Targeted Capture Sequencing Research Articles

Confirming the TMEM232 gene associated with atopic dermatitis through targeted capture sequencing

Atopic dermatitis (AD) is a common and complex skin disorder, and the 5q22.1 region had been reported to be associated with AD. To confirm the susceptibility gene for AD in the 5q22.1 region by haplotype and targeted capture sequencing. The haplotypes were reconstructed with the genotyping data of four SNPs and six deletions from 3624 Chinese Hans AD patients and 5076 controls. The targeted capture sequencing spanning 5q22.1 region was performed in the selected samples. The gene level enrichment analysis was done using loss of function variants. A total of 62 haplotypes were found, and the H15 haplotype had the strongest association with AD (P = 3.92 × 10−10, OR 0.17, 95% CI 0.09–0.32). However, no co-segregation mutation sites were found in the sequencing analysis within the 16 selected samples, while the enrichment analysis indicated that TMEM232 was significantly associated with AD (P = 7.33 × 10–5, OR 0.33, 95% CI 0.19–0.58). This study confirms previous findings that the TMEM232 gene is associated with AD by haplotype analysis and targeted capture sequencing.

Der(1;7)(q10;p10) Presents with a Unique Genetic Profile and Frequent ETNK1 Mutations in Myeloid Neoplasms

BackgroundDer(1;7)(q10;p10) (der(1;7) is an unbalanced translocation recurrently found in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and related disorders. Caused by a recombination between two homologous alphoid sequencing D1Z7 and D7Z1 on chromosomes 1 and 7, respectively, it results in monosomy 7q and trisomy 1q, which is implicated in the pathogenesis of der(1;7)-positive myeloid neoplasms. Previous studies reported frequent co-occurrence of +8 and del(20q), as well as RUNX1 mutations, the genetic and clinical characteristics of this abnormality has not fully been elucidated.MethodsIn this study, we enrolled a total of 153 cases myeloid neoplasms positive for der(1;7) from Japanese and German cohorts, in which co-occurring genetic lesions were analyzed using whole exome and/or targeted-capture sequencing. An additional 3,223 MDS and related neoplasm cases were also analyzed using targeted-capture sequencing to identify der(1;7)-specific genomic features.ResultsEthnicity was evaluated comparing the frequency of der(1;7) in 944 German MDS cases and 763 Japanese MDS cases. Der(1;7) cases were observed at a higher frequency in Japanese MDS cohort compared to German MDS cohort (73/763 cases versus 4/944 cases, p < 0.00001). Der(1;7) cases showed a strong male predominance (86.3%) (p<0.001). Of 153 myeloid neoplasm patients harboring der(1;7), 114 were diagnosed with MDS, 28 with AML, 5 with MDS-MPN and 1 with MPN. Targeted-capture sequencing revealed mutations in common myeloid drivers (n=61) in 96% of der(1;7) cases. The most frequently mutated gene was RUNX1 with 46%, followed by ETNK1 (24.5%) and EZH2 (24.5%). Of interest, ETNK1 mutation was identified as the most unique to der(1;7) when compared to myeloid neoplasm cases without der(1;7) (n=3,066) [odds ratio (OR)=15.06], followed by ETV6 (OR=9.35) and EZH2 (OR=6.52). To further examine the uniqueness of this mutation profile, the mutational profile of der(1;7) was compared to those myeloid neoplasm cases harboring amp(1q) (n=52) and monosomy 7 (n=105). Highly frequent ETV6 and ETNK1 mutations were highly unique to der(1;7) cases when compared to amp(1q) cases (OR=3.72, OR=2.57, respectively). BCOR and ETNK1 mutations were highly unique to der(1;7) cases when compared to monosomy 7 cases (OR=35.88, OR=4.29, respectively). Both amp(1q) and monosomy 7 cases showed a higher mutation rate in TP53 compared to der(1;7) cases (49.1% and 51%, respectively, vs 3.5 %) . From these mutational characteristics, ETNK1 was identified as being the most unique to der(1;7) when compared to amp(1q), monosomy 7 and other myeloid neoplasm cases. ETNK1-mutated der(1;7) cases were featured with eosinophilia (p < 0.0005), a lack of RAS pathway mutations and trisomy 8 when compared to ETNK1-wild type der(1;7) cases.Survival analysis was conducted to elucidate the difference in survival in der(1;7) cases (n=65) versus myeloid neoplasm cases (n=2066). Der(1;7)-harboring myeloid neoplasm cases had a median overall survival of 6.8 months (95% CI, 3.5 to 11.9) and non-der(1;7) harboring myeloid neoplasm cases were 11.8 months (95% CI, 10.5 to 12.6). Thus, der(1;7)-harboring myeloid neoplasm cases had poorer prognosis (p<0.001).ConclusionIn conclusion, der(1;7) is an unbalanced translocation that occurs predominantly in males and is seen more frequently in Japanese than Caucasian populations. Der(1;7) cases present with a mutational profile that is distinct from other myeloid neoplasm cases such as those with amp(1q) and monosomy7/del(7q), showing frequency of ETNK1 mutations. DisclosuresNannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria. Handa: Ono: Honoraria; BMS: Honoraria; Janssen: Honoraria; Daiichi Sankyo: Research Funding; Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding. Ohyashiki: Novartis Pharma: Other: chief clinical trial; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Ogawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

Efficient Screening Tests and Gene Mutation Spectrum for Hereditary Stomatocytosis in Japan

Background: Hereditary stomatocytosis (HSt) is a group of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. The most common subtype is dehydrated HSt (DHSt), and heterozygous mutations of the mechanosensitive calcium channel gene PIEZO1 have been associated with it most frequently. DHSt is suspected by screening tests such as erythrocyte morphology, cation concentration measurements inside and outside the erythrocyte membrane, or osmotic gradient ektacytometry; target-captured sequencing (TCS) is used for definitive diagnosis. We have shown that an increase in the residual red cells (%RRC) in a quantitative osmotic fragility test using a flow cytometer (FCM-OF) is useful as a screening test for DHSt.Purpose: We report the clinical findings and mutation spectrum of Japanese patients with DHSt confirmed by genetic testing.Methods: From April 2015 to June 2021, 27 patients who had a clinical diagnosis of DHSt and provided written consent were genetically tested. The clinical indications were hemolytic anemia with stomatocytes, accompanied by hemochromatosis, a family history, perinatal edema, and severe jaundice. Laboratory tests showed increased MCV, and subjects with an increased %RRC in FCM-OF were analyzed. TCS was performed using a hemolytic anemia-related gene panel.Results: Of the 27 patients, 14 had PIEZO1 variants, 3 had KCNN4 variants, and 2 had ABCB6 variants, for a total of 19 cases diagnosed as HSt. There was 1 SPTB mutation, 1 GCLC mutation, and 6 cases without mutations in genes known to be related to hemolytic anemia. There were 12 previously reported mutations (KCNN4: R352H, PIEZO1: V598M, T2014I, R2488Q, E2496ELE) and 5 novel mutations (KCNN4: P204R, A279T, PIEZO1: 427_428ins9AA, A1457V, K2323T).Notably, 5 E2496ELE mutations were found in unrelated individuals. There were no differences in age at diagnosis and severity of anemia between the E2496ELE mutation and other PIEZO1 mutations, but jaundice was more severe in patients with the E2496ELE mutation (p=0.007).The median age at diagnosis of the DHSt patients was 28 years (range: 2 months to 89 years); there were 6 men and 11 women. Three patients underwent splenectomy, and 2 patients with PIEZO1 mutations had postoperative thrombosis; 1 KCNN4 mutation had no complications, but no improvement in hemolytic anemia. Six patients had gallstones, 3 had fetal ascites, and 11 received red blood cell transfusions. Laboratory test results showed median Hb 10.4 g/dL (6.9-15.6), median MCV 99.7 fL (85-127.4), median MCHC 35.6% (33-39), and median T-Bil 3.4 mg/dL (0.5-37.9). The median ferritin level was 569.3 ng/mL (87.1-3895); of the 14 patients whose ferritin was measured at the time of diagnosis, 6 had already exceeded 1,000 ng/mL. The FCM-OF showed high values in all 16 cases tested.Discussion: Genetic testing was performed on cases in which DHSt was suspected based on clinical findings, smears, and FCM-OF; the diagnosis of DHSt was confirmed at a high percentage. As previously reported, the severity of hemolytic anemia was wide-ranging, and many cases of hemochromatosis were observed. The PIEZO1 mutation is the most common in the Japanese population, and the number of E2496ELE mutations is particularly conspicuous. Patients carrying E2496ELE mutations are reported to have a younger age at diagnosis and more severe hematological findings than other mutations; however, our results showed no significant differences in age at diagnosis or degree of anemia. Since the correlation between PIEZO1 gene mutation and phenotype has not yet been clarified, further research is considered necessary. DisclosuresNo relevant conflicts of interest to declare.

Population-Wide Introduction of Dose-Adjusted EPOCH-R Is Associated with Improved Outcome of High Grade B-Cell Lymphoma with MYC and BCL2 Rearrangements with Diffuse Large B-Cell Lymphoma Morphology

Population-Wide Introduction of Dose-Adjusted EPOCH-R Is Associated with Improved Outcome of High Grade B-Cell Lymphoma with MYC and BCL2 Rearrangements with Diffuse Large B-Cell Lymphoma Morphology

EPOR/JAK/STAT Signaling Pathway As Therapeutic Target of Acute Erythroid Leukemia

EPOR/JAK/STAT Signaling Pathway As Therapeutic Target of Acute Erythroid Leukemia

Phylogeny of Crataegus (Rosaceae) based on 257 nuclear loci and chloroplast genomes: evaluating the impact of hybridization.

BackgroundHawthorn species (Crataegus L.; Rosaceae tribe Maleae) form a well-defined clade comprising five subgeneric groups readily distinguished using either molecular or morphological data. While multiple subsidiary groups (taxonomic sections, series) are recognized within some subgenera, the number of and relationships among species in these groups are subject to disagreement. Gametophytic apomixis and polyploidy are prevalent in the genus, and disagreement concerns whether and how apomictic genotypes should be recognized taxonomically. Recent studies suggest that many polyploids arise from hybridization between members of different infrageneric groups.MethodsWe used target capture and high throughput sequencing to obtain nucleotide sequences for 257 nuclear loci and nearly complete chloroplast genomes from a sample of hawthorns representing all five currently recognized subgenera. Our sample is structured to include two examples of intersubgeneric hybrids and their putative diploid and tetraploid parents. We queried the alignment of nuclear loci directly for evidence of hybridization, and compared individual gene trees with each other, and with both the maximum likelihood plastome tree and the nuclear concatenated and multilocus coalescent-based trees. Tree comparisons provided a promising, if challenging (because of the number of comparisons involved) method for visualizing variation in tree topology. We found it useful to deploy comparisons based not only on tree-tree distances but also on a metric of tree-tree concordance that uses extrinsic information about the relatedness of the terminals in comparing tree topologies.ResultsWe obtained well-supported phylogenies from plastome sequences and from a minimum of 244 low copy-number nuclear loci. These are consistent with a previous morphology-based subgeneric classification of the genus. Despite the high heterogeneity of individual gene trees, we corroborate earlier evidence for the importance of hybridization in the evolution of Crataegus. Hybridization between subgenus Americanae and subgenus Sanguineae was documented for the origin of Sanguineae tetraploids, but not for a tetraploid Americanae species. This is also the first application of target capture probes designed with apple genome sequence. We successfully assembled 95% of 257 loci in Crataegus, indicating their potential utility across the genera of the apple tribe.

Exploring Somatic Alteration Associating With Aggressive Behaviors of Papillary Thyroid Carcinomas by Targeted Sequencing.

Wisely differentiating high-risk papillary thyroid carcinoma (PTC) patients from low-risk PTC patients preoperatively is necessary when comes to making a personalized treatment plan. It is not easy to stratify the risk of patients according to sonography or lab results before surgery. This study aims to seek out potential mutation gene markers that may be helpful in stratifying the risk of PTC. A custom panel of 439 PTC relevant and classic tumor metabolic pathway relevant genes was designed. Targeted capture sequencing was performed on 35 pairs of samples from 35 PTC tumors and 35 para-tumor thyroid tissues obtained during surgery. Variant calling and detection of cancer gene mutations were identified by bio-information analysis. Ingenuity Pathway Analysis (IPA) was performed to do functional enrichment analysis of high-frequency mutant genes. Immunohistochemistry (IHC) was performed on 6 PTC patients to explore the expression of protein associated with interested genes. Event-free survival (EFS) was calculated to determine which genes might affect the prognosis of patients. We have identified 32 high-frequency mutant genes in PTC including BRAF. RBL2 was found to be significantly correlated to event-free survival, FOXO1, MUC6, PCDHB9, NOTCH1, FIZ1, and RTN1 were significantly associated with EFS, while BRAF mutant was not correlated to any of the prognosis indicators. Our findings in this study might open more choices when designing thyroid gene panels used in FNA samples to diagnose PTC and predict the potentially aggressive behavior of PTC.

Unlocking inaccessible historical genomes preserved in formalin.

Museum specimens represent an unparalleled record of historical genomic data. However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

Histologic and genomic features of breast cancers with alterations affecting the SWI/SNF (SMARC) genes

The SWI/SNF family of proteins is a multisubunit ATPase complex frequently altered in human cancer. Inactivating mutations in SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCs) underpin a subset of tumors such as the malignant rhabdoid tumor and small cell carcinoma of the ovary, hypercalcemic type. Here, we investigated the genotypic and phenotypic characteristics of breast cancers harboring somatic genetic alterations affecting genes of the SMARC family. We analyzed a series of 6026 primary and metastatic breast cancers subjected to targeted-capture sequencing. SMARC core subunit (SMARCA4, SMARCB1, and SMARCA2) alterations were identified in <1% of all breast cancers, consisting of 27 primary and 30 recurrent/metastatic tumors. The majority of SMARC alterations were monoallelic mutations (47/57, 82%) and thus categorized into two groups: Class 1 alterations consisting of potentially pathogenic mutations and rearrangements and Class 2 alterations consisting of missense mutations and small in-frame deletions of unknown significance. Biallelic events in a SMARC gene were present in a minority of cases (10/57, 18%). Histologic patterns in the form of rhabdoid, composite rhabdoid, sarcomatoid or anaplastic features were observed in a subset of Class 1 primary and metastatic tumors (7/57, 12%). SMARC protein was preserved in nearly all tumors analyzed with immunohistochemistry (26/30, 87%). Four Class 1 tumors demonstrated altered SMARC protein expression in the form of loss (1/30, 3%) or mosaic pattern (3/30, 10%). Complete loss of SMARCA2 (BRM) was observed in a sole tumor with composite rhabdoid morphology, and biallelic hits in the SMARCA2 gene. The genomic landscape of both primary Class 1 and 2 breast cancers did not reveal any characteristic findings. In summary, SMARC alterations likely contribute to the biology of a rare subset of breast cancers in the form of biallelic or pathogenic alterations in SMARC, as evidenced by SMARC-deficient phenotype or altered expression of SMARC protein.

Admixture may be extensive among hyperdominant Amazon rainforest tree species.

Summary Admixture is a mechanism by which species of long‐lived plants may acquire novel alleles. However, the potential role of admixture in the origin and maintenance of tropical plant diversity is unclear. We ask whether admixture occurs in an ecologically important clade of Eschweilera (Parvifolia clade, Lecythidaceae), which includes some of the most widespread and abundant tree species in Amazonian forests.Using target capture sequencing, we conducted a detailed phylogenomic investigation of 33 species in the Parvifolia clade and investigated specific hypotheses of admixture within a robust phylogenetic framework.We found strong evidence of admixture among three ecologically dominant species, E. coriacea, E. wachenheimii and E. parviflora, but a lack of evidence for admixture among other lineages. Accepted species were largely distinguishable from one another, as was geographic structure within species.We show that hybridization may play a role in the evolution of the most widespread and ecologically variable Amazonian tree species. While admixture occurs among some species of Eschweilera, it has not led to widespread erosion of most species’ genetic or morphological identities. Therefore, current morphological based species circumscriptions appear to provide a useful characterization of the clade's lineage diversity.

Multi-region sequencing reveals genetic correlation between esophageal squamous cell carcinoma and matched cell-free DNA

PurposeThis study aimed to determine if both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) in patients with esophageal squamous cell carcinoma (ESCC). MethodsPaired multi-regional tumor tissues, cfDNA, and white blood cells (WBCs) were collected from five ESCC patients before treatment, as part of an ongoing prospective study (NCT02395705). Samples from Cohort 1 were sequenced by whole-exome sequencing and samples from Cohort 2 were sequenced by targeted capture sequencing. Somatic single-nucleotide variations (SNVs) were detected by comparing solid tumor or cfDNA with matched WBCs, with a minimum variant allele frequency (VAF) of 0.1% and P value <0.05. ResultsGenomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were sequenced successfully. In Cohort 1, a significant linear relationship between the tumor and cfDNA VAFs (R2= 0.78, P < 0.0001) was found. In Cohort 2, cfDNA could recover an average of 60.7% (31/51; range, 35.7–76.2%) of somatic mutations present in matched solid tumors. There was a significant positive correlation in VAFs between cfDNA and matched solid tumor tissues (R2= 0.92, P < 0.0001). ConclusionsBoth sequencing approaches revealed high intratumoral heterogeneity in ESCC, and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA.

Facilitating population genomics of non-model organisms through optimized experimental design for reduced representation sequencing

BackgroundGenome-wide data are invaluable to characterize differentiation and adaptation of natural populations. Reduced representation sequencing (RRS) subsamples a genome repeatedly across many individuals. However, RRS requires careful optimization and fine-tuning to deliver high marker density while being cost-efficient. The number of genomic fragments created through restriction enzyme digestion and the sequencing library setup must match to achieve sufficient sequencing coverage per locus. Here, we present a workflow based on published information and computational and experimental procedures to investigate and streamline the applicability of RRS.ResultsIn an iterative process genome size estimates, restriction enzymes and size selection windows were tested and scaled in six classes of Antarctic animals (Ostracoda, Malacostraca, Bivalvia, Asteroidea, Actinopterygii, Aves). Achieving high marker density would be expensive in amphipods, the malacostracan target taxon, due to the large genome size. We propose alternative approaches such as mitogenome or target capture sequencing for this group. Pilot libraries were sequenced for all other target taxa. Ostracods, bivalves, sea stars, and fish showed overall good coverage and marker numbers for downstream population genomic analyses. In contrast, the bird test library produced low coverage and few polymorphic loci, likely due to degraded DNA.ConclusionsPrior testing and optimization are important to identify which groups are amenable for RRS and where alternative methods may currently offer better cost-benefit ratios. The steps outlined here are easy to follow for other non-model taxa with little genomic resources, thus stimulating efficient resource use for the many pressing research questions in molecular ecology.

Using target capture to address conservation challenges: Population-level tracking of a globally-traded herbal medicine.

The promotion of responsible and sustainable trade in biological resources is widely proposed as one solution to mitigate current high levels of global biodiversity loss. Various molecular identification methods have been proposed as appropriate tools for monitoring global supply chains of commercialized animals and plants. Here, we demonstrate the efficacy of target capture genomic barcoding in identifying and establishing the geographic origin of samples traded as Anacyclus pyrethrum, a medicinal plant assessed as globally vulnerable in the IUCN Red List of Threatened Species. Samples collected from national and international supply chains were identified through target capture sequencing of 443low-copy nuclear makers and compared to results derived from genome skimming of plastome and DNA barcoding of standard plastid regions and ITS. Both target capture and genome skimming provided approximately 3.4million reads per sample, but target capture largely outperformed standard plant barcodes and entire plastid genome sequences. We were able to discern the geographical origin of Anacyclus samples collected in Moroccan, Indian and Sri Lankan markets, differentiating between plant materials originally harvested from diverse populations in Algeria and Morocco. Dropping costs of analysing samples enables the potential of target capture to routinely identify commercialized plant species and determine their geographic origin. It promises to play an important role in monitoring and regulation of plant species in trade, supporting biodiversity conservation efforts, and in ensuring that plant products are unadulterated, contributing to consumer protection.

Targeted capture sequencing identifies genetic variations of GRK4 and RDH8 in Han Chinese with essential hypertension in Xinjiang.

Essential hypertension is a common cardiovascular disease with complex etiology, closely related to genetic and environmental factors. The pathogenesis of hypertension involves alteration in vascular resistance caused by sympathetic nervous system (SNS) and renin angiotensin system (RAS). Susceptibility factors of hypertension vary with regions and ethnicities. In this study, we conducted target capture sequencing on 54 genes related to SNS and RAS derived from a collection of Han nationality, consisting of 151 hypertension patients and 65 normal subjects in Xinjiang, China. Six non-synonymous mutations related to hypertension were identified, including GRK4 rs1644731 and RDH8 rs1801058, Mutations are predicted to affect 3D conformation, force field, transmembrane domain and RNA secondary structure of corresponding genes. Based on protein interaction network and pathway enrichment, GRK4 is predicted to participate in hypertension by acting on dopaminergic synapse, together with interacting components. RDH8 is involved in vitamin A (retinol) metabolism and consequent biological processes related to hypertension. Thus, GRK4 and RDH8 may serve as susceptibility genes for hypertension. This finding provides new genetic evidence for elucidating risk factors of hypertension in Han nationality in Xinjiang, which in turn, enriches genetic resource bank of hypertension susceptibility genes.

Association of emergence of new mutations in circulating tumuor DNA during chemotherapy with clinical outcome in metastatic colorectal cancer

BackgroundThe understanding of molecular changes in mCRC during treatment could be used to personalise therapeutic strategies. The aim of our study was to explore the association of circulating tumour DNA (ctDNA) with clinical outcome in metastatic colorectal cancer (mCRC).MethodsSequential patients with mCRC receiving standard first-line chemotherapy were included prospectively. Both plasma ctDNA and serum CEA were assessed in samples obtained before treatment and after 4 cycles of chemotherapy (C4). Computed tomography (CT) scans were carried out at baseline and post-C4 (8–10 weeks) and were assessed using Response Evaluation Criteria In Solid Tumours version 1.1 (RECIST v1.1). Target-capture deep sequencing with a panel covering 1021 genes was performed to detected somatic mutations in ctDNA.ResultsA total of 20 patients were prospectively included and treated with either leucovorin, fluorouracil, and oxaliplatin (FOLFOX) (15/20) or leucovorin, fluorouracil, and irinotecan (FOLFIRI) (5/20). Median follow-up was 6.9 months (range 1.6–26.6). Somatic mutations for baseline ctDNA analysis were identified in 85% (17/20) of the patients. Mutation variations of ctDNA after chemotherapy were tested in 16/20 (80.0%) of the patients. In multivariate analyses, a high baseline molecular tumour burden index (mTBI) in ctDNA was associated with a higher risk of disease progression, as well as emergence of new mutations in ctDNA during chemotherapy. Patients with newly detected mutations had shorter progression-free survival (PFS) compared to those without (median 3.0 versus 7.3 months; hazard ratio (HR), 5.97; 95% confidence interval (CI), 0.70–50.69; P = 0.0003). Fold changes in mTBI from baseline to post-C4 were obtained in 80.0% (16/20) of the patients, which were also related to PFS. Patients with fold reduction in mTBI above 0.8-fold had longer PFS compared to those below (median 9.3 versus 4.1 months; HR, 4.51; 95% CI, 1.29–15.70; P = 0.0008).ConclusionsNewly detected mutations in ctDNA during treatment might potentially be associated with clinical outcome in mCRC and may provide important clinical information.

Abstract 2276: Genetic classification of colorectal cancer

Abstract Introduction: Comprehensive genetic research has classified colorectal cancer (CRC) into hypermutated CRC and non-hypermutated CRC. Among hypermutated CRC, microsatellite instable CRC (MSI-CRC) is reported as a distinct entity with good overall survival. However, there are still no widely accepted classification in non-hypermutated CRC. To classify CRC based on driver mutations and their association with clinical outcomes, we conducted targeted-capture sequencing of known driver mutations in over 3000 CRC samples. Methods: We enrolled a total of 3056 CRC patients with varying stages, including stage I (n=653), II (n=894), III (n=994) and IV (n=653). All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Targeted-capture sequencing was performed using RNA baits designed for detecting 169 driver genes in CRC. Additional baits for 1,630 SNPs were also included to assess genome-wide abnormalities (CNA). Results: The median age at diagnosis was 64 (32-95) with a median observation period of 1,819 days. The average depth of the sequencing was 839 ×. Hypermutated CRC accounted for 9.6%, which included MSI-CRC, POLE mutated CRC and POLD1 mutated CRC (8%, 1,5%, 0.1%, respectively). As previously reported, they showed excellent overall survival. APC (88%), TP53 (79%), KRAS (45%), and PIK3CA (15%) represented the most frequent mutational targets among non-hypermutated CRC. We applied non-negative matrix factorization (NMF) consensus clustering to the identified driver mutations and discovered seven robust subsets of tumors with discrete genetic signatures (Cluster1-7) among non-hypermutated CRC. Clusters are characterized by APC/TP53 mutations (without other mutations, Cluster1), PI3K pathway gene alterations (Cluster2), TGF-beta pathway gene alterations (Cluster3), cell cycle pathway gene alterations (Cluster4), APC/TP53/KRAS mutations (without other mutations, Cluster5), SOX9/ARID1A mutations (Cluster6) and FBXW7mutation (Cluster7). Cluster1 and 5, which only harbor the basic genetic alterations in CRC carcinogenesis, showed moderate overall survival. Cluster7 showed improved overall survival, which indicates a protective role of FBXW7 mutation. In contrast, Cluster3, 4 and 6 have worse prognosis, which each cluster defining alterations are predicted to have additional effects on the aggressiveness of CRC. We constructed a multivariate model considering age, sex, stage and genetic clusters as variables, with stageI and Cluster1 as reference. Although stage showed the strongest contribution to overall survival, Cluster3, 4 and 6 had a hazard ratio of 1.51, 1.64 and 1.54. Thus, the genetic clusters captured outcome differences that were independent of stage. Conclusions: We have revealed the genetic classification of non-hypermutated CRC in a large cohort. CRC are classified into 7 distinct genetic subtypes characterized by unique profiles of gene mutations and clinical outcomes. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yasuhito Nanya, Yusuke Shiozawa, Yasuhide Takeuchi, Yoichi Fujii, Yuichi Shiraishi, Kenichi Chiba, Tetsuichi Yoshizato, Satoshi Nagayama, Yoshiharu Sakai, Seishi Ogawa. Genetic classification of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2276.

Genetic signature of human longevity in PKC and NF-κB signaling.

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer's disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.

Revised Risk Stratification Criteria for Children with Newly Diagnosed Acute Myeloid Leukemia: A Report from the Children's Oncology Group

Introduction: Currently, most phenotypic, cytogenetic or molecular markers identified in children with acute myeloid leukemia (AML) are not used for risk stratification or treatment assignment. Historically, in the Children's Oncology Group (COG) pilot study AAML03P1, patients with core binding fusion (CBF) [t(8;21), inv(16)] or with NPM1 or CEBPA mutations had a more favorable outcome. Patients with FLT3 -ITD with high allelic ratio (FLT3 -ITD-HAR), monosomy 7, or -5/del(5q) had more adverse outcomes. In AAML03P1, 31% of patients were deemed low risk (LR), 9% were high risk (HR) and the remaining 60% were considered standard risk (SR) with event-free survival (EFS) of approximately 50% (Figure 1A). The same risk classification was carried forward to the successor Phase III COG study AAML0531 with similar outcomes. To develop an improved risk stratification strategy, we conducted a comprehensive retrospective evaluation of potential biomarkers that have been reported using available karyotype, immunophenotype and next generation sequencing (NGS) data from patients treated on AAML0531.Methods: AAML0531 enrolled 1,022 patients (age 0-29 years) and randomized to standard therapy with or without gemtuzumab ozogamicin. Centrally reviewed data karyotype and FLT3, NPM1 and CEBPA status were available for this study. An additional 740 individual diagnostic specimens were interrogated by NGS (whole genome, transcriptome or targeted capture sequencing) to identify additional somatic variants including cryptic fusions, single nucleotide (SNV) and copy number variants (CNVs). Post induction response by multidimensional flow cytometry (MDF), an important prognostic modality, was also analyzed for risk stratification assignment in SR patients. Information from genomic and MDF parameters were analyzed for association with event free (EFS) and disease free survival (DFS).Results: Table 1 shows the cytogenetic, molecular, and immunophenotypic markers used in our new risk stratification model. HR fusions detected by karyotype included 5 distinct KMT2A (MLL) fusion types, DEK-NUP214, NPM1 - MLF1, MECOM (3q26.2), and KAT6A (8p11.21). HR cryptic fusions detected by NGS include CBFA2T3-GLIS2, NUP98 family fusions (NUP98-NSD1, NUP98-KDM5A, NUP98-HMGB3 and NUP98-HOXD13) as well as ETS transcription factor family members (ETV6, ERG, FEV). NGS also identified HR MLL, t(6;9) or ETS fusions in 10% of patients who were initially reported as having a normal karyotype. HR stratification by immunophenotype analysis includes the RAM phenotype that was reported in younger patients with overlap with FAB-M7 as well as CBFA2T3-GLIS2 fusions.When karyotype, immunophenotype, and NGS analyses were retrospectively applied to patients treated in AAML0531, the distribution of patients in each risk group changed significantly compared to AAML03P1 (Figure 1A and 1B). According to the revised risk stratification, HR variants constituted 34% of all AML patients and had poor prognosis (EFS-26.3% ± 5.4%). Patients with CBF AML, NPM1 or CEBPA mutations were included in the LR cohort and those with RBM15-MLK1 fusion were relocated to the LR cohort and these collectively constitute 33% of AML cases (EFS-70.4% ± 5.6%). The SR cohort was reduced to 33% in this proposed risk stratification schema (EFS-48.6% ± 6.2%). Minimal residual disease (MRD) by MDF at end of first induction was factored for SR patients in the proposed risk classification (Figure 1C) and found to have prognostic significance; therefore SR patients were assigned to either HR (DFS-28.5% ± 5.2%) or LR (DFS-63.7% ± 4.5%) cohorts.Conclusions: Analysis of refined karyotyping and molecular data and incorporation of validated NGS data for patients in AAML0531 significantly altered the risk stratification used to predict relapse and assign patients to appropriate therapy. This report is the first to study a large number of patients assigned to a clinical trial and apply new standards for karyotyping, molecular and NGS. In future studies, we plan to study all patients on COG trials by NGS methods, validate additional novel biomarkers, and to identify new targets for future precision medicine-based clinical trials. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.1. Apoptotic associated pathway (FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.2. Epigenetic modifier pathway (GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.3. Translation initiation pathway (DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.4. Transcription factors (MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complexpathogenesis of this rare tumor. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Molecular Pathogenesis of Progressive Refractoriness in MLL -Rearranged AML

BackgroundDespite advanced therapeutics, many leukemia patients become refractory to additional therapy, accounting for a major cause of leukemic deaths. Among major human acute myeloid leukemias (AMLs), MLL -rearranged AML is characteristic of progressive refractoriness to chemotherapy and shorter period to relapse. To clarify the molecular pathogenesis of such characteristics, we analyzed genetic abnormalities accumulated in MLL -rearranged AML in additional to the genetic events like MLL -fusion and MLL -partial tandem duplication (MLL-PTD).MethodWe exploited a mouse model of recurrent leukemia generated by mouse progenitors transduced with human MLL / AF9. Mouse leukemic cells with MLL / AF9 were serially transplanted in a total of 58 mice in 4 generations. We investigated somatic genetic events in leukemic cells from mouse models of MLL -rearranged AML and patients with MLL -rearranged AML. Whole exome and targeted-capture sequencing results were combined and analyzed to detect somatic genetic events and whole transcriptome sequencing and microarray dataset to assess gene expression.ResultsIn two independent experiments using mouse with transduced MLL/AF9, leukemic cells obtained in the 1st (N=2) and 4th (N=9) transplants were analyzed by whole exome sequencing. The onset of leukemia became progressively earlier in later generation transplant. Median periods to relapse were 57 and 16 days in the 1st and the 4th transplants, respectively (P < 0.001). With regard to molecular pathogenesis, leukemic cells in the 4th transplants had 12−31 more mutations, in addition to the mutations found in those in the 1st transplants (6-44 mutations). Significantly, two RNA polymerases (Polr2a and Polr3d), which are functionally related to Mll, were mutated in two experiments during the 4th transplants, suggesting that genetic lesions in MLL-associated complex could play a synergetic role in survival advantage of MLL/AF9 positive cells.In addition, mutations in Ptpn11 (G60R) and Gnb2 (G77R), which correspond to the same residues in human proteins, were also detected. In the cohort of human hematological neoplasms analyzed by targeted-capture sequencing, PTPN11 (G60R) is located at the very well documented hotspot in MLL -rearranged human AML and GNB2 G77 residue is present close to that affected by other recurrent mutations in human AML. Of note is that a germline mutation of the identical position of orthologous protein GNB1 was most recently reported in a case with acute lymphoblastic leukemia. To confirm pathological significance of such a novel GNB2 mutation, we analyzed gene expression in human AML. When compared between human MLL -rearranged (N=176) and MLL -rearrangement (−) (N=692) AML cases, GNB2 transcripts were significantly upregulated in human MLL -rearranged AML cases. Functionally, we induced wild-type and G77R mutant GNB2 in human hematopoietic cell lines. Overexpression of wild-type GNB2 conferred cytokine independency to granulocyte macrophage colony-stimulating factor dependent leukemic cell line TF1. Induced GNB2 (G77R)and knocked down wild-type GNB2 in MLL -rearranged AML cell lines were used to assess and confirm the molecular pathogenesis associated with a novel candidate protein GNB2.ConclusionsOur results in serial transplant experiments provide insights into the molecular mechanism of progression and progressive refractoriness of human MLL -rearranged AML. This study confirms that GNB2 is a novel candidate protein whose overexpression and activated mutation might play a significant role in the pathogenesis of this leukemia. DisclosuresTakaori-Kondo:celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria. Makishima:Yasuda Medical Foundation: Research Funding.