAE group had a significant higher sensor 7 signal compared to those without TRAE (p=.042). The cross-validated accuracy for detecting TRAE was 67%. The ROC-AUC was .857 [.638-1]. Interpretation: eNose breath profiles at baseline may predict which patients will develop TRAEs. The small sample size increases the risk of overfitting, therefore another cohort of 34 immuno-monotherapy patients is being analyzed (15 with and 19 without TRAEs, pending). We demonstrated no new safety data. Disclosure: This study was sponsored by a research grant from AstraZeneca. Citation Format: M Benthe Muntinghe-Wagenaar, Hanneke Kievit, Lucie B. Hijmering-Kappelle, Birgitta I. Hiddinga, J Fred Ubbels, Robin Wijsman, Mechteld F. Brasz, Gitte Slingers, Rianne de Vries, Milou M. Schuurbiers, Harry J. Groen, Huib A. Kerstjens, Anthonie J. van der Wekken, Michel M. van den Heuvel, T Jeroen Hiltermann. A phase 1 study to detect adverse events after SBRT and immunotherapy by electronic nose in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 666.

A phase 1 study in patients with advanced non-small cell lung cancer treated with stereotactic radiotherapy to the primary tumor combined with durvalumab and tremelimumab.

e21121 Background: Cancers can be recognized by the immune system and immunotherapy (ICI) has become an important treatment for lung cancer. Compared to chemotherapy, combining an anti-CTLA-4 and PD-L1 inhibitor increases response- and survival rates independent of PD-L1 status. We hypothesized that adding stereotactic radiotherapy (SBRT) to increase the release of cancer cell antigens might improve treatment results even further. Because of accumulating tumor heterogeneity it may be important to irradiate the primary tumor (addressing trunk mutations), rather than its metastases (branch mutations). We assessed toxicity of adding SBRT to (dual) ICI. Methods: In 3 sequential cohorts, ICI regimes combined with SBRT were studied in stage IIIB/IV NSCLC patients progressing on chemotherapy. All patients were irradiated on an FDG avid part of the primary tumor (1x20 Gy on 9cc) 1 week after the 1st dose of ICI; in case of dual ICI the 1st dose of the 2nd ICI was administered within 1 week following SBRT. Following treatments both ICI were combined. The 1st cohort (n = 3) received durvalumab (D) monotherapy for up to 1 year. The 2nd cohort starting with tremelimumab (T) and the 3rd cohort starting with D (both n = 6) received a combination of D and T (4 cycles) followed by D monotherapy for up to 1 year. The 2nd and 3rd cohort only proceeded when dose limiting toxicities (DLT) occurred in ≤33% of cases in the previous cohort. DLT was defined as any grade ≥3 toxicity that occurred during the first 8 weeks of therapy. Treatment related adverse events (TRAEs) were categorized using NCI CTCAE version 4.3. Descriptive statistics were used to summarize baseline characteristics and TRAEs. Results: Fifteen patients were included from June 2018 to November 2020. Last study visit was in September 2021. Baseline characteristics of the groups were comparable. The median irradiated tumor volume was 9.13 cc (8.98-9.60 cc) with a mean lung dose of 0.44 Gy (0.14-0.73 Gy). The V20 was nihil (0-0.1%) and the V5 1.8% (0-4%). There was 1 DLT in cohort 3 (colitis grade 3). There was 1 low grade TRAE (pneumonitis grade 2) in cohort 1. In cohort 2, 6 low grade TRAEs (CTC 1-2) were observed and 2 high grade (CTC 3) in 1 patient, who discontinued treatment. In cohort 3, 4 low grade and 1 high grade TRAE occurred. Median progression free survival was 2 months, median overall survival 10 months. Conclusions: No additional toxicity was observed by adding SBRT to the primary tumor to (dual) ICI. Clinical trial information: 2017-002797-39. [Table: see text]

Mutation Analysis of Second Primary Tumors in Oral Cancer in Taiwanese Patients through Next-Generation Sequencing.

Head and neck cancer has poor overall survival. Patients with head and neck cancer more frequently develop second primary tumors than do patients with other cancers, leading to a poor prognosis. In this study, we used next-generation sequencing to analyze and compare mutations between first tumors and second tumors in oral cancer. We retrieved tumor tissues collected from 13 patients who were diagnosed twice as having cancer. We used driver gene and trunk mutations to distinguish between recurrent cancer and primary cancer in oral cancer. We observed unique driver gene mutations in three patients with an initial clinical diagnosis of recurrent cancer; hence, we believe that the corresponding patients had primary cancer. Four patients with an initial clinical diagnosis of primary cancer were found to actually have recurrent cancer according to our results. Genetic testing can be used to enhance the accuracy of clinical diagnosis.

Association of recurrent APOBEC3B alterations with the prognosis of gastric-type cervical adenocarcinoma

ObjectiveGastric-type cervical adenocarcinoma (GCA) is a rare and aggressive type of endocervical adenocarcinoma (ECA) with distinct histopathologic features and unfavorable treatment outcomes, but no genomic prognostic factor has been revealed. We aimed to systematically investigate the somatic alterations of GCA at genome-wide level and evaluate their prognostic value. MethodsWe performed whole-exome sequencing (WES) on 25 pairs of tumor and matched normal samples to characterize the genomic features of Chinese patients with GCA and investigated their relations to histopathological characterizations and prognosis. The prognostic value of the genomic alterations was evaluated in a total of 58 GCA patients. ResultsMutations were commonly observed in reported GCA-related driver genes, including TP53 (32%), CDKN2A (20%), SKT11 (20%), BRCA2 (12%), SMAD4 (12%), and ERBB2 (12%). Recurrent novel trunk mutations were also observed in PBRM1 (12%), FRMPD4 (12%), and NOP2 (8%) with high variant allele frequency. Moreover, enrichment of the APOBEC signature was attributed to frequent gain of somatic copy number alteration (SCNA) of APOBEC3B (20%), which perfectly matched the nuclear-positive staining of APOBEC3B through immunohistochemistry. In contrast, APOBEC3B alteration was absent in patients with conventional type of ECA (N = 52). Notably, positive APOBEC3B was consistently enriched in patients with favorable prognosis in both the discovery cohort and an additional 33 GCA patients, thus indicating a significant association with lower relapse risk of GCA independent of cancer stage (P = 0.02). ConclusionOur results can aid understanding of the molecular basis of GCA in the Chinese population by providing genomic profiles and highlighting the potential prognostic value of APOBEC3B for GCA through routine clinical IHC.

Intra-Tumor Heterogeneity of Colorectal Cancer Necessitates the Multi-Regional Sequencing for Comprehensive Mutational Profiling.

BackgroundThe panorama and details of quantitative intratumor heterogeneity have not been fully investigated in colorectal cancer (CRC) patients with solitary lesion without distal metastasis, and its influences on sequencing interpretation and therapeutic strategies have not been explored.MethodsCancer tissues and matched blood from 70 sporadic CRC patients were collected and were divided into two cohorts. Four individual tissue biopsies were obtained from each of the 47 patients (multi-sample cohort). One random cancer tissue biopsy was obtained from each of the rest 23 patients (single-sample cohort). A 10 mL of blood was collected from all patients and the circulating cell-free DNA (cfDNA) was extracted. A 605-gene panel was used for targeted sequencing with tissue and paired blood.ResultsMutational landscape revealed significantly higher mutational frequency in APC, CARD11 and CSMD3 in multi-sample cohort than single-sample cohort (P<0.05). The number of mutations and the ratio of trunk, shared and branch mutations showed extensive heterogeneity in multi-sample cohort, and the percentage of trunk mutations in major driver genes, including APC, TP53 and KRAS, was higher than 70%. A total of 929 mutations were detected in tissue/blood in multi-sample group, with 921(99.1%) from tissue and 472(50.8%) from blood (464 common mutations,49.9%). In contrast, 394 mutations were detected in tissue/blood in single-sample group, with 231 (58.6%) from tissue and 219 (55.6%) from blood (56 common mutations, 11.9%). The number of mutations of major driver genes detected in tissue was higher than that in blood in the multi-sample cohort, while it was similar in the single-sample group. Quantification analysis revealed differential correlation between tissue and blood VAF in trunk, shared and branch mutations. Meanwhile, VAF of trunk mutations was significantly higher than shared mutations and branch mutations. VAF exhibited significant differences among distinct stages, locations, differentiation and sex status.ConclusionCharacteristic extensive heterogeneity was revealed for solitary CRC without distal metastasis. Multi-regional biopsy was necessary for comprehensive mutation detection in CRC.

Disovery of Novel Mutations Exclusively Shared By Accelerated and Blast Crisis Phase CML Patients Using Whole Exome Sequencing:Implication in Hunt for Common Biomarker /Drug Target of CML Progression

Introduction:Chronic Myeloid Leukemia (CML) is resulted due t (9;22) which leads to fusion oncogene BCR-ABL. Although chronic phase (CP) CML is treatable, accelerated (AP) and blast phases (BP) have high degrees of drug resistance and subsequent treat failures 1. Mechanism of this disease progression in CML is poorly understood and few markers studied in this regard are found only in a subset of advanced phase CML patients 2,3. Therefore, this study was designed to find out common gene mutations (trunk mutations) associated with all advanced phase CML patients.Material and Methods:We investigated imatinib responder AP (N=13), BC (10) and CP (5) CML patients along with age-sex matched healthy controls using whole exome sequencing (WES). Illumina NGS instrument (HiSeq) generated bcl files which were converted to fastq files by using bcl2fastqtool 4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio 4. R package was employed to align specific gene mutants to disease phases 5. In order find a common CML progression biomarkers, gene mutations shared by all patients at a CML phase (so called trunk mutations) were selected and confirmed using Sanger sequencing.Results:Whole exome sequencing revealed novel gene mutations associated with different disease phases in CML. CP CML patients with long term CML response harbored mutations in TTN, PDLIM4, CELF2, ANO5, DOK2, MYO16, RAI1, LTBP3, CNNM3. On the other hand, specific genes were found mutated in AP (EV15L, FAM120B, ZNF208, PML, TMEM145, ASXL1, ABL1) and BC (ATXN3)CML patients.Discussion & Conclusions:All the genes mutated in AP and BC CML were either involved in other cancers or cellular processes like DNA repair, regulation of gene expression (for instance ATXN3 gene mutated in all BC-CML is involved in DNA repair and in repressing expression of some other genes6 which indicates role of damaged DNA repair in CML blast crisis). Mutations in long term CML responders indicate role of all are some of these genes in relation to sensitivity to tyrosine kinase inhibitors (TKIs). Very recently, it has been reported that CML patients with rs460089-GC (SLC22A4) genotype had stable major molecular response 7. Biomarkers of stable response can help identify patients who can be the candidates of cessation of TKIs which is one of the major focus of many CML trials. Identification of biomarkers of early CML progression can help identify and clinically intervene patients at high risk of transformation to advanced and blast phases of CML which can considerably minimize morbidities and mortalities associated with CML patients 2,3.

Mutation profile of non-small cell lung cancer revealed by next generation sequencing

BackgroundPrecision therapy for lung cancer requires comprehensive genomic analyses. Specific effects of targeted therapies have been reported in Asia populations, including Taiwanese, but genomic studies have rarely been performed in these populations.MethodWe enrolled 72 patients with non-small cell lung cancer, of whom 61 had adenocarcinoma, 10 had squamous cell carcinoma, and 1 had combined adenocarcinoma and squamous cell carcinoma. Whole-exome or targeted gene sequencing was performed. To identify trunk mutations, we performed whole-exome sequencing in two tumor regions in four patients.ResultsNineteen known driver mutations in EGFR, PIK3CA, KRAS, CTNNB1, and MET were identified in 34 of the 72 tumors evaluated (47.22%). A comparison with the Cancer Genome Atlas dataset showed that EGFR was mutated at a much higher frequency in our cohort than in Caucasians, whereas KRAS and TP53 mutations were found in only 5.56% and 25% of our Taiwanese patients, respectively. We also identified new mutations in ARID1A, ARID2, CDK12, CHEK2, GNAS, H3F3A, KDM6A, KMT2C, NOTCH1, RB1, RBM10, RUNX1, SETD2, SF3B1, SMARCA4, THRAP3, TP53, and ZMYM2. Moreover, all ClinVar pathogenic variants were trunk mutations present in two regions of a tumor. RNA sequencing revealed that the trunk or branch genes were expressed at similar levels among different tumor regions.ConclusionsWe identified novel variants potentially associated with lung cancer tumorigenesis. The specific mutation pattern in Taiwanese patients with non-small cell lung cancer may influence targeted therapies.

Genomic and immune intratumor heterogeneity in gastric linitis plastica.

e16518 Background: Gastric linitis plastica (LP) is a rare and aggressive type of gastric cancer (GC) for which the genomic landscape and architecture have gone largely undescribed. Methods: 4 LP patients were enrolled. 10 region tumor samples of each LP patient and matched peripheral blood were collected. Matched blood cells of each patient were also collected for removing germline background Whole-exome sequencing(WES), TCR sequencing, TCGA gastric cancer and several WES articles data were used to investigate intra and inter patient genomic and immune heterogeneity. Results: All 4 LP patients were female and were in stage III. LP biopsies were sequenced with median 290.6x effective depth. A total of 11,504 somatic mutations including 6,339 non-silent mutations were identified. The median non-silent tumor mutation burden (TMB) of biopsy samples was 3.23 mutations/Mb (range from 1.36 to 4.88), which was comparable to gastric adenocarcinoma(p = 0.3). Phylogenetic trees of 4 LP patients demonstrated clear evidence of branched evolution, and the phylogenetic trees varied extensively across the four cases. The percentages of trunk mutations of 4 LP were 12.8%, 5.4%, 5.4% and 30.7%, respectively, while the proportions of trunk neoantigens were 6.2%, 2.2%, 12% and 12.4, respectivelyWhen comparing LP to other multiregion WES studies, e.g., lung adenocarcinoma, renal cell carcinoma, and esophageal squamous cell carcinoma, LP was one of the most heterogeneous tumor types. The top mutational signatures in this cohort associated with spontaneous deamination, DNA mismatch repair (MMR), and small indels at repeats etc. Furthermore, profound TCR ITH was observed in all 4 LP patients. None of the T cell clones were shared among all tumor regions and 94.23-94.41% T cells were restricted to individual tumor regions. To quantify the TCR ITH, we utilized the Morisita overlap index (MOI), which ranged from 0.34 to 0.56 across different regions within the same tumors suggesting marked inter-individual TCR repertoire heterogeneity and profound intratumor TCR heterogeneity. Conclusions: Based on whole-exome sequencing and TCR sequencing, we demonstrate that LP is highly heterogeneous for mutations, neoantigens and T cells, which contributes to its poor prognosis.

Whole-exome mutational landscape of metastasis in patient-derived hepatocellular carcinoma cells

In order to explore the genomic basis for liver cancer metastasis, whole-exome sequencing (WES) was performed on patient-derived hepatocellular carcinoma (HCC) cell lines with differential metastatic potentials and analyzed their clonal evolution relationships. An evolutionary tree based on genomic single nucleotide polymorphism (SNP) was constructed in MegaX software. The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55% (range, 15.38%–18.17%). C: G > T: A and T: A > C: G somatic transitions were the two most frequent substitutions. In these metastatic HCC cell lines, non-silent gene mutations were found in 21.88% of known driver genes and 10 classical signaling pathways. The protein interaction network was constructed by STRING, and hub genes were found in the shared trunk mutation genes and the heterogeneous branch mutations respectively. In cBioPortal database, some of the selected hub genes were found to be associated with poor overall survival (OS) of HCC patients. Among the mutated HCC driver genes, a novel KEAP1 mutation with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells. In conclusion, WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo, and they do indeed have a genetic relationship at the genomic level.

Clonal Architecture of &lt;i&gt;EGFR&lt;/i&gt; Mutation Predicts the Efficacy of EGFR-Tyrosine Kinase Inhibitors in Advanced NSCLC: A Prospective Multicenter Study

Background: Although EGFR mutations in lung cancer are identified most often as trunk mutations in the early stage, cancer evolution often follows a branched trajectory. Whether EGFR would always be the dominant clone in the advanced stage is unknown. Methods: This prospective multicenter clinical trial recruited treatment naive patients with stage IIIB-IV non–small cell lung cancer from 14 centers in China. Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1021 genes. Clonal dominance analysis was performed on the basis of PyClone. Findings: Overall, 300 patients were recruited and 132 patients treated with first line targeted therapy were followed. The median follow-up time was 10 months (IQR 6–13). Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The mPFS was longer for these patients than for the 22 patients who had EGFR non-dominant clone (11 months vs 10 months, hazard ratio [HR] 0.46, 95% CI 0.24–0.88, p = 0.02). The difference was more significant if patients who were defined as having EGFR dominant clone by both tumor tissue and plasma (n = 43) versus those not (n = 8) (11 vs 6 months, HR = 0.13, 95% CI 0.04–0.50, p = 0.003). Moreover, multivariate cox proportional hazard ratio analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors. Interpretation: In advanced NSCLC,EGFR mutations do not always make up a dominant clone. Clonal architecture of EGFR in the pretreatment ctDNA is associated with the efficacy of EGFR-TKIs in NSCLC. Trial Registration: The trial has been registered with the number NCT03059641. Funding Statement: Geneplus-Beijing Co. Ltd. Declaration of Interests: RC, PL, LC and XY were employees of Geneplus-Beijing Co. Ltd. All other authors declare no conflict of interests. Ethics Approval Statement: This study was performed under a protocol (KS1707) approved by the Institutional Review Board of Shanghai Chest hospital (Shanghai, China).

Abstract GMM-023: PATIENT DERIVED XENOGRAFTS (PDXS) AND PDX DERIVED TUMOR CELLS (PDTC) ALLOW THE IDENTIFICATION OF ACTIONABLE CANCER GENES AND TREATMENT OPTIONS FOR PLATINUM REFRACTORY/RESISTANT OVARIAN CARCINOMAS

Abstract Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments even after the incorporation of anti-angiogenic therapies. Besides anti-PARP inhibitors, matching individual critical genomic alterations with the best available drugs has not advanced as in other cancers, likely because a handful of cancer-related genes are mutated at high frequency, while many more are found mutated at much lower frequencies. This so called “mutation tail” is not only long but also mostly unexplored. We used Patient Derived Xenografts (PDXs) to identify actionable cancer genes and PDX Derived Tumor Cells (PDTCs) to accelerate the discovery of treatment options. We envisioned that the alleged weakness of PDX models, i.e. lack of human stromal and immune cells, might be instrumental to identify mutations in cancer and to test approved or experimental targeted drugs as monotherapy or in different combinations to link biomarkers to treatments. Forty-nine PDX lines from metastatic epithelial ovarian carcinomas have been propagated and fully characterized as far as histology, immunohistochemistry of epithelial and high-grade serous-specific markers and presence of TP53 and BRCA1/2 mutations. Copy number variations (CNV) analysis and Whole Exome Sequencing (WES) were carried out PDX lines derived from naïve metastatic high-grade epithelial ovarian carcinomas, which came out to be refractory/resistant to platinum drugs. We studied non-synonymous mutations with allele frequencies ≥0.1. Only mutations in cancer genes listed in databases were further analyzed. SNPdb allowed ruling out polymorphisms. SIFT and PROVEAN softwares predicted deleterious or damaging effects onto the protein sequences. DGIdb helped selecting actionable genes. We identified in one PDX line, a possibly loss-of-function mutation of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) had an allele frequency=0.9 in early and late passages. Moreover, in two micro-dissected FFPE samples of the source tumor this mutation had an allele frequency nearly identical to that of the mutated TP53. Hence, PIK3R1W624R could be a trunk mutation in the PDX line and possibly in the human counterpart. Treatment options were assayed ex-vivo, on short-term cultures of PDTCs of the PIK3R1W624R PDX line. Buparlisib, a pan-class I PI3K inhibitor, showed the ability to block proliferation of PDTCs and the growth in vivo of PDXs in regression preclinical trial. These data proofed-the-concept that a PDX-based pipeline is able to unveil actionable pathways for the treatment of advanced/metastatic ovarian cancer. Citation Format: Martina Olivero, Jessica Erriquez, Maddalena Arigoni, Sonia Capellero, Concetta D'Ambrosio, Gloria Mittica, Fulvio Borella, Dionyssios Katsaros, Silvana Privitera, Enrico Berrino, Tiziana Venesio, Giorgio Valabrega, Raffaele Calogero and Maria Flavia Di Renzo. PATIENT DERIVED XENOGRAFTS (PDXS) AND PDX DERIVED TUMOR CELLS (PDTC) ALLOW THE IDENTIFICATION OF ACTIONABLE CANCER GENES AND TREATMENT OPTIONS FOR PLATINUM REFRACTORY/RESISTANT OVARIAN CARCINOMAS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-023.

Clinical Sequencing Analysis of the Mutation Profiles Associated with Extreme Radioresistance

Precision medicine using genetic data of individual tumors is emerging in the field of cancer treatment. However, mutation profiles associated with the response of tumors to radiotherapy have not been fully elucidated. We hypothesized that the patients who exhibited extreme resistance to radiotherapy carry relevant mutation profiles. The patient with uterine cervical cancer that repeatedly locally-recurred after multiple rounds of curative radiotherapy, was selected as analytical subject. Biopsy samples were obtained from treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy. Exon sequencing of 409 cancer-related genes was performed using a next-generation sequencer. Prevalence of candidate mutation profiles in clinical malignancy specimens was investigated using cBioportal. Contribution of candidate mutation profiles on radioresistance was examined by (i) meta-analysis of published in vitro radiosensitivity data combined with genomics data of Cancer Cell Line Encyclopedia (CCLE), and (ii) isogenic cell-based experiments using gene knock-in and siRNA techniques. The Differences between two groups were examined by Mann–Whitney test. The sequencing analysis identified activating mutations in PIK3CA (E545K) and KRAS (G12D), and putative biallelic inactivating mutations in SMAD4 (R361C and R261H) as trunk mutation signatures that persisted over the clinical course. Mining of published genomics data revealed that simultaneous mutations of KRAS and SMAD4 have not been reported previously for uterine cervical cancer (n = 0/343); the mutation signature was also rare among various types of malignancies (1.2%, n = 279/22928). Interestingly, this mutation signature was most prevalent in pancreatic cancer (23.7%, n = 195/820 cases) that is known to be clinically radioresistant. These data indicated that simultaneous mutations of KRAS and SMAD4 contributed to extreme radioresistant nature of the tumors analyzed in this study. Mining of CCLE genomics data identified 12 cancer cell lines carrying simultaneous mutations of KRAS and SMAD4. Meta-analysis of 94 publications identified by systemic literature review demonstrated that SF2, surviving fraction after 2 Gy-irradiation as assessed by clonogenic assays, was significantly higher for the KRAS/SMAD4-mutated cell lines compared to that for the background-matched control cell lines (P = 0.010). Experiments using KRASG12D-knock in- and SMAD4-knock down-SW48 cells demonstrated that SF2 for the KRAS/SMAD4-modulated cells was significantly higher than that for the parental cells (P = 0.034). These data indicate that simultaneous mutations in KRAS and SMAD4 are associated with radioresistance. The mutation signature is potentially useful as the biomarker for radioresistance in the practice of precision medicine.

Genomic Landscape and Immune Microenvironment Features of Preinvasive and Early Invasive Lung Adenocarcinoma

BackgroundUnderstanding the genomic landscape and immune microenvironment features of preinvasive and early invasive lung adenocarcinoma may provide critical insight and facilitate development of novel strategies for early detection and intervention. MethodsA total of 80 tumor tissue samples and 30 paired histologically normal lung tissue samples from 30 patients with adenocarcinoma in situ (AIS) (n = 8), minimally invasive adenocarcinoma (MIA) (n = 8), and invasive adenocarcinoma (IAC) (n = 14) were subjected to multiregion whole exome sequencing and immunohistochemistry staining for CD8 and programmed death ligand 1 (PD-L1). ResultsAll tumors, including AIS, exhibited evidence of genomic intratumor heterogeneity. Canonical cancer gene mutations in EGFR, erb-b2 receptor tyrosine kinase 2 gene (ERBB2), NRAS, and BRAF were exclusively trunk mutations detected in all regions within each tumor, whereas genes associated with cell mobility, gap junction, and metastasis were all subclonal mutations. EGFR mutation represented the most common driver alterations across AIS, MIA, and IAC, whereas tumor protein p53 gene (TP53) was identified in MIA and IAC but not in AIS. There was no difference in PD-L1 expression among AIS, MIA, and IAC, but the CD8 positivity rate was higher in IAC. Tumors positive for both PD-L1 and CD8 had a larger proportion of subclonal mutations. ConclusionsMutations in EGFR, ERBB2, NRAS, and BRAF are early clonal genomic events during carcinogenesis of lung adenocarcinoma, whereas TP53 and cell mobility, gap junction, and metastasis-related genes may be late events associated with subclonal diversification and neoplastic progression. Genomic intratumor heterogeneity and immunoediting are common and early phenomena that may have occurred before the acquisition of invasion.

Abstract 2725: The single-cell atlas of driver mutations in acute myeloid leukemia

Abstract Assessment of intratumor genetic heterogeneity by next-generation sequencing (NGS) is confounded by tumor purity and zygosity of mutations. Furthermore, bulk NGS cannot visualize the cell-level co-occurrence or exclusivity among multiple mutations, causing the inaccurate inference of subclonal architecture. Here, we performed single cell DNA sequencing (scDNA seq) in 82 bone marrow samples from 70 patients with acute myeloid leukemia (AML) using a novel microfluidics-based platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). All samples were concurrently sequenced by the bulk NGS using 295 gene exome capture sequencing. Selected cases were also analyzed by SNP array (Illuminia Omni 2.5 array) to obtain allele-specific copy number data. In total, 319,406 cells (median 3,755 cells/sample) were genotyped with median allele drop-out rate of 8.7% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 24x/cell. The scDNA seq detected 230 of 238 (97%) bulk NGS-confirmed mutations. RUNX1 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss-of-heterozygosity of RUNX1 in these cases. Additionally, in cases with homozygous FLT3-ITD mutations, SNP array detected allele-specific copy number gain of mutant loci, which likely resulted in homozygous calls. scDNA seq data unambiguously resolved the single-cell level co-occurrence of driver mutations in AML such as DNMT3A/FLT3-ITD/NPM1, SRSF2/IDH2, and ASXL1/RUNX1, confirming the cooperative function of these driver mutations. On the other hand, the data also revealed mutually exclusive relationships at cellular level between IDH1/IDH2, FLT3-ITD/TKD, NRAS/KRAS, NRAS/PTPN11, and SRSF2/EZH2 mutations, which supports the functional redundancy of these genes in leukemogenesis. Inference of phylogenetic trees using SCITE algorithm (Jahn et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the analyzed cases had a linear evolution pattern where the founder mutations linearly acquired sub-clonal mutations in a step-wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 11 patients revealed the remodeling of clonal architecture at relapse. Patients who had 2 or more major subclones at baseline had significantly worse overall survival than those with one subclone (2-year OS 13 vs. 70 months, p = 0.0493). For the first time our data provides a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single-cell level using high-throughput scDNA seq genotype information. Citation Format: Kiyomi Morita, Feng Wang, Katharina Jahn, Yuanqing Yan, Robert Durruthy-Durruthy, Anup Parikh, Jairo Matthews, Latasha Little, Curtis Gumbs, Jianhua Zhang, Xingzhi Song, Erika Thompson, Keyur Patel, Carlos Bueso-Ramos, Courtney DiNardo, Farhad Ravandi, Elias Jabbour, Michael Andreeff, Jorge Cortes, Marina Konopleva, Guillermo Garcia-Manero, Hagop Kantarjian, Dennis J. Eastburn, P Andrew Futreal, Niko Beerenwinkel, Koichi Takahashi. The single-cell atlas of driver mutations in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2725.