Malignancy-related Genes Research Articles

Prognostic significance of CHCHD2P9 and ZNF204P in breast cancer: exploring their expression patterns and associations with malignancy-related genes.

Non-coding RNAs (ncRNAs) have a crucial impact on diverse cellular processes, influencing the progression of breast cancer (BC). The objective of this study was to identify novel ncRNAs in BC with potential effects on patient survival and disease progression. We utilized the cancer genome atlas data to identify ncRNAs associated with BC pathogenesis. We explored the association between these ncRNA expressions and survival rates. A risk model was developed using candidate ncRNA expression and beta coefficients obtained from a multivariate Cox regression analysis. Co-expression networks were constructed to determine potential relationships between these ncRNAs and molecular pathways. For validation, we employed BC samples and the RT-qPCR method. Our findings revealed a noteworthy increase in the expression of AC093850.2 and CHCHD2P9 in BC, which was correlated with a poor prognosis. In contrast, ADAMTS9-AS1 and ZNF204P displayed significant downregulation and were associated with a favorable prognosis. The risk model, incorporating these four ncRNAs, robustly predicted patient survival. The co-expression network showed an effective association between levels of AC093850.2, CHCHD2P9, ADAMTS9-AS1, and ZNF204P and genes involved in pathways like metastasis, angiogenesis, metabolism, and DNA repair. The RT-qPCR results verified notable alterations in the expression of CHCHD2P9 and ZNF204P in BC samples. Pan-cancer analyses revealed alterations in the expression of these two ncRNAs across various cancer types. This study presents a groundbreaking discovery, highlighting the substantial dysregulation of CHCHD2P9 and ZNF204P in BC and other cancers, with implications for patient survival.

Abstract 5649: PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors

Abstract Background: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. Methods: PRRX1 expression in human Malignant peripheral nerve sheath tumors (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro, and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA seq, and structural prediction were used to identify proteins interacting with PRRX1. Results: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumor malignancy-related gene sets including mTORC1, KRAS, and SRC signaling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. Conclusion: Targeting the PRRX1-TOP2A interaction in malignant tumors with high PRRX1 expression might provide a novel tumor-selective therapeutic strategy. Significance: MPNST is a malignant tumor with a poor prognosis and still lacks definitive treatment. Our results could hold promise as an alternative therapeutic strategy to inhibit the interaction between PRRX1 and TOP2A, and may improve the prognosis for malignant tumor patients with high expression of PRRX1 and TOP2A such as MPNSTs. Citation Format: Shota Takihira, Eiji Nakata, Daisuke Yamada, Tatsunori Osone, Tomoka Takao, Takeshi Takarada, Akira Hirasawa, Takuto Itano, Tomohiro Fujiwara, Toshiyuki Kunisada, Toshifumi Ozaki. PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5649.

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours

BackgroundPaired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated.MethodsPRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1.ResultsHigh expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1–TOP2A interaction.ConclusionTargeting the PRRX1–TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

High Prevalence of MYD88 and CD79B Mutations and PD-L1 Alterations in Bone Marrow Large B Cell Lymphoma Associated with Hemophagocytic Lymphohistiocytosis

Introduction: Bone marrow large B cell lymphoma associated with hemophagocytic lymphohistiocytosis (HLH) is a rare disease with a highly aggressive clinical course. Until now, the molecular genomics of bone marrow large B cell lymphoma associated with HLH remains undetermined. Methods: Cases of bone marrow large B cell lymphoma with HLH were included. The inclusion criteria were as follows: (1) diagnosis of large B cell lymphoma presenting initially in the bone marrow; (2) no overt lymph node involvement or tumor formation; (3) fulfillment of five or more of the eight HLH-2004 diagnostic criteria. (4) cases with available samples. Genomic DNA was extracted from bone marrow cells or the plasma. A panel comprising 475 hematological malignancy-related genes were used for targeted gene sequencing. The prepared library was sequenced using Illumina HiSeq 4000. Molecular classification was predicted by using the LymphGen algorithm. Results: A total of 23 cases were included. The median age was 63. 71.4% of patients were the non-GCB subtype (10/14) based on the Hans algorithm and 50% (9/18) of cases showed CD5 expression. Somatic mutations were identified in all the cases. The most common disrupted genes were PIM1 (12/23, 52.2%), PD-L1 (9/23, 39.1%), MYD88 (8/23, 34.8%), BCL6 (6/23, 26.1%), CD79B (6/23, 26.1%), IRF4 (6/23, 26.1%) and TET2 (6/23, 26.1%) (Figure 1). For nine cases with PD-L1 aberrations, seven cases had PD-L1 structural variants (SVs) and one case showed PD-L1 amplification (Figure 2). All the six cases with BCL6 aberrations showed BCL6 SVs (Figure 2). For six cases with BCL6 SVs, four cases showed immunoglobulin heavy chain ( IGH)- BCL6 translocations and two cases harbored IKZF1- BCL6 translocations. Regarding molecular classification, eight cases (34.8%) were classified as the MCD subtype, two cases the BN2 subtype, two cases the ST2 subtype, and two cases the composite subtype. Conclusion: To the best of our knowledge, this is the first study exploring the molecular genetics of bone marrow large B cell lymphoma with HLH. Our study suggesting bone marrow large B cell lymphoma with HLH showed frequent MYD88/CD79B and PD-L1 alterations. Our study provided clues for the pathogenesis of bone marrow large B cell lymphoma with HLH and identified potential druggable targets for future clinical trials.

Integrated analysis of single-cell and Bulk RNA sequencing reveals a malignancy-related signature in lung adenocarcinoma.

Lung adenocarcinoma (LUAD), the most common histotype of lung cancer, may have variable prognosis due to molecular variations. The research strived to establish a prognostic model based on malignancy-related risk score (MRRS) in LUAD. We applied the single-cell RNA sequencing (scRNA-seq) data from Tumor Immune Single Cell Hub database to recognize malignancy-related geneset. Meanwhile, we extracted RNA-seq data from The Cancer Genome Atlas database. The GSE68465 and GSE72094 datasets from the Gene Expression Omnibus database were downloaded to validate the prognostic signature. Random survival forest analysis screened MRRS with prognostic significance. Multivariate Cox analysis was leveraged to establish the MRRS. Furthermore, the biological functions, gene mutations, and immune landscape were investigated to uncover the underlying mechanisms of the malignancy-related signature. In addition, we used qRT-PCR to explore the expression profile of MRRS-constructed genes in LUAD cells. The scRNA-seq analysis revealed the markers genes of malignant celltype. The MRRS composed of 7 malignancy-related genes was constructed for each patient, which was shown to be an independent prognostic factor. The results of the GSE68465 and GSE72094 datasets validated MRRS's prognostic value. Further analysis demonstrated that MRRS was involved in oncogenic pathways, genetic mutations, and immune functions. Moreover, the results of qRT-PCR were consistent with bioinformatics analysis. Our research recognized a novel malignancy-related signature for predicting the prognosis of LUAD patients and highlighted a promising prognostic and treatment marker for LUAD patients.

Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer

BackgroundIn vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines.MethodsWe established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein.ResultsAs a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1.ConclusionsOur study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

KDM2A plays a dual role in regulating the expression of malignancy-related genes in esophageal squamous cell carcinoma

KDM2A is a histone demethylase, which primarily catalyzes the demethylation of H3K36me2. Abnormal expression of KDM2A is observed in many types of cancers; however, the molecular events connected to KDM2A expression remain unclear. We report that KDM2A performs an oncogenic function in esophageal squamous cell carcinoma (ESCC) and is robustly expressed in ESCC cells. ShRNA-mediated knockdown of KDM2A resulted in a significant inhibition of the malignant phenotype of ESCC cell lines, whereas ectopic expression of KDM2A showed the opposite effect. We also analyzed the function of KDM2A using a CRISPR-CAS9 depletion system and subsequent rescue experiment, which also indicated a cancerous role of KDM2A. Interestingly, analysis of the gene expression network controlled by KDM2A using RNA-seq revealed an unexpected feature: KDM2A could induce expression of a set of well-documented oncogenic genes, including IL6 and LAT2, while simultaneously suppressing another set of oncogenes, including MAT2A and HMGCS1. Targeted inhibition of the upregulated oncogene in the KDM2A-depleted cells led to a synergistic suppressive effect on the malignant phenotype of ESCC cells. Our results revealed the dual role of KDM2A in ESCC cells, which may have therapeutic implications.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy‐related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele‐lacking (HLA[−]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%–49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%–37.3%) in the eight patients without HLA(−) granulocytes, 8.8% (4.1%–9.8%) in the five patients with HLA(−) cells accounting for >90% of granulocytes, and 7.8 (2.1%–8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%–88.7%]) in the five AA patients without HLA(−) granulocytes possessing somatic mutations (c‐kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%–88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

Identification of key genes associated with papillary thyroid microcarcinoma characteristics by integrating transcriptome sequencing and weighted gene co-expression network analysis.

Papillary thyroid microcarcinoma (PTMC) is the most prevalent histological type of thyroid carcinoma. Despite the overall favorable prognosis of PTMC, some cases exhibit aggressive phenotypes. The identification of robust biomarkers may improve early PTMC diagnosis. In this study, we integrated high-throughput transcriptome sequencing, bioinformatic analyses and experimental validation to identify key genes associated with the malignant characteristics of PTMC. Total RNA was extracted from 24 PTMC samples and 7 non-malignant thyroid tissue samples, followed by RNA sequencing. The differentially expressed genes (DEGs) were identified and used to construct co-expression networks by weighted gene co-expression network analysis (WGCNA). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and protein-protein interaction networks were constructed. Key modules and hub genes showing a strong correlation with the malignant characteristics of PTMC were identified and validated. The green-yellow and turquoise modules generated by WGCNA were strongly associated with the malignant characteristics of PTMC. Functional enrichment analysis revealed that genes in the green-yellow module participated in cell motility and metabolism, whereas those in the turquoise module participated in several oncogenic biological processes. Nine real hub genes (FHL1, NDRG2, NEXN, SYNM, COL1A1, FN1, LAMC2, POSTN, and TGFBI) were identified and validated at the transcriptional and translational levels. Our preliminary results indicated their diagnostic potentials in PTMC. In this study, we identified key co-expression modules and nine malignancy-related genes with potential diagnostic value in PTMC.

TP53 Allelic Status in Myeloid Malignancies

IntroductionTP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53.The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing.Methods:We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status.Results:18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years.23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis.Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases.Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF >50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations.Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well.Conclusion:In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PNH-Phenotype Cells and HLA Class I Allele-Lacking Leukocytes Shared By Family Members Who Developed Acquire Aplastic Anemia: Studies on Six Pairs of Patients in Different Families

[Background] Aplastic anemia (AA) is a syndrome characterized by pancytopenia and bone marrow hypoplasia caused by various factors. When AA occurs in family members, congenital AA is usually suspected. However, some anecdotal reports have described the occurrence of AA in family members who did not show any signs or laboratory findings compatible with congenital AA and instead represented an acquired feature, such as a good response to immunosuppressive therapy. Studying such familial AA without evident congenital features may help to clarify the genetic or environmental factors that are closely associated with susceptibility to AA. [Objectives/Methods] To characterize non-congenital AA that occurred in family members, we reviewed the clinical records of about 3000 AA patients who were referred to our clinic for the examination of glycosylphosphatidylinositol-anchored protein-deficient (PNH-type) blood cells from 1999 to 2017 and found 6 pairs of AA patients in 6 different families (Table). Three pairs were parents and children, and the other three pairs were siblings. None of the families had malformations or family histories except for the affected pairs. All patients' samples were subjected to high-sensitivity flow cytometry for detecting PNH-type cells and HLA genotyping. Two pairs of patients (Cases 7 and 8, and Cases 9 and 10) who were diagnosed after 2015 were also examined for the presence of HLA class I-lacking leukocytes using monoclonal antibodies specific to class I HLA alleles, and for the presence of myeloid malignancy-related gene abnormalities using a next-generation sequencer. [Results] PNH-type granulocytes were detected in 9 of 12 cases at frequencies of 0.016% to 30.381%. Five pairs shared HLA-DRB1*15:01, which is known to be associated with PNH, while three pairs shared HLA-B*40:02, an HLA class I allele most frequently lost as a result of 6pLOH or allelic mutations. HLA-A allele-lacking leukocytes were detected in one of the two pairs studied (Figure). Unexpectedly, the lacked HLA alleles in the pair (Cases 7 and 8) were not B*40:02but A*02:06. No somatic mutations were found in 61 myeloid malignancy-related genes of the granulocytes from the 2 cases. All of 12 patients responded to immunosuppressive therapy (IST, 6 with cyclosporine alone and 4 with ALG/ATG+/-cyclosporine) and achieved transfusion independence. [Conclusions] The high prevalence of markers for immune pathophysiology, including PNH-type cells and HLA allele-lacking cells, the high frequency of DRB*15:01 and B*40:02, and a good response to IST in the 6 family pairs suggests that non-congenital familial AA is a typical type of immune-mediated bone marrow failure affected by the genetic background. The lack of the same HLA class I allele (A*02:06) in the mother (Case 7) and her daughter (Case 8) in their granulocytes suggests the involvement of a common environmental factor that induces the generation of cytotoxic T lymphocytes specific to common antigens presented by the HLA-A allele. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Moderate Increase in the WT1 mRNA Copy Number in Peripheral Blood of Patients with Long-Standing Acquired Aplastic Anemia without Apparent Signs of Myelodysplastic Syndrome

Background: The copy number of Wilms tumor 1 mRNA (WT1) correlates well with the acute myeloid leukemia (AML) cell burden and is being used as a reliable marker of residual AML cells. WT1 has also been shown to be useful for distinguishing high-risk myelodysplastic syndrome from low-risk myelodysplastic syndrome (MDS) as well as diagnosing disease progression with time in patients with low-risk MDS. However, few studies have so far focused on the role of measuring WT1 in diagnosing clonal evolution in patients with acquired aplastic anemia (AA). Although normal values of WT1 are defined as <50 copies/µgRNA, the reference values in AA patients without a propensity toward progression to MDS also remain unclear. To address these issues, we retrospectively studied 36 patients with bone marrow failure whose WT1 copy numbers in peripheral blood (PB) were serially measured to determine their correlation with the clinical picture and prognosis of the patients. Patients & Methods: PB samples of 36 patients (male/female, 19/17; a median age, 53.5 [range, 23-85] years) were subjected to WT1 measurement using a quantitative PCR method. The diagnoses of the patients were acquired AA in 29, MDS-multilineage dysplasia (MLD) evolved from AA in 4, and hemolytic paroxysmal nocturnal hemoglobinuria (PNH) in 3. Among 29 AA patients, 2 was untreated, and 22 were in convalescence while 5 were refractory after 0.5-457 months of immunosuppressive therapy (IST) and/or androgen therapy. WT1 was serially measured over time for all the patients except 5 whose initial examination produced negative (<50 copies/µgRNA) results. Myeloid malignancy-related gene abnormalities were screened in 11 patients showing an increase in the WT1 copy number. Results: WT1 at the initial examination was negative in 12 and positive in 17 (59%) patients with AA ranging from 59 to 520 copies. The 17 patients with increased WT1 had a disease duration of 122 months (11-495) but did not shown any signs of progression to MDS. The WT1 levels in patients possessing PNH-type cells (<50-330, median 62) were comparable to those without PNH-type cells (<67-520, median 89. There was a significant difference in the WT1 level between the 10 patients with a disease duration <3 years (<50-190, median <50) and the 19 patients with a disease duration ≥3 years (<50-520, median, 110, P=0.032). All 6 patients with WT1>200 had a disease duration ≥3 years. In contrast, the WT1 levels in the 4 patients who relapsed with cytopenia due to progression to MDS were as high as 690-5,700 (median, 2,400). Deep sequencing revealed somatic mutations of myeloid malignancy-related genes in 4 of 11 patients studied, which included EZH2 in an MDS patient (WT1 2,000), BCOR and DNMT3A in another MDS patient (WT1 2,800), and ASXL1 in a PNH patients (WT1 96). Conclusions: The WT1 copy numbers in PB of AA patients were higher than in AML patients with minimal residual diseases, and the cut-off value that helps diagnose MDS evolving from AA was considered to be around 600 copies. The high WT1 levels in long-standing AA patients without any signs of progression to MDS may represent an increased proliferative potential of hematopoietic stem/progenitor cells that support sustained hematopoiesis with their limited numbers. An increase in the WT1 copy number was associated with somatic mutations in some MDS patients but not in AA patients. Although AA patients with increased WT1 copy numbers need to be carefully followed, a moderate increase in the WT1 copy number does not portend myeloid malignancies unless the number steadily increases. DisclosuresIshiyama:Alexion Pharma GK: Research Funding.

Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML

To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR1) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML). DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR1) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics. 34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR1 rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR1 (P=0.032) and the RFS of patients who got CR1 after one course of induction chemotherapy was significantly longer than that of patients without CR1 [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR1 in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048). CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

Clinical and prognostic values of TP53 mutation in patients with acute myeloid leukemia

目的探讨伴TP53基因异常急性髓系白血病（AML）患者的临床特征及预后。方法回顾性分析2010年1月至2019年1月上海长海医院血液科新诊断的有初发靶向二代测序（NGS）数据的265例AML患者临床资料。采用包含210个血液肿瘤相关基因的靶向测序技术进行突变分析。分析TP53基因突变和（或）缺失与临床特征之间的关系及其对患者生存的影响。结果20例（7.5%）患者伴TP53基因异常，其中错义突变17例（6.4%），移码（缺失）基因突变2例（0.7%），剪切位点突变1例（0.4%）。共检测到23种TP53基因突变，其中4个位于DNA结合结构域第3~4号外显子，16个位于DNA结合结构域第5~8号外显子，2个位于第10号外显子，1个为剪切子突变。伴TP53基因异常患者平均突变基因个数（6.2个）与无TP53基因异常组（6.4个）差异无统计学意义（P=0.770）。TP53基因异常患者的中位年龄为52（26~72）岁，高于无TP53基因异常患者的45（14~75）岁（P=0.008）；复杂核型比例（45.0%，9/20）显著高于无TP53异常组（6.1%，15/245）（P<0.001）；中位总生存（OS）时间[14.1（95%CI 6.78~21.42）个月]较无TP53异常组[31.4（95%CI 13.20~49.59）个月]显著缩短（P=0.029）。20例伴TP53基因异常患者中4例采用“地西他滨+CAG”方案诱导治疗，15例采用“3+7”方案治疗，中位生存时间分别为30.0个月和12.5个月（P=0.018）。多因素分析中，TP53、DNMT3A、USH2A基因异常及初发WBC>12.45×109/L是影响OS的独立预后不良因素。结论伴TP53基因异常AML患者中错义突变常见，突变位点主要分布于DNA结合结构域。TP53基因异常与高龄、复杂核型相关，且常与多个基因突变相伴出现。以地西他滨为基础的诱导化疗及异基因造血干细胞移植可能会提高患者生存率。

High throughput sequencing for detection of 82 kinds of hematologic malignancy related gene mutations in patients with chronic myeloid leukemia resistant to tyrosine kinase inhibitors

High throughput sequencing for detection of 82 kinds of hematologic malignancy related gene mutations in patients with chronic myeloid leukemia resistant to tyrosine kinase inhibitors

Spectrum of Gene Mutations and Clinical Significance in Myeloid Malignancies Patients with ASXL1 Mutations

AbstractIntroduction: With the development of next generation sequencing (NGS), the interrelation between genetic and epigenetic abnormality in myeloid malignancies has attracted significant attention. Clinical reports provide strong evidence that while the specific gene mutations are the initial event for the myeloid malignancies, the concomitant gene mutations contribute to the disease progression. Although ASXL1 mutations have been found in myeloid malignancies, the impact of co-mutation with ASXL1 on the disease progression remains largely unknown. In the current study, we aim to investigate the clinical significance of the association between ASXL1 mutations and a spectrum of gene mutations in a large cohort of patients with myeloid malignancies.Methods: Targeted sequencing including 112 hematopoietic malignancy-related genes was used to analyze the gene mutations in patients with ASXL1 mutations. The impact of gene mutations on clinical characteristics and prognosis was further analyzed. The correlation between clinical/laboratory features and the gene mutations was performed by the χ2 test, and differences in values and in ranks were assessed by Student t-tests. Overall survival rate was assessed by the Kaplan-Meier method and calculated by the Log-rank test.Results: A cohort of 138 myeloid malignant patients harboring ASXL1 mutations was recruited to the current study, including patients with myelodysplastic syndromes (MDS) (37.68%, n = 52), myeloproliferative neoplasms (MPN) (21.01%, n = 29), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (7.25%, n = 10), and acute myeloid leukemia (AML) (34.06%, n = 47). In addition, to ASXL1 mutations, 89 genes were mutated in these patients, and 96.4% (133) of the patients were accompanied by at least one gene mutation. Among those mutated genes, 55.8% (77/138) was epigenetic genes, 65.9% (91/138) was signal transduction pathway genes, 28.2% (39/138) was spliceosome related genes, 36.9% (51/138) was transcription factor genes, and 18.8% (26/138) was cell cycle and apoptosis related genes. The most common co-mutated genes were RAS pathway related genes (25.4%, 35/138) and SETBP1 (21.7%, 30/138). Patients with ASXL1 and RAS pathway co-mutations (ASXL1mutRASmut) had significantly lower levels of hemoglobin and platelets compared to ASXL1 mutated patients without RAS pathway mutation (ASXL1mutRASwt) (hemoglobin 81 (33-152) g/L vs. 96 (18-195) g/L, P=0.012; and platelets (51 (8-695)×109/L vs. 75 (3-3149) × 109/L, P=0.032, respectively). Importantly, MDS patients with ASXL1mutRASmut were more likely to be associated with high International Prognostic Scoring System (IPSS) scores (P=0.016). Moreover, the median survival time of these ASXL1mutRASmut patients (mean = 17 months, 1-35 months) was significantly shorter than that of ASXL1mutRASwt patients (mean = 21 months, 2-75 months) (P=0.031).Conclusions: Our study provides a comprehensive overview of the association between the clinical features and prognosis with genes co-mutated with ASXL1 in patients with myeloid malignancies. We conclude that concomitant mutations of ASXL1 with RAS pathway genes associate with high risk of myeloid transformation and lower overall survival rates. DisclosuresNo relevant conflicts of interest to declare.

High throughput sequencing combined with DNA-PCR for detection of 51 kinds of hematologic malignancy related gene mutations in patients with myelodysplastic syndromes

High throughput sequencing combined with DNA-PCR for detection of 51 kinds of hematologic malignancy related gene mutations in patients with myelodysplastic syndromes

Transcriptome-wide elucidation of liposomal formulations for anticancer drug delivery.

Although widely used in chemotherapy, free doxorubicin (Dox) might enhance cell malignancy undesirably. Liposomal Dox (Doxlipo) has been clinically approved for the treatment of breast cancer due to reduced systematical toxicity and increased tumor targeting, yet the transcriptome-wide elucidation of the Doxlipo formulations remains elusive. To this end, we explored the impact of two Dox liposomal formulations, Doxlipo mainly containing hydrogenated soy phosphatidylcholine or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, on the transcriptional pattern of MCF-7 cells. The two types of Dox liposomal formulations with different drug release kinetics were investigated to reveal the relationship between the formulation and tumor malignancy. Interestingly, we found that liposomal formulation significantly altered the transcriptional pattern of a wide range of genes. Under equivalent dosage of Dox, free Dox substantially changed the expression of ANK1, ACTA2, GPR87, GDF15, FZD6, and WNT4 in MCF-7 cells. Notably, free Dox induced much higher expression of ABCB1 and significantly enhanced the cell migration behavior in comparison with HSPC Doxlipo under a similar level of cytotoxicity. Finally, siRNA targeting GPR87 was codelivered with cationic Doxlipo to reduce the expression of malignancy-related genes. Our study, for the first time, provides an overview of the influence of formulation on the malignancy at transcriptional level and reveals the relationship between cytotoxicity and cell malignancy from the formulation aspect, offering valuable reference for the future formulation design for anticancer drug delivery.

Long-term exposure of MCF-7 breast cancer cells to ethanol stimulates oncogenic features.

Alcohol consumption is a risk factor for breast cancer. Little is known regarding the mechanism, although it is assumed that acetaldehyde or estrogen mediated pathways play a role. We previously showed that long-term exposure to 2.5 mM ethanol (blood alcohol ~0.012%) of MCF-12A, a human normal epithelial breast cell line, induced epithelial mesenchymal transition (EMT) and oncogenic transformation. In this study, we investigated in the human breast cancer cell line MCF-7, whether a similar exposure to ethanol at concentrations ranging up to peak blood levels in heavy drinkers would increase malignant progression. Short-term (1-week) incubation to ethanol at as low as 1–5 mM (corresponding to blood alcohol concentration of ~0.0048–0.024%) upregulated the stem cell related proteins Oct4 and Nanog, but they were reduced after exposure at 25 mM. Long-term (4-week) exposure to 25 mM ethanol upregulated the Oct4 and Nanog proteins, as well as the malignancy marker Ceacam6. DNA microarray analysis in cells exposed for 1 week showed upregulated expression of metallothionein genes, particularly MT1X. Long-term exposure upregulated expression of some malignancy related genes (STEAP4, SERPINA3, SAMD9, GDF15, KRT15, ITGB6, TP63, and PGR, as well as the CEACAM, interferon related, and HLA gene families). Some of these findings were validated by RT-PCR. A similar treatment also modulated numerous microRNAs (miRs) including one regulator of Oct4 as well as miRs involved in oncogenesis and/or malignancy, with only a few estrogen-induced miRs. Long-term 25 mM ethanol also induced a 5.6-fold upregulation of anchorage-independent growth, an indicator of malignant-like features. Exposure to acetaldehyde resulted in little or no effect comparable to that of ethanol. The previously shown alcohol induction of oncogenic transformation of normal breast cells is now complemented by the current results suggesting alcohol's potential involvement in malignant progression of breast cancer.

Ethnic Difference in Genomic Profiles of Chronic Lymphocytic Leukemia in Korea: Targeted Exome Sequencing and Molecular Cytogenetics

Background: Chronic lymphocytic leukemia (CLL) is considered as typical malignancy with ethnic difference, which is one of the common leukemia in western countries, while extremely rare in Asian countries. In addition to incidence rates, mean age of CLL in Asian is younger than that of Caucasian. Recently, many report using next generation sequencing revealed predictive mutations with prognostic implication and treatment response in Caucasian. Yet, genetic profiles of CLL and its prognostic significance have rarely been reported in Asian. To investigate whether genomic profile of CLL in Korea and prognostic impact of adverse mutation shows ethnic difference from Caucasian, we performed target capture sequencing of 87 hematologic malignancy related genes and fluorescent in situ hybridization for prognostic cytogenetic aberrations.Methods: A total of 71 CLL patients were enrolled (median age at diagnosis 61 years, range 23-81 years). Conventional cytogenetic study (n=60) and FISH for chromosome 12 enumeration, 13q14.3 deletions, and 17p13 deletions (n=51) were performed. Target exome sequencing for an 87-gene panel was performed using IlluminaHiSeq 2500 (n=48). Mutations were selected by running algorithms including SIFT, Polyphen2, and CADD, and normal variant was filtered by by 1000Gp, ESP6500, and the in-house Korean database (n=250). Candidate mutations were confirmed by Sanger sequencing. We assessed disease progression, survival and response to treatment.Results: Overall, 37 of 48 patients (77.1%) had at least one mutation; the mean number of mutations per patient was 2.1 (range 0-6). The average coverage of target regions was 200-fold. Targeted exome-sequencing analysis revealed 78 substitutions and insertion/deletions. The most common recurrent mutation (>5% frequency) was ATM (20.8%), followed by TP53 (14.6%), SF3B1 (10.4%), KLHL6 (8.3%), LAMB4 (6.25%), BCOR (6.25%) and NOTCH1 (6.25%). Compared to Caucasian, the ATM, KLHL6 and BCOR mutationsshowed higher frequency in Korean, while NOTCH1, LRP1B, CHD2, POT1 and TGM7 mutations were more frequent in Caucasian. Of note, the ATM mutation exhibited a 2-fold higher incidence (20.8% vs. 9%) in Koreans compared to Caucasians. Frequencies of TP53, SF3B1, MYD88, FAT4, SAMHD, DDX3X, ITPKB, and MED12 mutations were similar between Korean and Caucasian. The LAMB4, SH2B3, RUNX1, SCRIB, KIT, GATA2, CEBPA, TCF12, STAG2, ZRSR2, SETBP1, CSF1R, and SETBP1 mutations have not been reported in Caucasians. Mutations with adverse prognostic implication in Korean CLL was different from those of Caucasian. The TP53, MYD88, SF3B1, ITPKB, SAMHD1, and EGR2 mutations were associated with poor prognosis (P =0.023, P =0.005, P =0.032, P =0.011, P =0.049 and P =0.032, respectively, conventional Cox regression model, P <0.05). Aberrant karyotypes were observed in 28.3% (17/60) of patients by G-banding and in 66.7% (34/51) of patients by FISH. Compared to the reports of western countries, frequency of cytogenetic aberrations by FISH is similar and 13q14 deletion (45.8%, 22/48) was the most frequent chromosomal aberration, follow by trisomy 12 (30.0%, 15/50) and 17p deletion (23.5%, 12/51).Conclusions: The somatic mutation profiles and adverse prognostic genes of Korean CLL differed from that of Caucasian CLL patients, while cytogenetic profiles were similar. Novel mutations discovered in this study must be validated through large cohort studies and may offer clues to the mechanisms underlying the ethnic difference in CLL incidence. In the future, therapeutic strategies targeting these genes should be evaluated and considered. To our knowledge, this is the first comprehensive mutation analysis of CLL patients in Asians using next generation sequencing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.