Tumor-associated Mutations Research Articles

MDM4 exon skipping upon dysfunctional ribosome assembly

Recent studies revealed how nucleolar stress enhances MDM4 exon skipping and activates p53 via the ribosomal protein L22 (RPL22; eL22). Tumor-associated L22 mutations lead to full-length MDM4 synthesis, overcoming tumor suppression by p53. This forum article explores how MDM4 splicing patterns integrate stress signaling to take p53-dependent cell fate decisions.

Long noncoding RNA LINC01550 inhibits colorectal cancer malignancy by suppressing the Wnt/β-catenin signaling pathway.

Colorectal cancer (CRC) is a common gastrointestinal malignancy. Long noncoding RNAs (lncRNAs) are associated with the progression of various cancers, including CRC. Herein, we explored the function of lncRNA LINC01550 in CRC. LINC01550 expression in CRC was analyzed using The Cancer Genome Atlas (TCGA). The diagnostic value of LINC01550 was evaluated using ROC curves. The relationship between clinicopathological variables and LINC01550 expression was explored, and its prognostic value was assessed using Kaplan-Meier and Cox regression analyses. The relationship between LINC01550 expression and immune cell infiltration was analyzed using CIBERSORT. Tumor-associated mutations and drug sensitivity were compared between high and low LINC01550 expression groups. The effects of LINC01550 overexpression on CRC cells were investigated using CCK-8, flow cytometry, wound healing, Transwell, qRT-PCR, and western blot assays. LINC01550 was downregulated in CRC tissues, and the low expression of LINC01550 was correlated with advanced stage and metastasis. CRC patients with low LINC01550 expression had poorer overall survival. LINC01550 expression was an independent risk factor for CRC prognosis. APC and TP53 mutations were more frequent in the low LINC01550 expression group, while the high LINC01550 expression group was significantly more sensitive to 5-fluorouracil, irinotecan, trametinib, gemcitabine, rapamycin, and XAV939. LINC01550 overexpression suppressed the proliferation, migration, invasion, and epithelial-mesenchymal transition of HCT-116 and HT-29 cells and promoted apoptosis. LINC01550 exerted these effects by inhibiting Wnt/β-catenin signaling. Our results suggest LINC01550 as a diagnostic and prognostic predictor in CRC that acts as a tumor suppressor and a potential therapeutic target.

TP53 and/or BRCA1 Mutations Based on CtDNA Analysis as Prognostic Biomarkers for Primary Triple-Negative Breast Cancer.

Precise biomarkers for predicting the therapeutic efficacy of molecularly targeted drugs are limited at the protein level; thus, it has been important to broadly scrutinize individual cancer driver gene mutations for effective cancer treatments. Multiplex cancer genome profiling can comprehensively identify gene mutations that are therapeutic targets using next-generation sequencing (NGS). In addition, circulating tumor DNA (ctDNA) is a DNA fragment released into the blood by tumor cell-derived cell death or apoptosis. Liquid biopsy with ctDNA is a novel clinical test for identifying genetic mutations in an entire population noninvasively, in real-time, and heterogeneously. Although there are several reports on ctDNA, fewer have evaluated ctDNA with NGS before an initial treatment for breast cancer patients. Therefore, we examined whether analyzing tumor-associated gene mutations in primary breast cancer based on ctDNA could serve as a biomarker for prognosis and optimal treatment selection. Ninety-five primary breast cancer patients treated at our department from January 2017 to October 2020 were included. Pretreatment plasma samples were subjected to NGS analysis of ctDNA, and correlations with patients' clinicopathological characteristics were evaluated. Fifty-nine (62.1%) patients were positive for ctDNA. ctDNA tended to be positive in hormone receptor-negative, and TP53 (34%), BRCA1 (20%), and BRCA2 (17%) gene mutations were more frequent. Regarding recurrence-free survival, the prognosis was poor in the TP53 and/or BRCA1 mutation-positive groups, especially in triple-negative breast cancer (TNBC) patients. In conclusion, the results of this study indicate that ctDNA with liquid biopsy could identify the poor prognosis group before treatment among TNBC patients and for those for whom optimal treatment selection is desirable; additionally, optimal treatment could be selected according to the ctDNA analysis results.

Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate β-Catenin Signaling.

Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.

Clinical utility of plasma cell-free DNA in pancreatic neuroendocrine neoplasms.

In advanced pancreatic neuroendocrine neoplasms (PanNEN), there are little data detailing the frequency of genetic alterations identified in cell free DNA (cfDNA), plasma-tissue concordance of detected alterations, and clinical utility of cfDNA. Patients with metastatic PanNENs underwent cfDNA collection in routine practice. Next-generation sequencing (NGS) of cfDNA and matched tissue when available was performed. Clinical actionability of variants was annotated by OncoKB. Thirty-two cfDNA samples were analyzed from 25 patients, the majority who had well-differentiated intermediate grade disease (13/25; 52%). Genomic alterations were detected in 68% of patients and in 66% of all cfDNA samples. The most frequently altered genes were DAXX (28%), TSC2 (24%), MEN1 (24%), ARID1B (20%), ARID1A (12%), and ATRX (12%). Twenty-three out of 25 (92%) patients underwent tumor tissue NGS. Tissue-plasma concordance for select genes was as follows:DAXX (95.7%), ARID1A (91.1%), ATRX (87%), TSC2 (82.6%), MEN1 (69.6%). Potentially actionable alterations were identified in cfDNA of 8 patients, including TSC2 (4; level 3b), ATM (1; level 3b), ARID1A (2; level 4), and KRAS (1; level 4). An ETV6:NTRK fusion detected in tumor tissue was treated with larotrectinib; at progression, sequencing of cfDNA identified an NTRK3 G623R alteration as the acquired mechanism of resistance; the patient enrolled in a clinical trial of a second-generation TRK inhibitor with clinical benefit. In metastatic PanNENs, cfDNA-based NGS identified tumor-associated mutations in 66% of plasma samples with a high level of plasma-tissue agreement in PanNEN-associated genes. Clonal evolution, actionable alterations, and resistance mechanisms were detected through circulating cfDNA genotyping.

Mutant p53 and ELK1 co-drive FRA-1 expression to induce metastasis in breast cancer.

Tumor-associated p53 mutations induce activities different from wild-type p53, thus causing loss of the protein's tumor inhibition function. The cells carrying p53 mutations have more aggressive characteristics related to invasion, metastasis, proliferation, and cell survival. By comparing the gene expression profiles of mutant p53 (mutp53) and mutp53 silenced cohorts, we found that FOS-related antigen-1 (FRA-1), which is encoded by FOSL1, is a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition, is elevated in the presence of p53 mutations. Mechanistically, mutant p53 cooperates with the transcription factor ELK1 in binding and activating the promoter of FOSL1, thus fostering lung metastasis. This study reveals new insights into how mutant p53 contributes to metastasis in breast cancer.

CNSC-43. TARGETING NEURONAL EXCITABILITY FOR NF1 LOW-GRADE GLIOMA THERAPY

Abstract While neuronal activity has previously been implicated in malignant glioma growth control, we recently demonstrated that neuron excitability can also regulate low-grade (optic) glioma initiation and progression. In this prior study, we showed that NF1 germline mutation-dependent neuronal excitability sustains optic glioma growth through HCN channel-mediated midkine activation of a T cell-microglia stromal circuit. Relevant to clinical translation, lamotrigine modulation of HCN channel function reduced neuronal hyperexcitability, midkine production and optic glioma (OPG) growth. To establish an obligate role for neuronal activity and midkine (Mdk) production in OPG growth, we leveraged a series of unique genetically engineered mouse models. First, we demonstrate that midkine is necessary for gliomagenesis, as Nf1-OPG; Mdk-/- mice do not develop tumors and have restored retinal pathology, as evidenced by increased retinal ganglion cell (RGC) numbers and retinal nerve fiber layer thickness. Second, we show that midkine production and neuronal hyperactivity are interdependent, such that only tumor-associated germline Nf1 mutations result in increased RGC excitability and midkine production, whereas those mutations that do not cause optic gliomas lack RGC hyperexcitability or elevated midkine expression. Third, lamotrigine treatment both during and after Nf1-OPG development attenuates midkine levels and tumor proliferation. Moreover, this anti-tumoral effect is durable for months after the cessation of treatment. Fourth, oral treatment of Nf1-OPG-bearing mice with clinically relevant doses of lamotrigine (2.5-7.5mg/kg/day) during OPG development blocks tumor formation. Together, these data establish midkine and neuronal hyperexcitability as key drivers of Nf1 optic glioma growth, relevant to planned clinical trials of this anti-seizure medication for children with NF1-OPG.

Associations Between Cancer Predisposition Mutations and Clonal Hematopoiesis in Patients With Solid Tumors.

Clonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors. We used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants. We observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH comutation patterns in ATM, TP53, and CHEK2 suggested biallelic inactivation as a potential mediator of clonal expansion. Moreover, we observed that CH-PPM1D mutations, similar to somatic tumor-associated PPM1D mutations, were depleted in patients with P/LP germline mutations in the DNA damage response (DDR) genes ATM, CHEK2, and TP53. Patients with solid tumors and harboring P/LP germline mutations, CH mutations, and mosaicism chromosomal alterations might be at an increased risk of developing secondary leukemia while germline variants in TP53 were identified as an independent risk factor (hazard ratio, 36; P < .001) for secondary leukemias. Our results suggest a close relationship between inherited variants and CH mutations within the DDR genes in patients with solid tumors. Associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities in patients with cancer harboring these germline mutations.

Detecting variation of clonal hematopoiesis using machine-learning in liquid biopsy.

e15027 Background: Tumor‐derived DNA released to the plasma of cancer patients which could be captured and detected by ultra-deep sequencing. NGS provides the technical probability of somatic mutations detection from circulating cell‐free DNA (cfDNA) in plasma samples, while clonal hematopoiesis (CH) mutations affect the accuracy of liquid biopsy for clinical diagnosis or choice of drug and tracing of minimal residual disease (MRD). CH mutation screening of cfDNA samples still an unsolved issue, herein we propose a method that based on multi-variable tree model which screen the CH mutation effectively. Methods: We sequenced 1992 cfDNA (raw depth 35000x) and matched peripheral blood lymphocyte (PBL, raw depth 2000x) samples by 1123 genes target capture sequencing from cohorts which include 962 non-small cell lung cancers (NSCLC), 631 gastric cancers (GC), 379 other non-hematological cancers and 40 healthy normal control. First, we compared the coherence patterns of cfDNA-PBL between CH genes (ASXL1, DNMT3A, TET2) hotspot mutation and tumor associated mutation (EGFR L858R T790M, KRAS G12D G13D, TP53 R43H R141H). Next, we labeled canonical mutations by: point mutation and indels; top frequent tumor genes (from cbioportal collected TCGA cancers); published frequent CH genes; known hotspots and unknown significance genes. Furthermore, hotspot tumor mutation and hotspot CH mutations and other mutations were labeled. kNN classify all mutation to labeled mutations groups, and decided the next steps tree model’s node thresholds build a classify tree model screen the confident CH and somatic mutations. Finally, we validated 6 TP53 CH mutation from healthy normal control by ddPCR. Results: Previous published imbalance test model: 'fisher test p.value &gt; 0.5 &amp; 0.5 &lt; OR &lt; 1.5' only covered 23.2%, 26.9%, 17.5% top 10 hotspot CH mutations (ASXL1, DNMT3A, TET2) correspondingly, while 0.75 quantiles both g_VAF and t_VAF less than 1% and alter reads count less than 20. In terms of tumor associated mutations only 3/155,6/123,7/108,7/71,323/998,16/189 exceed the imbalance test models threshold, especially L858R with median t_VAF 3.4% and t_alter_count 60. Population frequency distribution of CH and tumor genes mutation were different significantly (median 0.776% and 0.569%; p.value &lt; 1.44e-05, F-test). After assign hotspot and CH mutations classify by kNN, The tree model screened 42.9% and 83.6% CH mutations in cancer and healthy normal samples respectively. TP53 were common both in tumor and CH mutation ambiguously, tree model screened 6 TP53 CH mutation (c.12G &gt; A, R43H, G113D, R70C, c.16C &gt; T, A80T) from normal plasma sample all validated by ddPCR. Conclusions: CH mutations were ubiquitous in plasma sample and abundance pattern different from common tumor mutations significantly. CH mutation could be screened by optimal condition assembled decision trees effectively.

Genomic landscape and gene expression profiles of feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a cancer of the squamous cell lining in the oral cavity and represents up to 80% of all oral cancers in cats, with a poor prognosis. We have used whole exome sequencing (WES) and RNA sequencing of the tumor to discover somatic mutations and gene expression changes that may be associated with FOSCC occurrence. FOSCC offers a potential comparative model to study human head and neck squamous cell carcinoma (HNSCC) due to its similar spontaneous formation, and morphological and histological features. In this first study using WES to identify somatic mutations in feline cancer, we have identified tumor-associated gene mutations in six cats with FOSCC and found some overlap with identified recurrently mutated genes observed in HNSCC. Four samples each had mutations in TP53, a common mutation in all cancers, but each was unique. Mutations in other cellular growth control genes were also found such as KAT2B and ARID1A. Enrichment analysis of FOSCC gene expression profiles suggests a molecular similarity to human OSCC as well, including alterations in epithelial to mesenchymal transition and IL6/JAK/STAT pathways. In this preliminary study, we present exome and transcriptome results that further our understanding of FOSCC.

Clinical implications of myeloid malignancy‑related somatic mutations in aplastic anemia

Aplastic anemia (AA) is a potentially fatal bone marrow failure syndrome characterized by a paucity of hematopoietic stem cells and progenitor cells with varying degrees of cytopenia and fatty infiltration of the bone marrow space. Recent advances in genomics have uncovered a link between somatic mutations and myeloid cancer in AA patients. At present, the impact of these mutations on AA patients remains uncertain. We retrospectively investigated 279 AA patients and 174 patients with myelodysplastic syndromes (MDS) and performed targeted sequencing of 22 genes on their bone marrow cells using next-generation sequencing (NGS). Associations of somatic mutations with prognostic relevance and response to treatment were analyzed. Of 279 AA patients, 25 (9.0%) patients had somatic mutations, and 20 (7.2%) patients had one mutation. The most frequently mutated genes were ASXL1(3.2% of the patients), DNMT3A (1.8%) and TET2 (1.8%). In the MDS group, somatic mutations were detected in 120 of 174 (69.0%) patients, and 81 patients (46.6%) had more than one mutation. The most frequently mutated genes were U2AF1 (24.7% of the patients), ASXL1 (18.4%) and TP53 (13.2%). Compared with MDS patients, AA patients had a significantly lower frequency of somatic mutations and mostly one mutation. Similarly, the median variant allele frequency was lower in AA patients than in MDS patients (6.9% vs. 28.4%). The overall response of 3 and 6 months in the somatic mutation (SM) group was 37.5% and 66.7%, respectively. Moreover, there was no significant difference compared with the no somatic mutation (N-SM) group. During the 2-years follow-up period, four (20%) deaths occurred in the SM group and 40 (18.1%) in the N-SM group, with no significant difference in overall survival and event-free survival between the two groups. Our data indicated that myeloid tumor-associated somatic mutations in AA patients were detected in only a minority of patients by NGS. AA and MDS patients had different gene mutation patterns. The somatic mutations in patients with AA were characterized by lower mutation frequency, mostly one mutation, and lower median allelic burden of mutations than MDS. Somatic mutations were a common finding in the elderly, and the frequency of mutations increases with age. The platelet count affected the treatment response at 3 months, and ferritin level affected the outcome at 6 months, while somatic mutations were not associated with treatment response or long-term survival. However, our cohort of patients with the mutation was small; this result needs to be further confirmed with large patient sample.

Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.

Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs),incontrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.

Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices

Single-cell sequencing provides promising information in tumor evolution and heterogeneity. Even with the recent advances in circulating tumor cell (CTC) technologies, it remains a big challenge to precisely and effectively isolate CTCs for downstream analysis. The Cell RevealTM system integrates an automatic CTC enrichment and staining machine, an AI-assisted automatic CTC scanning and identification system, and an automatic cell picking machine for CTC isolation. H1975 cell line was used for the spiking test. The identification of CTCs and the isolation of target CTCs for genetic sequencing were performed from the peripheral blood of three cancer patients, including two with lung cancer and one with both lung cancer and thyroid cancer. The spiking test revealed a mean recovery rate of 81.81% even with extremely low spiking cell counts with a linear relationship between the spiked cell counts and the recovered cell counts (Y = 0.7241 × X + 19.76, R2 = 0.9984). The three cancer patients had significantly higher TTF-1+ CTCs than healthy volunteers. All target CTCs were successfully isolated by the Cell Picker machine for a subsequent genetic analysis. Six tumor-associated mutations in four genes were detected. The present study reveals the Cell RevealTM platform can precisely identify and isolate target CTCs and then successfully perform single-cell sequencing by using commercially available genetic devices.

MRD Monitoring of Non-DTA Genes By Digital Droplet PCR in the Early Period after Allogeneic Hematopoietic Stem Cell Transplantation Can Effectively Predict Patients at High Risk of Relapse

Background: Hematological malignancies relapse is the major cause of death after allogeneic hematopoietic stem cell transplantation (HSCT). The detection of measurable residual disease (MRD) after HSCT is the effective and important predictor of relapse. Digital droplet polymerase chain reaction (ddPCR), as the third generation PCR, is a novel technology for absolute quantification of nucleic acids developed in recent years. Compared with real-time quantitative PCR (qPCR, first generation) and next-generation sequencing (NGS), ddPCR has better sensitivity, specificity and repeatability, which provide a new platform for the detection of MRD. The purpose of this study was to evaluate whether early MRD monitoring by ddPCR after HSCT can effectively predict the recurrence of primary disease. Methods: We retrospectively analyzed patients with hematological malignancies who underwent allogeneic HSCT and monitored post-transplant MRD by ddPCR at the BMT Center of the First Affiliated Hospital of Zhejiang University School of Medicine from August 2018 to February 2021. Patients who met the following criteria were included in our study: (1) at least one hematological tumor-associated mutations or fusion genes for MRD monitoring at the time of diagnosis; (2) underwent allogeneic HSCT and the stem cell engraftment was successful; (3) received bone marrow MRD detection by ddPCR at least once in the early period (+30 days to +120 days) after HSCT; (4) bone marrow morphology was in remission status within +30 days to +120 days. Results: 100 patients were included in our study. The median follow-up time of patients was 20 months (range 5-46), and the median age was 41 years (range 14-67). Among them, 59 (59.0%) were male, and the most frequent primary diseases were AML (N=59, 59.0%), ALL (N=21, 21.0%), MDS (N=16, 16.0%), respectively. A total of 43 (43.0%) patients were found to be MRD+ after HSCT by early ddPCR detection. The difference in the 2-year cumulative incidence of relapse (2y CIR, 32.5% vs. 19.6%, P= 0.103), 2-year relapse-free survival (2y RFS, 67.0% vs. 79.8%, P=0.110), and overall survival (OS, 63.0% vs. 84.1%, P=0.566) between the MRD+ and MRD- groups were not statistically significant. 30 patients had at least one mutation related to clonal hematopoiesis gene: DNMT3A, TET2, and ASXL1 (DTA), whereas 97 patients had at least one non-DTA gene. When DTA group and non-DTA group were considered separately, MRD+ were 33.3% in DTA gene group and MRD+ were 36.1% in non-DTA gene group. The 2y CIR (0% vs. 21.5%, P=0.160), 2y RFS (100% vs. 78.5%, P=0.170) and OS (90.0% vs. 85.0%, P=0.737) after HSCT was not statistically different between DTA MRD+ group and DTA MRD- group, suggesting that DTA was not suitable as monitoring genes for relapse after transplantation. However, in the non-DTA group, patients with MRD+ had higher 2y CIR (39.8% vs. 18.4%, P=0.014) and inferior 2y RFS (60.2% vs. 80.8%, P=0.017) compared with MRD- patients. To explore whether ddPCR and multiparameter flow cytometry (MFC) can complement each other and further improve the prediction accuracy of relapse, we conducted a joint analysis on the bone marrow MRD detected simultaneously by non-DTA ddPCR and MFC, and divided into four parts: ①MFC MRD+ and non-DTA MRD+ (F+/M+, n=7); ②MFC MRD- and non-DTA MRD+ (F-/M+, n=26); ③MFC MRD+ and non-DTA MRD- (F+/M-, n=8); ④MFC MRD- and non-DTA MRD- (F-/M-, n=56). The sensitivity of ddPCR was higher than that of MFC. The 2y CIR of the four groups was (F+/M+ 71.4% vs. F-/M+ 34.5% vs. F+/M- 53.1% vs. F-/M- 12.2%, P<0.001), and the 2y RFS was (F+/M+ 28.6% vs. F-/M+ 65.5% vs. F+/M- 46.9% vs. F-/M- 87.3%, P<0.001) and OS was (F+/M+ 31.3% vs. F-/ M+ 61.8% vs. F+/M- 87.5% vs. F-/M- 83.8%, P=0.219). Through multivariate analysis, F+/M+ is an independent risk factor for relapse after HSCT (hazard ratio, 9.531, 95%CI, 2.884 to 31.496). It suggested that F+/M+ detected in the early stage after transplantation had higher CIR and inferior RFS. Conclusion: MRD monitoring for non-DTA gene in the early period after HSCT based on ddPCR has a prognostic effect on the recurrence of primary disease and overall survival after transplantation. When ddPCR is combined with MFC to monitor MRD, it is more accurate in predicting relapse and may help instruct preemptive treatment and then reduce the risk of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitotic Checkpoint Imbalances in Familial Cancer

Numerical chromosomal aberrations are highly frequent in cancer cells. However, tumor-associated mutations in regulators of the mitotic machinery that controls chromosome segregation are rather rare. By sequencing families with hereditary cancer, Chen and colleagues report two novel heterozygous mutations in CDC20, a coactivator of the anaphase-promoting complex (APC/C) and a target of the spindle assembly checkpoint (SAC) that prevents chromosome missegregation during mitosis. CDC20 mutations result in partial SAC functionality and segregate with tumor susceptibility in families with aneuploid ovarian cancers and other malignancies. The expression of these mutations in a knock-in mouse model accelerates the development of Myc-induced lymphomas and mortality, strongly supporting the notion that partial dysfunction of mitotic regulators may have profound implications in spontaneous and hereditary cancer. See related article by Chen et al., p. 3499.

Pan-cancer analysis of LncRNA XIST and its potential mechanisms in human cancers

BackgroundX-inactive specific transcript (XIST), it has been found, is abnormal expression in various neoplasms. This work aims to explore its potential molecular mechanisms and prognostic roles in types of malignancies. MethodsThis research comprehensively investigated XIST transcription across cancers from Oncomine, TIMER 2.0 and GEPIA2. Correlations of XIST expression with prognosis, miRNAs, interacting protens, immune infiltrates, checkpoint markers, mutations of tumor-associated genes and promoter methylation were also analyzed by public databases. In addition, 98 BRCA samples were collected to investigate XIST expression and evaluate its clinicopathological value. ResultsIn public databases, compared to normal tissues, XIST was lower in BRCA, CESC, COAD and so on, but increased in KIRC and PRAD. Databases also showed that XIST was a good indicator of prognosis in BRCA, COAD and so on, but a bad one in KIRC, KIRP and so on. From starBase, we found 29 proteins interacting with XIST, and identified 4 miRNAs which might be sponged by XIST in cancers. Furthermore, XIST was linked with immune infiltration, especially T cell CD4+, and was related to over 20 immune checkpoint markers. Moreover, several tumor-associated gene mutations and promoter methylation were negatively related to its expression. In addition, IHC showed that XIST in BRCA was obviously lower in comparison of normal tissues and was negatively related to lymph node invasion and TNM stage. ConclusionIn summary, abnormal expression of XIST influenced prognosis, miRNAs and immune infiltration across cancers, especially BRCA.

Abstract 538: Cell-free whole genome sequencing for the detection of cancer in patients with germline BRCA1/2 mutations

Abstract Background: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by a germline likely/pathogenic variant in BRCA1 or BRCA2, leading to increased risk of developing breast, ovarian, prostate, and pancreatic cancers. While HBOC patients often undergo regular surveillance including breast imaging, other cancers have no effective screening or biomarkers. One emerging technology is the analysis of circulating tumor DNA (ctDNA), fragments of DNA that are shed from the tumor into the bloodstream. In sporadic cancer, several studies have demonstrated the advantages and sensitivity of cell-free whole genome sequencing (cfWGS) compared to targeted panel sequencing. However, the use of cfWGS for cancer detection in HBOC has not been explored. Methods: Matched tumor, germline, and plasma were collected from 5 HBOC patients (3 breast, 2 ovarian) as part of the Canada-wide CHARM consortium (https://charmconsortium.ca). Tumor (40x), germline (20x), and plasma (20x) underwent whole genome sequencing (WGS) and subsequent variant calling pipeline using an ensemble of 6 variant callers. Insertions, deletions, and structural variants were not included in downstream analyses. Plasma tumor fraction prediction and copy number alterations were performed using ichorCNA. Detection of tumor associated mutations in plasma was compared to MRDetect software. Results: Variant calling identified an average of 11,170 tumor (7,649-16,706) and 1,215 (544-3,205) plasma SNVs per patient. Somatic BRCA1/2 mutations were detected in 0/5; whereas deletion of the intact BRCA1/2 allele was identified in 5/5 patients. An average of 24.6% (8.3%-75.9%) of plasma mutations where shared with SNVs identified in matched tumor. Targeted panel deep-sequencing (20,000X) was only able to detect mutations in TP53 in 3/5 cases (0.8% - 8.6%). Plasma derived copy number alterations correlated well with tumor derived copy number alterations in 3/5 cases and ichorCNA predicted tumor fractions ranged from 0.038 to 0.196. Conclusions: Traditionally, targeted panel sequencing has been used to identify tumor associated mutations in plasma. While this technique is sensitive, major disadvantages include the narrow breadth of the captured regions and the inability to detect deletion events such as those that occur in BRCA1/2. Here we show that plasma WGS is effective for detecting tumor associated mutations in HBOC and is more sensitive than targeted panel sequencing. We are currently expanding our cohort to an additional 20 matched tumor pairs, including patients with pre-diagnosis plasma timepoints. Citation Format: Derek Wong, Adriana Aguilar-Mahecha, Josiane Lafleur, Clarissa Chan, Kirsten Farncombe, Maia Norman, Leslie Oldfield, Prabhjit Basra, Stephanie Pederson, Johanna Wellum, Stephenie Prokopec, Mark Basik, Raymond H. Kim, Trevor Pugh. Cell-free whole genome sequencing for the detection of cancer in patients with germline BRCA1/2 mutations [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 538.

Next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) in patients (pts) with advanced hepatocellular carcinoma (HCC).

4110 Background: HCC is often diagnosed based on high-quality cross-sectional imaging, and when a biopsy is pursued, acquisition of tissue may be of limited quantity and quality or complicated by underlying medical comorbidities. NGS of tumor derived circulating cfDNA represents an investigational tool for non-invasive molecular profiling, that has the potential to aid in diagnosis, prognosis, and in monitoring disease status. Although prior reports have evaluated such technologies, few studies have included tumor tissues to confirm histology and to explore plasma-tissue gene concordance. Methods: The primary objective of this retrospective cohort study was to define genomic alterations in circulating cfDNA and to explore plasma-tissue genotype concordance in HCC pts. HCC pts underwent collection of cfDNA for NGS using the MSK-ACCESS 129-gene assay between August 2019 and February 2021. Matched tissue-based NGS with the FDA authorized MSK-IMPACT gene assay was completed when tumor tissue was available. Clinical actionability of sequence variants was annotated by OncoKB, an FDA recognized knowledge base. Clinicopathologic characteristics were extracted, and all data were reported with descriptive statistics. Results: 51 unique patients with 53 plasma samples had an HCC histological diagnosis. Pts were male (39, 76%), median age 69 (42-87), viral hepatitis-related (24, 47%), and advanced stage (Stage III:9, 18%; Stage IV:38, 74.5%). Extrahepatic disease and macrovascular involvement were observed in 28 (55%) and 19 (38%) pts, respectively. 22 (43%) pts had AFP ≥400 ng/mL. 49 (92.5%) of 53 plasma samples had detectable genomic alterations. Median cfDNA yield after extraction was 39.43 ng (range: 7.93-287.68). The most frequently mutated genes occurring in &gt; 10% of patients were TERT (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%) and TSC2 (14%). The most common oncogenic pathways that contained alterations were WNT-β-Catenin (45%) and PIK3-AKT-TOR (25%). 37 (73%) pts underwent tissue sequencing with MSK-IMPACT with a median time of 9.0 months to the time cfDNA testing. MSK-ACCESS identified mutations observed in tumor in most cases: TERT (20/22; 91%), TP53 (16/17; 94%), CTNNB1 (11/12; 92%), ARID1A (6/6; 100%) and TSC2 (6/7; 86%). In 18 (49%) of 37 paired samples, additional mutations in cfDNA not seen in tumor were detected and included KRAS, EGFR, and TP53 alterations. Potentially actionable mutations were identified through cfDNA in 37% of cases including TSC1/2 (18%), BRCA1/ 2 (8%) and PIK3CA (8%). Conclusions: Circulating cfDNA genotyping with MSK-ACCESS identifies previously reported HCC tumor genomic profiles and revealed tumor-associated mutations in 92.5% of plasma samples. Ongoing efforts will explore predictive and prognostic implications of NGS at different HCC stages as well as kinetics of treatment response.

Efficacy of liquid biopsy for disease monitoring and early prediction of tumor progression in EGFR mutation-positive non-small cell lung cancer.

15–40% of non-small cell lung cancer (NSCLC) patients harbor epidermal growth factor receptor (EGFR)-sensitizing mutations. Tyrosine kinase inhibitors (TKIs) provide significant clinical benefit in this population, yet all patients will ultimately progress. Liquid biopsy can reliably identify somatic tumor-associated EGFR mutations in plasma. This study aimed to assess the feasibility and value of the quantitative assessment of EGFR driver mutations in plasma in EGFR-mutated NSCLC patients treated with EGFR-TKIs as a tool to evaluate therapeutic response to TKIs and monitor for disease progression. The study included 136 patients with tissue biopsy-confirmed EGFR-sensitizing, mutation-positive lung adenocarcinoma with plasma collected prior to TKI treatment and at least two post-initiation TKI treatment/follow-up blood samples. Plasma samples were tested with the cobas® EGFR Mutation Test v2 (cobas EGFR Test), and semi-quantitative index (SQI) values for each identified mutation were reported by the assay software. The most common baseline EGFR mutations detected in tissue were L858R (53.7%) and exon 19 deletion (39.7%). Plasma cell-free DNA analysis detected EGFR mutations in 74% of the baseline samples. Objective response rate by RECIST 1.1 was achieved in 72% of patients, while 93% had a molecular response (defined as disappearance of the EGFR mutation from plasma). 83% of patients had molecular progression (MP; 1.5X SQI increase or new T790M mutation), and 82% who had a clinical response had clinical progression. On average, MP occurred 42 days prior to clinical progression. Patients who progressed while on first-line TKI showed MP of the original EGFR-sensitizing mutations prior to the emergence of a T790M mutation, which was detected in 27% of the EGFR plasma-positive patients. Longitudinal monitoring of EGFR mutational load in plasma is feasible and can predict both response and clinical progression in EGFR-mutated NSCLC patients treated with EGFR-TKIs, as well as detect treatment-emergent EGFR mutations.

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors.

The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.