Somatic Alterations Research Articles

BIOM-17. WHOLE GAIN OF CHROMOSOME 19, NOT CO-GAIN OF CHROMOSOMES 19 AND 20, IS A FAVORABLE PROGNOSTIC BIOMARKER IN IDH-WILDTYPE GLIOBLASTOMA

Abstract BACKGROUND IDH-wildtype glioblastoma carries an abysmal prognosis, but long-term survivors exist. Previous studies have suggested that co-gain of chromosomes 19 and 20 (+19/+20) is a favorable prognostic marker. We sought to validate this claim and investigate whether gain of chromosome 19 (+19) is a superior prognostic indicator. METHODS We collected somatic copy number alteration data for primary glioblastoma tumors from TCGA (N = 356) and paired primary (N = 169) and recurrent (N = 167) glioblastoma tumors from GLASS. We assessed the frequency of these events and conducted survival analyses with univariate and multivariate Cox proportional hazard models to examine the association between +19/+20 and +19 and patient outcomes. RESULTS Among primary glioblastomas, the frequency of +19/+20 (20.8% vs. 23.7%) and +19 (34.6% vs. 36.7%) did not differ between TCGA and GLASS. However, the presence of +19/+20 was significantly reduced in GLASS recurrent tumors compared to primary tumors (14.3%, p = 0.04), while +19 without +19/+20 was unchanged (13.8%), indicating that +19/+20 is selected against in recurrent glioblastoma. Tumors harboring +19/+20 did not exhibit improved overall survival (OS) or progression-free survival (PFS) in TCGA (p = 0.21 and p = 0.14, respectively), nor did they demonstrate improved OS in GLASS (p = 0.32). Conversely, tumors with +19 showed significantly better OS and PFS in TCGA (HR = 1.4, p< 0.01; HR = 1.5, p< 0.01, respectively) and improved OS in GLASS (HR = 1.6, p< 0.01). In multivariate survival analyses, +19 remained a prognostic factor independent of +19/+20, patient sex, age, MGMT promoter methylation status, and KPS in TCGA (p< 0.05) and +19/+20 and age in GLASS (p = 0.01). CONCLUSION Although previous studies have suggested that +19/+20 is a positive prognostic biomarker in glioblastoma, our findings indicate that this assertion should be reevaluated. Our results demonstrate that +19 is associated with favorable outcomes in the TCGA and GLASS datasets, whereas +19/+20, while possibly selected against in recurrent glioblastomas, is not.

PATH-34. CLINICOPATHOLOGIC CHARACTERIZATION OF DIFFUSE PEDIATRIC-TYPE HIGH-GRADE GLIOMA, H3-WILDTYPE AND IDH-WILDTYPE: AN AGGRESSIVE AND GENOMICALLY COMPLEX GROUP OF TUMORS AFFECTING CHILDREN AND ADULTS

Abstract Diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG) was newly introduced in the 2021 WHO CNS5. pHGG is further divided into distinct methylation subtypes (MYCN, A/B, RTK1, RTK2). Compared to adult-type diffuse gliomas, relatively little is known about pHGG, especially in the adult population and we therefore aimed to more fully understand the clinicopathological spectrum of pHGG. Our cohort of methylation-profiling-confirmed pHGGs (n=299) included pediatric (n=211) and adult (n=88) populations (age range=0-79 years). The mean age was highest in the A/B subtypes (27.7 years) and lowest in the MYCN subtype (13.5 years). Median survival (18 months for the entire cohort), MGMT promoter methylation status, and history of prior radiation did not differ between the pediatric and adult populations. MGMT promoter methylation was highest in RTK1C (51%) and lowest in subtype A (0%). All those with history of prior radiation (n=37) were classified as RTK1, with subclass distribution differing between patients with history of prior radiation (RTK1A=7.3%, RTK1B=41.8%, RTK1C=50.9%) versus those without (RTK1A=41.8%, RTK1B=21.5%, RTK1C=36.7%). Interestingly, the genomic somatic copy number alteration (SCNA) load did not differ for RTK1 based on prior radiation history status. However, adult pHGG did harbor a higher SCNA load than pediatric pHGG (p< 0.01). The higher SCNA load was driven by genomic copy number losses rather than amplifications, including adult pHGG harboring more CDKN2A/B and RB1 deletions. Amplifications of MET, CDK4, PDGFRA, MYC, and KIT were present across all subtypes, but were enriched in RTK1. MYCN amplification was enriched in the MYCN subtype. EGFR, MDM4, and CDK6 amplifications did not have subtype predilection. Overall, while the pediatric and adult pHGG have similar outcomes, there are differences in their genomic structures, most notably in adult pHGG harboring a higher SCNA load. Overall, pHGG represent a biologically diverse set of clinically aggressive tumors that warrant further intensive study.

EPCO-22. LONGITUDINAL MOLECULAR CHARACTERIZATION IDENTIFIES TREATMENT DRIVEN EVOLUTION OF OLIGODENDROGLIOMA

Abstract Oligodendroglioma is characterized by oligodendrocyte-like histology, mutation in the IDH genes, along with codeletion of chromosome arms 1p and 19q. Treatment for oligodendrogliomas combines surgery, followed by observation, and chemotherapy and/or radiotherapy when tumors progress. Most oligodendrogliomas eventually recur with a more aggressive phenotype, leading to patient mortality but molecular mechanisms remain poorly understood. To investigate the molecular trajectory of oligodendroglioma, we collected more than 270 longitudinal samples from over 130 oligodendroglioma patients, to perform whole genome or whole exome sequencing, bulk RNA, and single-nucleus RNA sequencing (n = 30 samples). Common mutations in IDH1/2, TERT promoter, CIC, and FUBP1 were observed. About half of the CIC and FUBP1 mutations occurred in the initial sample and persisted stably throughout recurrence. The majority of PIK3CA and PIK3R1 mutations were either shared or present in recurrences only implicating the PI3K pathway in oligodendroglioma recurrence. Mutational signature analysis revealed 34.5% of tumors treated with alkylating agents acquired SBS11/SBS31 associated hypermutation. By integrating signatures with clonality estimates, we approximated the timing of mutational processes and found that clonal timings of treatment related mutations correlate with the treatment administration date. We found that aging signature-related mutations were mostly clonal, and treatment signature-related mutations were more likely to be sub-clonal, supporting the late emergence of these mutations following treatment. We observed increased somatic copy number alterations in oligodendroglioma recurrences, reflected in notable chromosome 4 losses. Associating treatment with snRNAseq derived cellular states, we observed a significant increase in the fraction of proliferating stem-like cells in the hypermutator versus non-hypermutator recurrent samples. These findings shed light on the molecular changes occurring in oligodendrogliomas following treatment and provide insights that may aid in targeting treatment resistance.

MODL-50. CLONAL EXPANSION OF SPONTANEOUS AND INJECTED TUMORS IN SYNGENEIC MOUSE MODELS OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) tumors are highly heterogenous and plastic. To better understand how tumors respond to treatment, it is important to use immunocompetent mouse models. We've created various mouse GBM models with common genetic mutations found in different subtypes of GBM, including classical, proneural, mesenchymal, and BRAF-driven. METHODS Using a combination of CRISPR and PiggyBac Transposon constructs, we injected plasmid mixtures into the ventricles of the mouse brain during the post-natal period. Electroporation was then used to enable the plasmids to enter nearby neural stem cells, which led to the spontaneous formation of tumors (spontaneous model). We collected tumor cells from these models and injected them into the adult mouse brain to create the injected mouse GBM model. Here we use spatial and single cell transcriptome profiling approaches to characterize these models at endpoint. Spatial transcriptome data was collected using the Visium 10X platform and tumor spots were deconvoluted from non-malignant spots using consensus non-negative matrix factorization. Copy number alterations (CNAs) were inferred using non-malignant spots and single-cell clusters as a reference. RESULTS We found that copy number states are largely homogenous within each sample, supporting an early population expansion in vivo. CNAs found in spontaneous tumors are also found in the matched injected tumors, which in turn harbour additional CNAs, suggesting that further selection occurs in vitro. Some of the CNAs include relevant cancer genes, suggesting the observed clonal expansions are potentially driven by additional somatic alterations that cooperate with the engineered driver mutations. Studying these models using orthogonal methods will allow us to discover alterations that synergize with the original drivers and delineate principles of GBM initiation and progression in an immune-competent environment.

Acquired copy number variation in prostate tumours: a review of common somatic copy number alterations, how they are formed and their clinical utility.

Prostate cancer is one of the most commonly diagnosed cancers in men and unfortunately, disease will progress in up to a third of patients despite primary treatment. Currently, there is a significant lack of prognostic tests that accurately predict disease course; however, the acquisition of somatic chromosomal variation in the form of DNA copy number variants may help understand disease progression. Notably, studies have found that a higher burden of somatic copy number alterations (SCNA) correlates with more aggressive disease, recurrence after surgery and metastasis. Here we will review the literature surrounding SCNA formation, including the roles of key tumour suppressors and oncogenes (PTEN, BRCA2, NKX3.1, ERG and AR), and their potential to inform diagnostic and prognostic clinical testing to improve predictive value. Ultimately, SCNAs, or inherited germline alterations that predispose to SCNAs, could have significant clinical utility in diagnostic and prognostic tests, in addition to guiding therapeutic selection.

Tracing cancer evolution and heterogeneity using Hi-C

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

Characterization of the genomic alterations in poorly differentiated thyroid cancer

Poorly differentiated thyroid carcinoma (PDTC) is a subtype of thyroid cancer that has a high rate of metastasis or recurrence and a relatively poor prognosis. However, there are few studies that have been conducted on PDTC at the whole protein-coding gene scale. Here, we performed genomic profiling of 15 patients with PDTC originated from follicular thyroid carcinoma using whole exome sequencing and also performed gene functional enrichment analysis of differentially expressed genes (DEGs) for three patients. Further, we investigated genetic variants associated with PDTC progression and the characteristics of clinical pathology. We revealed somatic genomic alterations in the RAF1, MAP2K2, and AKT2 genes that were not reported in previous studies. We confirmed frequent occurrences in the RAS gene in patients with PDTC; the genetic alterations were associated with the RAS-RAF-MEK-ERK/JNK, PI3K-AKT-mTOR signaling pathways, and the cell cycle. DEG analysis showed that immune response was lower in cancer tissues than in normal tissues. Through the association analysis of somatic mutations and the characteristics of clinical pathology from patients with PDTC, the somatic mutations of ABCA12, CLIP1, and ATP13A3 were significantly associated with a vascular invasion phenotype. By providing molecular genetic insight on PDTC, this study may contribute to the discovery of novel therapeutic target candidates.

Nonlobular Invasive Breast Carcinomas with Biallelic Pathogenic CDH1 Somatic Alterations: A Histologic, Immunophenotypic, and Genomic Characterization

CDH1 encodes for E-cadherin, and its loss of function is the hallmark of invasive lobular carcinoma (ILC). Albeit vanishingly rare, biallelic CDH1 alterations may be found in nonlobular breast carcinomas (NL-BCs). We sought to determine the clinicopathologic characteristics and repertoire of genetic alterations of NL-BCs harboring CDH1 biallelic genetic alterations. Analysis of 5842 breast cancers (BCs) subjected to clinical tumor-normal sequencing with an FDA-cleared multigene panel was conducted to identify BCs with biallelic CDH1 pathogenic/likely pathogenic somatic mutations lacking lobular features. The genomic profiles of NL-BCs with CDH1 biallelic genetic alterations were compared with those of ILCs and invasive ductal carcinomas (IDCs), matched by clinicopathologic characteristics. Of the 896 CDH1-altered BCs, 889 samples were excluded based on the diagnosis of invasive mixed ductal/lobular carcinoma or ILC or the detection of monoallelic CDH1 alterations. Only 7 of the 5842 (0.11%) BCs harbored biallelic CDH1 alterations and lacked lobular features. Of these, 4/7 (57%) cases were ER-positive/HER2-negative, 1/7 (14%) was ER-positive/HER2-positive, and 2/7 (29%) were ER-negative/HER2-negative. In total, 5/7 (71%) were of Nottingham grade 2, and 2/7 (29%) were of grade 3. The NL-BCs with CDH1 biallelic genetic alterations included a mucinous carcinoma (n = 1), IDCs with focal nested growth (n = 2), IDC with solid papillary (n = 1) or apocrine (n = 2) features, and an IDC of no special type (NST; n = 1). E-cadherin expression, as detected by immunohistochemistry, was absent (3/5) or aberrant (discontinuous membranous/cytoplasmic/granular; 2/5). However, NL-BCs with CDH1 biallelic genetic alterations displayed recurrent genetic alterations, including TP53, PIK3CA (57%, 4/7; each), FGFR1, and NCOR1 (28%, 2/7, each) alterations. Compared with CDH1 wild-type IDC-NSTs, NL-BCs less frequently harbored GATA3 mutations (0% vs 47%, P = .03), but no significant differences were detected when compared with matched ILCs. Therefore, NL-BCs with CDH1 biallelic genetic alterations are vanishingly rare, predominantly comprise IDCs with special histologic features, and have genomic features akin to luminal B ER-positive BCs.

Somatic Genetic Rescue Involving CSF3R and Other Novel Phosphothyrosine Kinase Receptor Mutations Occurring in Myeloid Malignancies

Somatic gene rescue (SGR), via acquisition and selection of somatic (SM) genetic hits, neutralizes functional defects resulting from hypomorphic germline (GL) mutations. However, this process may be maladaptive and lead to the development of leukemia. Acquisition of SM gain-of-function CSF3R mutations in the context of severe congenital neutropenia (SCN) illustrates such a scenario. Here, we further explored this and similar mechanisms in a cohort of 7121 adults with myeloid neoplasia and bone marrow failure (AML=1473, MDS=2248, other=3400). CSF3R mutations were found in 97 patients. In total, there were 37 and 56 patients with SM and GL mutations, respectively; biallelic GL and SM were 4. Among SM alterations, 18 were pathogenic or likely pathogenic including 8 nonsense truncating variants in class III isoform specific region of the cytoplasmic domain and 10 classical missense mutations in the juxtamembrane region, previously defined in the context of post-SCN AML and CNL. We thus hypothesized that these CSF3R SM mutations may also represent SGR in adult patients harboring hypomorphic GL variants and investigated the presence of SCN-associated alterations ( ELANE, WAS, GFI1) and in other phosphothyrosine kinase receptors (PTKR) such as CSF1R, CSF2RA, CSF2RB, FLT3 and KIT. Indeed, we found biallelic WAS p.D264H GL and CSF3R p.A119T SM in a patient with aplastic anemia (AA). Biallelic CSF3R were present in 4 patients: a) p.Q749X GL withp.M696T SM, b) E835K GL with p.T618I SM, c) p.Y752X GL withp.Q754E SM and d) biallelic p.Y752X GL with p.Q754E GL configuration with T615A SM. Compound heterozygous configuration of CSF3R SM included CSF1R SM variants( CSF3R p.M696T SM & CSF1R p.R294Q GL in a 64 year old. patient with MPN, CSF3R p.M696T SM & CSF1R c.1626+3G>A GL in a 61 year old patient with MDS) and CSF2RB ( CSF3R p.T618I SM & CSF2RB p.V890I GL in a 69 year old female patient with AML with leukopenia). When we analyzed the remaining 66 GL CSF3R variants in 56 carriers (in addition to the aforementioned biallelic combinations), we identified 9 compound heterozygous mutants involving CSF1R SM (n=1), CSF2RA (n=1), CSF2RB (n=3), FLT3(TKD/ITD, n=4/1). Specifically, we found the following configurations and disease associations: a) CSF3R p.V372E GLCSF2RB p.P842L SM in a 49 yo. AA patient who subsequently developed AML with FLT3 TKD, b) MDS patients with CSF3R p.E149 GLCSF1R p.G413S SM, c) CSF3R p.R440Q GL& CSF2RA p.R164Q SM and d) CSF3R p.E405K GL& CSF2RB c.1152+6G>A SM and e) AML patients with CSF3R p.E405K GL& CSF2RB p.R432C SM; f) CSF3R p.L619S GLandg,h)2 AML cases with CSF3R p.E808K GL of which all 3 acquired compound heterozygosity with FLT3 TKD, i) CSF3R p.E808K GLCSF3R p.E835K GL in a 71 year old patient with MDS with FLT3ITD AML. In addition to FLT3 GOF mutations, the most common SM hits observed in CSF3RGLcarriers were ASXL1 (n=12), TET2 (n=12), and SF3B1 (n=12). Identification of somatic and GL alterations in PTKR genes other than CSF3R, led us to systematically analyze these genes as targets of GL and SM CSF3R alterations. In total, we identified 72 GL and 73 SM CSF1R variants, 7 SM and 19 GL CSF2RA and 47 GL and 69 SM CSFR2B. Among these, we found 3 patients with compound heterozygous CSF2RBSMCSF3RGL, one with CSF2RBGLCSF3RGLas described above. We also identified one patient with CSF2RASMCSF3RGL. Finally, we identified two patients with CSF1RGL and CSF3RSM, and one with CSF1RSMCSF3RGLmutations. In summary, our study provides evidence for a tremendous complexity of interaction between less penetrant GL alterations of receptor genes and somatic GOF mutations, which may in some instances, correspond to the result of SGR. It is likely that the degree of LOF corresponds to the selection pressure for somatic rescue lesions.

Compound Heterozygosity Involving Other Dead ( DDX) and Deah ( DHX) Box Helicases Explains Seemingly Monoallelic Somatic and Germline DDX41 Mutations

DDX41 mutations typify a seemingly distinct group of AML/MDS that appear to differ in many features from wild type entities. Classical DDX41 mutant myeloid neoplasia (MN) harbors biallelic mutations whereby, canonical germline (GL), mostly frameshifts (fs, chiefly at amino acid position 140) predispose to the acquisition of somatic canonical R525H and possible other somatic variants in the contralateral allele. Yet, in many instances, only GL or somatic mutations can be found. We stipulated that somatic DDX41 is not present in mature leukocytes but would be detectable in hematopoietic stem cells (HSCs). Indeed, higher VAF is detected in HSCs vs neutrophils likely due to differentiation arrest. However, it is also possible that other somatic mutations ( e.g., in other RNAhelicases) and conversely in cases with R525H without GL DDX41 might be present. In either case, upon our original discovery based on lower ATPase activity of R525H mutants 1 we stipulated that this mutation would be hypomorphic, an indirect evidence that has been, to date, not further substantiated. Here, we propose that R525H may indeed be functional via some unknown mechanisms, such as a GOF variant selected in the mode of somatic gene rescue (SGR) as a maladaptive response to relieve the GL LOF fs alteration. To that end, we studied the molecular makeup of a cohort of 1,893 MN patients (including public datasets 2,3) using NGS. In total, 101 patients with MN harbored DDX41 lesions. Among these, 35 patients had somatic hits other than R525H, detected as a seemingly monoallelic configuration and 15 patients harbored biallelic hits involving D140fs (n=3), M1I/L (n=2), and other pathogenic GL variants (n=10). A total of 51 patients carried GL mutations without matching a second allele hit; including D140 as monoallelic mutation in 4 patients, fs/del/splice sites in 10 patients, and missense variants in 37 patients (among this, M1I/L accounted for 24%). When we studied these monoallelic GL cases, we found a second somatic alteration in other DDX/ DHX helicases in carriers of seemingly monoallelic fs/splice sites. We found somatic hits in DDX10 (n=7), DDX25 (n=2), DDX52 (n=1), DHX16 (n=4) and DHX34 (n=1) suggesting that neoplastic progression occurring in these cases was due to compound heterozygosity involving other helicases substituting for the absence of canonical biallelic configuration. Of note is that no hit in any other helicases was detected in cases with sole GL D140, leaving unexplained the somatic cause driving neoplastic progression in these cases. However, upon manual inspection, R525H was detected in 2 patients with D140 and one patient with M1I albeit at very low VAFs%. Ultra low input NGS of pre-sorted bone marrow cellular fractions showed higher clonal burden of somatic R525H in HSCs, revealing the truly biallelic nature of these cases. 4 We then sought to explain the absence of GL in cases with R525H and other pathogenic lesions. For instance, we found one suspicious GL variant in DDX20 in one patient with R525H and one in DDX31 in one patient with non-R525 hit. These results suggest that monoallelic DDX41 cases may have compound heterozygosity (somatic/GL vs GL/somatic) for DDX41 and other DDX genes. These compound heterozygous cases prompted us to screen other patients with wild type DDX41 for the presence of DDX/DHX mutations. In total, we identified 38 monoallelic cases. Of those, 22 were GL with the most frequent being DDX54 (n=4), DDX11, and DDX20 (n=3; for both), followed by others. We are currently exploring whether these cases might harbor cryptic R525H, which might have been not resolved in bulk sequencing. In summary, we show that the canonical DDX41 biallelic constellation is a more ubiquitous phenomenon in the disease evolution to MN and involves other DDX/DHX helicases. In fact, a proportion of seemingly monoallelic cases might be explained by compound heterozygosity of other RNA helicases. Moreover, most GL DDX41 are cryptic biallelic or compound heterozygous, while cases with somatic canonical DDX41 should be investigated for the presence of GL DDX41 or other RNA helicases using ultra low input NGS. We propose that somatic DDX41 hits correspond to maladaptive SGR of hypomorphic GL mutants in analogy but in reverse fashion compared to GL SAMD9/9L. Studies to be presented at ASH will further substantiate this possibility.

Somatic Mutational Landscape and Transcriptomic Profiles of Lymphoproliferative Disorders with Epstein-Barr Virus Infection in Activated PI3K Delta Syndrome

Introduction: Germline gain-of-function variants in PIK3CD and PIK3R1 cause activated PI3K delta syndrome (APDS) types 1 and 2, respectively. Lymphoproliferative disorder (LPD) and susceptibility to various infections, including Epstein-Barr virus (EBV), are well-known features of APDS. EBV-associated LPDs are frequently observed in patients with APDS, and oligoclonal LPD might transform malignant lymphoma (ML). However, somatic molecular alterations in LPD and ML are not fully elucidated. Here, we report clinical characteristics and somatic mutational landscape and transcriptomic profiles of LPDs with EBV infection in APDS. Methods: LPD and ML in APDS diagnosed during 2011-2022 were recruited all over Japan. Clinical characteristics and outcomes were analyzed. EBV infection status was analyzed by EBER ISH and RNA sequencing (RNA-seq). Whole-exome sequencing (WES), RNA-seq, and single cell RNA-seq (scRNA-seq) were also performed. Results: Five LPD and 5 ML (borderline ML: n = 2, diffuse large B-cell lymphoma (DLBCL): n = 2, classical Hodgkin lymphoma: n = 1) cases were enrolled. Seven cases were APDS1 and 3 cases were APDS2. The median follow-up period was 2.9 (0.5-8.4) years. LPD cases were treated with rituximab monotherapy (n = 1), prednisolone (n = 3), and hematopoietic cell transplantation (HCT) (n = 1). Rituximab-combined chemotherapy was performed in borderline ML and DLBCL (n = 4) or ABVD regimen in Hodgkin lymphoma cases (n = 1). Sirolimus was used in two ML cases. HCT was performed in 9 cases. Two-year overall survival and event-free survival (events: relapse and death) were 78.8% and 70.0% in total LPDs, respectively, and 60% and 40% in ML cases. EBV was negative in 3 cases and positive in 2 cases of LPD cases, and positive in 4 cases, and negative in 1 case of ML cases. The median of 18 (10-48) and 42 (18-189) somatic variants were detected by tumor/germline pair WES in LPD (n = 5) and ML (n = 7, 2 samples were from the same patient at different time points), respectively. The recurrent variants were identified in ZFHX3 (n = 4), TRPM2 (n = 3), BEND3, CDH23, UBR4, ARAF, TET2, FBXO10, MAP4K1, KMT2C, KMT2D and PIK3R1 (n = 2) in LPD and ML cases. In transcriptome analysis, genes associated with lymphocyte activation including B-cell receptor signaling were upregulated in LPD, cell-cell adhesion, cellular response to TNF, and IL-18 signaling pathway were upregulated in ML samples. We performed correlation analysis to elucidate a relationship between EBV infection status and host transcriptomic profiles using RNA-seq data. Interestingly, in addition to cell-cell adhesion and inflammatory machinery, the upregulated molecules of the p53 pathway and negative regulation of the immune system were correlated with transcripts associated with EBV latency, such as EBNA, LMP-1, and LMP-2. In single-cell transcriptome analysis, LPD sample consisted of two large clusters of T cells and B cells with 10 subsets, and EBV-positive DLBCL sample consisted of one independent large cluster with 9 subsets. In LPD sample, components indicative of the normal lymphocyte differentiation process, including the germinal center, were preserved. The molecules involved in lymphocyte activation were upregulated in LPD subsets. In DLBCL sample, the genes associated with mitotic G2-M phases, chromatin organization and RNA metabolism were upregulated in the subsets in which EBV transcripts were more actively transcribed. Discussion and Conclusions: Our retrospective analysis demonstrated that the outcome of LPD and ML in APDS is not satisfactory. scRNA-seq data revealed that cells in LPD still retain the signature of normal lymph node. However, once transformed with EBV, EBV infection may affect epigenetic machineries including chromatin organizer followed by active RNA transcription involved in cell-cell adhesion, inflammatory machineries, molecules of p53 pathway and negative regulation of immune system in the host genome. As the outcome of ML is inferior to LPD, preemptive HCT would be an option for LPD cases.

Genetic Profile of Myeloid Neoplasms Developed with Fanconi Anemia and Aldehyde Degradation Deficiency Syndrome

Background Fanconi anemia (FA) is an inherited bone marrow failure syndrome which develops on the basis of defective cellular responses to DNA damage. Aldehyde degradation deficiency (ADD) syndrome has been recently found as a disease caused by the defect of degradation of endogenous formaldehyde, which shares similar clinical features with FA. Although genomic abnormalities in these diseases have been associated with the high propensity of myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the mechanism of the early development of MDS/AML remains unclear. Therefore, in this study, we aimed to understand the mechanism of the development of hematological malignancies by analyzing the genomic abnormalities seen in these diseases. Method We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) for 2 AML and 6 MDS developed in Japanese patients with FA (n = 6) and ADD syndrome (n = 2). Skin or bone marrow fibroblasts derived from the same patients were also studied as germline controls. We investigated somatic mutations, structural variants (SVs), and copy number alterations (CNAs) by WES and WGS. Mutational signature was analyzed to understand the mutational processes in these diseases. Result The number of somatic mutations and CNAs in AML identified by WES were 24 and 5 per patient on average, respectively, which were higher than those in MDS (6.8 mutations and 1.8 CNAs per patient) (P=0.07 and 0.06, respectively). However, no significant difference was observed for the number of SVs identified by WGS between MDS (35 counts per patient) and AML (52 counts per patient). Somatic mutations affected known driver genes in myeloid malignancies, such as RUNX1 (n = 1), ASXL1 (n = 1), SRSF2 (n = 1) and NRAS (n = 1) (Figure1). SVs also involved known driver genes in myeloid malignancies, such as CBL (n = 1), KDM6A (n = 1), PHF6 (n = 1), and RUNX1 (n = 1). In total, RUNX1 was most frequently altered (2/6, 33%). CNAs were more frequently accumulated in recurrent regions, including +1q (n = 5), +3q (n = 4), and -7q (n = 2) (Figure1). As previously reported, MDM4 and EVI1 were contained in +1q and +3q, respectively. Higher cancer cell fraction implied that most of these copy number changes were clonal, which suggested the early acquisition of copy number changes and their driver role in leukemic transformation. For mutational signatures, three known signatures were identified, which included clock-like SBS1 and SBS5 caused by endogenous mutational processes, and SBS3 related to defective homologous recombination. In addition, a novel mutational signature (Sig-A) was also detected. SBS3 and Sig-A were mainly detected in patients with FA and ADD syndrome, respectively, indicating that the different mutational processes are operating in the two diseases. Conclusion WES and WGS revealed the genetic landscape of FA and ADD syndrome mutation accumulation. Myeloid neoplasms related with FA and ADD syndrome were characterized by a unique pattern of driver mutations and CNAs, such as RUNX1 mutations/SVs, +1q and +3q. Although the two diseases share some clinical features, our study has revealed the different mutational processes in these diseases. Further investigation is warranted to reveal the underling mechanisms of mutation accumulation and the role of driver mutations in these diseases.

Genomic Profiles and Associated Survival Prognosticators in Black Patients with Acute Myeloid Leukemia

Background Our knowledge of the genomics of acute myeloid leukemia (AML), which serves as the basis of clinically used prognostic biomarkers and therapeutic advances (including targeted therapies), is almost exclusively based on data from patients (pts) of European ancestry. Methods We analyzed the exomes and transcriptomes of 100 Black AML pts at diagnosis who received intensive induction chemotherapy on Alliance for Clinical Trials in Oncology (Alliance) protocols. African Ancestry was confirmed by SNP analysis. We established their genetic landscape including somatic gene mutations, structural variants, gene fusions and associated gene expression. Somatic mutation frequency was compared to whole-exome sequencing of 741 White pts at diagnosis from the Beat AML cohort analyzed by identical workflow. Survival of Black pts was compared to 1,519 White pts treated on the same Alliance protocols. Multivariable analysis was used to identify clinical and molecular features associated with outcomes. Results We identified 162 recurrently mutated genes in Black AML pts. We detected different mutation frequencies for AML-associated genes based on ancestry, and mutations in genes not previously implicated in AML, including PHIP alterations in 7% of this cohort. 38 genes were mutated in 3-5% of Black pts but in <1% of White pts . Of the 162 recurrently mutated genes in Black pts, only 41 (25%) were recurrently mutated in White pts, while 121 genes (75%) were mutated in ≤1 White patient. Only 1/741 White AML pts did not have a mutation in at least 1/ 56 recurrently mutated AML genes assessed by clinical NGS panels. By comparison, 10% of Black pts had no mutations in these genes. Analysis of gene fusions showed recurrent, cryptic CBFA2T3 fusions in 4 pts and a recurrent GGNBP2:: MYO19 fusion involving chromosome 17 not previously described in AML in 2 pts. The CBFA2T3 fusions did not cluster with CBF-AMLs by gene expression, suggesting biological differences. t-SNE visualization of gene expression showed that Black and White AML pts clustered together by known driver mutations. We identified a t-SNE cluster enriched for pts with myelodysplasia-related (MR) mutations. Notably, Black pts in this cluster were significantly younger than White pts [50 vs 58-years (y), p=0.04], suggestive of intrinsic and/or extrinsic dysplasia-causing stressors. When matched for age, performance status and study date, Black pts had higher relapse rates (63% vs 48%, p=0.05), worse disease-free (DFS, 3-y rates, 30% vs 47%, p=0.01) and overall survival (OS, 3-y rates, 31% vs 47%, p=0.007) compared to White pts. In a multivariable analysis, MR mutations (HR=2.06, p=0.03) and mutations in NPM1 (HR=2.29, p=0.009) and NRAS (HR=1.95, p=0.04) were associated with shorter DFS. Using the recently published iScore as a proxy for inflammation, we detected an enrichment of high inflammation in Black pts harboring NPM1 mutations compared to Whites (iScore high vs low; Black, 69% vs 31%, White, 45% vs 55%; p=0.08). Furthermore, when we classified pts into LSC17 prognostic groups, only 13% of Black pts in the prognostically favorable low LSC17 group were NPM1-mutated, compared with to 38% of White pts, suggesting ancestry-associated differences in the biological impact of NPM1 mutations. Mutations in IDH1/2 associated with shorter OS (HR=1.73, p=0.05) in Black pts identifying ancestry-specific risk markers. Finally, we show that the current ELN 2022 risk stratification system can be significantly improved by including NPM1, NRAS and IDH1/2 mutations as ancestry-specific adverse risk markers for Black pts ( Figure 2). Mutations in NPM1, NRAS, and IDH1/2 were enriched in a cohort of 43 Black relapsed/refractory (R/R)-AML pts via MSK-IMPACT supporting their adverse prognostic risk in Black pts. Conclusions Our work demonstrates ancestry-related differences in the genomic profiles of AML pts and calls for additional studies of ancestry-diverse populations to understand the landscape of somatic genetic alterations in AML. Further, our results emphasize the need to include ancestry-associated variants in clinical testing panels and to refine genetic risk assessment of AML by incorporating ancestry-specific prognostic biomarkers. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224. https://acknowledgments.alliancefound.org.

Modified Staging System for Waldenström Macroglobulinemia (MSS-WM): A Multi-Institutional Externally Validated Prognostic Model for Active/Symptomatic Waldenström Macroglobulinemia

Background: Waldenström Macroglobulinemia (WM) is an indolent lymphoma with heterogeneous outcomes. The recently developed, Revised International Prognostic Scoring System for WM (rIPSS-WM) did not examine the prognostic impact of MYD88L265P, a somatic alteration present in most patients with WM. Moreover, rIPSS-WM could only be partially validated in an external cohort. Here, we report the performance of rIPSS-WM and propose a refined prognostic model with external validation. Methods: We reviewed medical records of treatment-naïve patients with active WM consecutively seen at Mayo Clinic, Rochester (MCR) between 01/01/1996 and 12/31/2017. We applied the rIPSS-WM model in patients with data available for all required parameters, including age, serum beta-2 microglobulin (β2M), serum lactate dehydrogenase (LDH) and serum albumin at diagnosis to assess the prognostic utility of this model. We then identified the dichotomized clinical and laboratory parameters prognostic for overall survival (OS) by univariate analyses (UVA) in our cohort. The significant parameters were analyzed to independently predict OS in a multivariable analysis (MVA) and to create a prognostic model, which was subsequently validated in an independent multi-institutional cohort. Results: We identified 889 patients with active WM at MCR, with a median follow-up of 8.2 [95% confidence interval (CI): 7.5-9] years. The estimated median OS for this cohort was 10.6 (95%CI: 9.7-11.4) years. Patients with available data in this cohort for the validation of rIPSS-WM (n=241) were risk stratified into very low (n=46), low (n=64), intermediate (n=58), high (n=46) and very-high risk groups (n=27) per rIPSS-WM, with the respective 5-year OS rates of 96%, 76%, 72%, 77% and 32%, demonstrating overlapping survival curves for the low, intermediate and the high-risk groups. Because rIPSS-WM could not be validated, we used the data from the MCR cohort and identified age at diagnosis of active WM, LDH >upper limit of normal (ULN), β2M >3µg/mL and albumin <3.5g/dL as statistically significant prognostic markers for OS on UVA. Notably, the presence of MYD88 L265P did not impact OS [median 11.4 years (95%CI: 10.1-not reached (NR) for MYD88 L265P genotype (n=231) versus 10 years (95% CI: 8.1-NR) for MYD88 WT genotype (n=65); p=0.33]. On MVA, LDH >ULN (HR 2.34), serum albumin <3.5 g/dL (HR 1.5) and age groups 65-75 years (HR 1.4) and >75 years (HR 2.6) were independently prognostic. Based on comparable HRs, we assigned a score of 1 point each to serum albumin <3.5 g/dL and age group 66-75 years and similarly, 2 points each for age >75 years and serum LDH >ULN per their impact on OS. Patients with the composite scores of 0, 1 and 2 were categorized as low risk, low- intermediate risk and intermediate risk, respectively. Due to similar OS for the scores of 3 to 5, these were combined to form the high-risk group of our proposed model: Modified Staging System for WM (MSS-WM). The median OS was 14.6 years [95%CI: 9.1 years-NR] for low-risk (score 0, n=71, 21%), 11.2 years (95% CI: 9.1-15.2 years) for low-intermediate risk (score 1, n=110, 32%), 8.1 years (95% CI: 6.4-12.2) for intermediate risk (score 2, n=81, 24%) and 5.5 years (95 % CI: 3.9-11 years) for the high-risk group (score ≥3, n=79, 23%); p<0.0001. In this derivation cohort, the 5-year OS rate was 93%, 82%, 69% and 55% and 10-year OS rate was 60%, 53%, 45% and 30% for the low, low-intermediate, intermediate, and high-risk cohort, respectively ( Figure 1A). We also validated MSS-WM using competing risk analysis (p=0.001) and in the cohort of rituximab treated patients (p<0.0001). We then used data from an independent series of 335 treatment-naïve symptomatic patients from academic institutions (n=5) across the US and Europe for external validation of the MSS-WM model. The median follow-up for the validation cohort was 6.3 years (95% CI: 5.5-7.2 years). The 5-year OS was 93%, 90%, 75% and 57% and the estimated 10-year OS was 91%, 80%, 64% and 35% for the low risk (n= 86, 26%), low-intermediate (n=107, 32%), intermediate (n=86, 26%) and high-risk (n=59, 18%) cohorts, respectively (p<0.0001, Figure 1B). Conclusions: Our proposed model, MSS-WM, is a simple, clinically useful, externally validated model that reliably risk stratifies previously untreated patients with active WM into four groups that have distinct outcomes based on the composite scores derived from the patients' age, serum albumin and serum LDH at diagnosis.

Indicators of Response to the Wee1 Inhibitor Adavosertib in Acute Myeloid Leukemia

Wee1 is a nuclear kinase that regulates cell cycle progression by inhibiting Cdk1, which is essential for G2 to M phase transition. Inhibition of Wee1 leads to high Cdk1 activity, causing cells to bypass proper DNA repair at the G2/M checkpoint, resulting in mitotic catastrophe and cell death, plus increased Cdk2 activity, causing abnormal DNA replication and DNA double-stranded breaks. Inhibitors of Wee1 are being investigated for the treatment of a variety of cancers including AML. In this study, we aimed to identify biomarkers for Wee1 inhibitor (Wee1i) response in AML using ex vivo drug sensitivity data, bulk RNA sequence, exome sequence, and associated clinical data for a large set of primary AML samples tested against the Wee1i adavosertib. Mononuclear cells from bone marrow aspirates (n=136) or peripheral blood samples (n=11) were collected from AML patients using approved protocols and after informed consent. 101 samples were collected at the time of diagnosis and 35 samples were from relapsed/refractory (R/R) patients. For ex vivo drug-sensitivity testing, freshly isolated cells were tested against a library of up to 634 oncology drugs (215 approved drugs) including adavosertib. The drugs were pre-plated in 5 concentrations in a 10,000-fold concentration range and incubated with cells for 72h. Cell viability was assessed using the CellTiter Glo (CTG) assay, and a drug sensitivity score (DSS) calculated based on the area under the dose response curve. Somatic alterations of the drug tested samples were identified via exome sequencing of DNA from the AML samples and matched skin biopsy controls. Gene expression data were obtained from bulk RNA sequence data of the AML samples. Out of 136 samples analyzed, the median DSS for adavosertib was 8.45. We found positive and significant correlations between sensitivity to adavosertib and two factors: 1) malignant cell percentage (R= 0.31, p-value= 0.0097), and 2) final relative viability percentage (R=0.58, p-value= 4.3e-13). Correlation analysis between the DSS of adavosertib and 633 other drugs revealed several strong positive and significant correlations. These included drugs with specific mechanisms of actions including HSP90 inhibitors (n=5), JAK2 inhibitors (n=4), topoisomerase II inhibitors (n=4), CDK4/6 inhibitors (n=3), mitotic inhibitors, vinca alkaloid microtubule depolymerizers (n=3), and PLK1 inhibitors (n=3). Since adavosertib also targets PLK1 in addition to Wee1, the correlation with PLK1 inhibitor activity confirms the similar target profiles. Based on the specific adavosertib DSS thresholds, the samples were separated in two groups. Samples sensitive to adavosertib (n=35) had DSS between 21.3 (max) and 11.9 (3rd quartile). Samples resistant to adavosertib (n=34) had DSS between 0.0 (min) and 5.65 (1st quartile). Differential gene expression analysis between the sensitive vs resistant samples showed that multiple isoforms of the PCDH and HOXB genes were upregulated in adavosertib sensitive samples. The GLI2, PI15, and SKIDA1 genes were also upregulated in the sensitive samples (Figure A). In contrast, genes such DNTT, PF4V1, TRH, CD1B, S100A16, and CD34 were overexpressed in resistant samples (Figure B). Gene set enrichment analysis revealed positive enrichment of pathways related to MYC targets v2 in the sensitive samples. On the other hand, hallmark pathways associated with cell cycle, apoptosis, RAS signaling, and p53 showed significant negative enrichment scores in the sensitive samples. No statistically significant associations were observed between drug response and frequently occuring somatic mutations in our dataset. However, genetic abberations related MLLT4-MLL/MLL-fusions were only observed in sensitive samples. Collectively, our current findings show that there may be specific gene expression signatures associated with Wee1i sensitivity such high expression of PCDH and HOXB. Also, the positive enrichment of the MYC target v2 pathway in adavosertib sensitive samples suggests that highly proliferating tumor cells might be more sensitive to Wee1i. In addition, high CD34 expression in resistant samples suggests that immature stem and progenitor cells may be less susceptible to Wee1i. In summary, targeting of Wee1 in AML - as studied in NCT05682170 azenosertib study - may be effective for a molecularly defined subset of patients.

Multi-Omic Insights into the Mutational Landscape and Dysregulated Transcriptional Programs of Autoimmune B-Cells in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with diverse manifestations, from mild and sporadic to severe and life-threatening. SLE heterogeneity arises from various factors, including genetic, epigenetic, and immune niche-related features (Kaul et al., 2016; Jenks et al., 2018). However, the exact interactions and hierarchy between these features remain unclear. Recent studies have found somatic mutations in genes typically mutated in B-cell lymphomas, in subsets of B-cells that are associated with SLE pathogenesis (Piotrowski et al., 2015; Pullabhatla et al., 2018; Singh et al., 2020; Brown et al., 2022). These mutations may support B-cell clonal bursts, but direct information from clinical specimens on the impact of these mutations on B-cell lineages and their influence on SLE progression or malignant transformation is missing. To define the somatic mutational landscape of B-cells in SLE, we first profiled peripheral blood mononuclear cells obtained from a pilot cohort of four SLE patients using whole-exome sequencing (WES; 200X depth). Following GATK best practices (Van der Auwera and O'Connor, 2020), we identified somatic alterations in DDX11 [variant allele frequency (VAF): 10.2%], DNMT3A [VAF: 3.4%]; PMS2 [VAF: 47.9%], and BICD1 [VAF: 47.3%]; these genes have been linked to lymphoid malignancies and SLE pathogenesis (Piotrowski et al., 2015; Pullabhatla et al., 2018; Niroula et al., 2021; Saeed et al., 2021). We validated the presence of the top-identified mutations using droplet digital PCR technology on sorted B-cell subsets (naïve, memory, and double-negative [DN; CD19 hiIgD loCD27 lo]) from these SLE patients. DN cells are a heterogeneous subtype of autoreactive memory B-cells that play a crucial role in SLE pathogenesis being the precursors of autoantibody producing plasma cells (Wei et al., 2007; Jenks et al., 2018). To investigate the transcriptional and epigenetic alterations in the mutation-harboring B-cells, we then conducted single-cell multi-omic sequencing. We profiled sorted activated B-cells (CD19 hiIgD lo) from the four SLE cases, to largely exclude non-autoreactive naïve B-cells. To maintain a comparison with naïve B cells, we included a small proportion of these cells in the input. In total, we analyzed 35,339 cells, and identified different B-cell subsets with distinct gene expression patterns, including DN cells ( n = 1,437 cells; ZEB2 hiTRAF5 lo, Jenks et al., 2018). To uncover, in an unbiased way, transcriptional differences between B-cell subsets, we catalogued variation in transcriptomic signatures derived from the scRNA data using consensus non-negative matrix factorization. We found a unique gene expression program exclusively present in DN cells ( Panel A), which includes key genes implicated in immune regulation and the development of autoimmunity, such as PTPN22 (Chung & Criswell, 2007; Tizaoui et al., 2021) . Pathway analysis of the DN-specific program revealed an enrichment of complement system genes (FDR = 0.074; Hallmark), which are crucial for immune regulation and tolerance, such as CBLB (Tang et al., 2019). Notably, in DN cells from one of the SLE samples carrying lymphoma/SLE-associated mutations in BICD1, PMS2, DDX11, we observed an altered expression of this gene program compared to DN cells from other samples ( P-value = 3.6x10 -11; Panel A, inset), suggesting that a malfunctioning complement system in DN cells may lead to an overly active immune response (Weinstein et al., 2021). In line with this notion, using chromatin accessibility profiles as a measure of transcription factor (TF) regulatory activities, we showed increased activity of TFs involved in interferon-dependent innate immune response and in B-cell activation and differentiation (RFX and OCT-1, respectively; FDR < 0.05), in the DN B-cells of this sample ( Panel B). In summary, we uncovered a unique transcriptional program associated with autoimmune DN B-cells. This program showed enrichment in key genes and pathways involved in immune response regulation. The disruption of this program, accompanied by the somatic SLE-associated mutations, may lead to overactivation of DN B cells, ultimately contributing to autoimmunity. Further analyses involving a larger cohort of clinical specimens are needed to directly link B-cell genotypes with their transcriptional and epigenetic programs and to elucidate the role of the identified mutations in SLE pathobiology.

Deep sequencing reveals recurrent somatic mutations and distinct molecular subgroups in gastric cancer in Mizo population, North East India

In India, Mizoram has the highest incidence of gastric cancer (GC) which might be associated with environmental factors such as diet, Helicobacter pylori (H.pylori) and Epstein-Barr virus (EBV) infections, and somatic genomic alterations. We performed PCR cum sequencing and fragment analysis for detection of H. pylori/EBV infection and microsatellite Instability (MSI) in GC patients (N = 68). Somatic mutations were identified by targeted and exome sequencing. We found 87% of GC patients infected with H. pylori and or EBV. Pathogenic infections were mostly mutually exclusive with only 16% of coinfection. TP53, MUC6, and ARID1A were significantly mutated. Two molecular subgroups with distinctive mutational profiles were identified: (1) patients harboring mutations in TP53 and (2) patients harboring mutations in RTK/RAS/PI3-K signaling pathway and chromatin-remodeling genes. Therefore, EBV and H. pylori infections and somatic mutations in the genes involved in RTK/RAS/PI3K signaling pathway, chromatin-remodeling, and TP53 might drive GC development and progression in Mizo patients.

Detection of Somatic Alterations in Peritoneal Lavages of Early-Stage Endometrial Carcinoma Patients

Detection of Somatic Alterations in Peritoneal Lavages of Early-Stage Endometrial Carcinoma Patients

COSMIC:a curated database of somatic variants and clinical data for cancer.

The Catalogue Of Somatic Mutations In Cancer (COSMIC), https://cancer.sanger.ac.uk/cosmic, is an expert-curated knowledgebase providing data on somatic variants in cancer, supported by a comprehensive suite of tools for interpreting genomic data, discerning the impact of somatic alterations on disease, and facilitating translational research. The catalogue is accessed and used by thousands of cancer researchers and clinicians daily, allowing them to quickly access information from an immense pool of data curated from over 29 thousand scientific publications and large studies. Within the last 4 years, COSMIC has substantially expanded its utility by adding new resources: the Mutational Signatures catalogue, the Cancer Mutation Census, and Actionability. To improve data accessibility and interoperability, somatic variants have received stable genomic identifiers that are associated with their genomic coordinates in GRCh37 and GRCh38, and new export files with reduced data redundancy have been made available for download.

Identification of Potential Genomic Alterations Using Pan-Cancer Cell-Free DNA Next-Generation Sequencing in Patients With Gastric Cancer.

Molecular cancer profiling may lead to appropriate trials for molecularly targeted therapies. Cell-free DNA (cfDNA) is a promising diagnostic and/or prognostic biomarker in gastric cancer (GC). We characterized somatic genomic alterations in cfDNA of patients with GC. Medical records and cfDNA data of 81 patients diagnosed as having GC were reviewed. Forty-nine and 32 patients were tested using the Oncomine Pan-Cancer Cell-Free Assay on the Ion Torrent platform and AlphaLiquid 100 kit on the Illumina platform, respectively. Tier I or II alterations were detected in 64.2% (52/81) of patients. Biomarkers for potential targeted therapy were detected in 55.6% of patients (45/81), and clinical trials are underway. ERBB2 amplification is actionable and was detected in 4.9% of patients (4/81). Among biomarkers showing potential for possible targeted therapy, TP53 mutation (38.3%, 35 variants in 31 patients, 31/81) and FGFR2 amplification (6.2%, 5/81) were detected the most. Next-generation sequencing of cfDNA is a promising technique for the molecular profiling of GC. Evidence suggests that cfDNA analysis can provide accurate and reliable information on somatic genomic alterations in patients with GC, potentially replacing tissue biopsy as a diagnostic and prognostic tool. Through cfDNA analysis for molecular profiling, it may be possible to translate the molecular classification into therapeutic targets and predictive biomarkers, leading to personalized treatment options for patients with GC in the future.