High bone marrow angiopoietin-1 expression is an independent poor prognostic factor for survival in patients with myelodysplastic syndromes

Background:

Angiogenic factors have an essential role in normal and pathologic angiogenesis. However, the clinical implication of angiogenic factor expression in myelodysplastic syndromes (MDS) remains unclear.

Methods:

In this study, we sought to investigate the prognostic impact of the expression of genes encoding angiopoietin-1 (Ang-1), Ang-2, the receptor Tie2, vascular endothelial growth factor-A (VEGF-A) and VEGF-C in the bone marrow (BM) in 208 patients with newly diagnosed primary MDS.

Results:

BM Ang-1 expression was significantly higher in MDS patients, especially those with higher-risk subtypes, than in normal controls. With a median follow-up time of 32.9 months, the disease transformed to acute leukaemia more frequently in the patients bearing higher Ang-1 expression than in those with lower expression (31.5% vs 18.6%, P=0.023). The MDS patients with higher Ang-1 expression had shorter overall survival than those with lower expression (median 20.8±4.5 months vs 63.3±17.8 months, P<0.001). Multivariate analyses showed that higher Ang-1 expression was an independent unfavourable prognostic factor for overall survival. There was no impact of the expression of other angiogenic factors on survival.

Conclusion:

BM Ang-1 expression may serve as a new biomarker to predict clinical outcome in MDS patients.     

The Frequency of SF3B1 Mutations in Thai Patients with Myelodysplastic Syndrome

Genetic mutations in genes encoding critical component of RNA splicing machinery including SF3B1 are frequentlyidentified and recognized as the pathogenesis in the development of myelodysplatic syndrome (MDS). In this study,PCR sequencings specific for SF3B1 exon 13, 14, 15, and 16 were performed to analyse genomic DNA isolated frombone marrow samples of 72 newly diagnosed MDS patients. We found that 10 of 72 (14%) patients harbor SF3B1missense mutations including E622D (1/72), R625C/G (2/72), H662Q (1/72), K666T (1/72), K700E (4/72) and G740E(1/72), respectively. Mutations were predominantly located on exon 14 and 15 of SF3B1 coding sequence. Interestingly,patients with SF3B1 mutations exhibited higher platelet counts (195×109/L VS. 140×109/L, p-value = 0.025) as well aslower hemoglobin levels (81 g/L VS. 92 g/L, p-value = 0.009) and associated with ring sideroblast phenotype (p-value< 0.001) when compared with patients without the SF3B1 mutation. In summary, we reported the frequency of SF3B1mutations in Thai patients with different subtypes of MDS. SF3B1 mutations were predominantly occurred in MDS-RSand considered as favourable prognosis value. This study further highlighted the clinical important of SF3B1 mutationsanalysis for the classification of MDS.     

SF3B1 mutation in pancreatic cancer contributes to aerobic glycolysis and tumor growth through a PP2A–c‐Myc axis

Hot spot gene mutations in splicing factor 3b subunit 1 (SF3B1) are observed in many types of cancer and create abundant aberrant mRNA splicing, which is profoundly implicated in tumorigenesis. Here, we identified that the SF3B1 K700E (SF3B1^K700E) mutation is strongly associated with tumor growth in pancreatic ductal adenocarcinoma (PDAC). Knockdown of SF3B1 significantly retarded cell proliferation and tumor growth in a cell line (Panc05.04) with the SF3B1^K700E mutation. However, SF3B1 knockdown had no notable effect on cell proliferation in two cell lines (BxPC3 and AsPC1) carrying wild‐type SF3B1. Ectopic expression of SF3B1^K700E but not SF3B1^WT in SF3B1‐knockout Panc05.04 cells largely restored the inhibitory role induced by SF3B1 knockdown. Introduction of the SF3B1^K700E mutation in BxPC3 and AsPC1 cells also boosted cell proliferation. Gene set enrichment analysis demonstrated a close correlation between SF3B1 mutation and aerobic glycolysis. Functional analyses showed that the SF3B1^K700E mutation promoted tumor glycolysis, as evidenced by glucose consumption, lactate release, and extracellular acidification rate. Mechanistically, the SF3B1 mutation promoted the aberrant splicing of PPP2R5A and led to the activation of the glycolytic regulator c‐Myc via post‐translational regulation. Pharmacological activation of PP2A with FTY‐720 markedly compromised the growth advantage induced by the SF3B1^K700E mutation in vitro and in vivo. Taken together, our data suggest a novel function for SF3B1 mutation in the Warburg effect, and this finding may offer a potential therapeutic strategy against PDAC with the SF3B1^K700E mutation.     

Frequencies of SF3B1, NOTCH1, MYD88, BIRC3 and IGHV mutations and TP53 disruptions in Chinese with chronic lymphocytic leukemia: disparities with Europeans

We studied 307 consecutive Chinese with chronic lymphocytic leukemia (CLL) in diverse disease-stages before and after diverse therapies for mutations in several CLL-related genes. Mutation frequencies were SF3B1, 5%, NOTCH1, 8%, MYD88, 8%, BIRC3, 2%, TP53, 15% and IGHV, 60%. Several of these frequencies differ from those reported in persons of predominately European descent with CLL. Biological and clinical associations were detected including SF3B1 and NOTCH1 mutations with un-mutated IGHV, MYD88 mutations with mutated IGHV, SF3B1 mutations with fludarabine-resistant CLL and NOTCH1 mutation with advanced Binet disease stage and with +12. The NOTCH1 correlation with briefer survival was confirmed in multivariate analyses but the SF3B1 correlation was confounded by concurrent mutations in TP53 and germline IGHV. We show differences in incidence and prognostic impact of mutations in Chinese and CLL compared with persons of predominately European descent with CLL. These data may give insights into the etiology and biology of CLL and suggests different risk stratification models may be needed for different CLL populations.     

Mutational analysis of splicing machinery genes SF3B1, U2AF1 and SRSF2 in myelodysplasia and other common tumors

Recurrent somatic mutations in splicing machinery components, including SF3B1, U2AF1 and SRSF2 genes have recently been reported in myelodysplastic syndromes (MDS). Such a recurrent nature strongly suggests that these mutations play important roles in tumor development. To see whether SF3B1, U2AF1 and SRSF2 mutations occur in other human tumors besides MDS, we analyzed the hotspot mutation regions of these genes in 2,345 tumor tissues from various origins (61 MDS, other 616 hematologic tumors, 1,421 epithelial tumors and 247 non‐epithelial stromal tumors) by single‐strand conformation polymorphism analysis. We found SF3B1, U2AF1 and SRSF2 mutations in 5 (8.2%), 12 (19.7%) and 8 (13.1%) of 61 MDS, respectively. We also confirmed these mutations in other myeloid neoplasia, including de novo acute myelogenous leukemia (AML), chronic myelomonocytic leukemia and MDS/myeloproliferative disorder. In addition, we discovered that the SRSF2 gene was mutated in two childhood acute lymphoblastic leukemias (childhood ALL) (1.5%). In solid tumors, we found SF3B1 mutations in gastric and prostate cancers, and U2AF1 mutation in a borderline mucinous tumor of ovary, but the overall incidences of the hotspot mutation regions were very low (0.2%). Our data suggest that SF3B1, U2AF1 and SRSF2 mutations occur not only in myeloid lineage tumors but also in lymphoid lineage tumors. The data suggest that the splicing gene mutations play important roles in the pathogenesis of hematologic tumors, but rarely in solid tumors.     

Alteration of SF3B1 and SRSF2 Genes in Myelodysplastic Syndromes Patients in Upper Northern Thailand

Background: The frequency and pattern of mutation in SF3B1 and SRSF2 RNA splicing machinery genes werefound to vary among myelodysplastic syndrome (MDS) patients in different populations. There have been less reportsof incidence of these gene mutations in Thailand especially in upper northern Thailand. This study therefore had aimsto investigate the frequency and pattern of mutation in mutational hotspot of SF3B1 and SRSF2 genes among MDSpatients in upper northern Thailand and to investigate the clinical features associated with the mutations. Methods:Fifty-five MDS patients who underwent treatment at Maharaj Nakorn Chiang Mai Hospital participated in this study.The detection of SF3B1 and SRSF2 hotspot mutations was carried out using polymerase chain reaction followed bySanger sequencing. In addition, clinical features of individual patients with these gene mutations were also investigated.Results: SF3B1 mutations (SF3B1mut) were found in 9 patients (16.4%) including E622D (1/9), R625C (1/9), H662Q(1/9), K700E (5/9), and Q699H co-mutation with K700E (1/9). SRSF2 mutations (SRSF2mut) were found in 4 patients(7.3%) which included P95H (3/4) and P95L (1/4). The SF3B1mut was associated with lower hemoglobin levels (p = 0.023)and higher platelet counts (p = 0.047) when compared with MDS patients without SF3B1mut, while SRSF2mut tendedto occur in patients with a higher percentage of bone marrow blasts (p = 0.074). Conclusion: The findings confirmedthe difference in frequency of SF3B1 and SRSF2 mutations among different populations. Specifically, we found aco-mutation of Q699H and K700E that has not been previously reported in MDS patients in the COSMIC database.It was also found that SF3B1mut was strongly associated with low hemoglobin level, and high platelet counts whereasSRSF2mut was mostly clustered in MDS with excess blasts subsequently increasing the probability of progression toacute myeloid leukemia.     

Correlation of tumor necrosis factor α (TNFα) with high Caspase 3-like activity in myelodysplastic syndromes

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor α (TNFα) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNFα signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNFα in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8±13.9 at 0 h vs. 177.8±55.2 units/mg protein at 4 h, n=14, P=0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76%±0.25% vs. 3.99%±1.1%, n=14, P=0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNFα levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (ρ=0.517, n=13, P=0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (ρ=0.593, n=13, P=0.033). Additionally when TNFα-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNFα-positive marrows (189.6±66.2 vs. 25.0±14.6 units/mg protein, respectively, P=0.043). The increase in AI%, though not statistically significant, was also higher in the TNFα-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNFα-induced apoptosis were evaluated. TNFα treatment significantly increased AI% (P<0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 μM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 μM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 μM/l) as well as PTX (250 μM/l) significantly curtailed the AI% induced by TNFα The present studies thus identify the downstream effectors of TNFα-inducible apoptosis in MDS and so also the suppressors of TNFα apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.     

Therapeutic Outcomes and Prognostic Impact of Gene Mutations Including TP53 and SF3B1 in Patients with Del(5q) Myelodysplastic Syndromes (MDS)

BackgroundGenetic alterations are increasingly being recognized to play an important role in both diagnosis and prognosis of MDS. In general, MDS patients with SF3B1 mutations (MT) are known to have favorable outcomes whereas those with TP53 mutations have dismal survivals. However, it is unclear if the impact of these mutations applies to all subtypes of MDS including del(5q) which is known for its response to lenalidomide and better prognosis.Materials and MethodsWe retrospectively reviewed 132 del(5q) MDS patients who were treated at the Moffitt Cancer Center (2001-2019).ResultsAmong patients who received lenalidomide (n = 98), 50%, 42.9%, and 7.1% achieved hematologic improvement or better, no response, and disease progression/death with a median overall survival (mOS) of 93.2, 72.4, and 25.6 months, respectively (P < .0001). The mOS was 73.3 months but only 25.6 months after patients stopped lenalidomide. TP53 was the most common mutation accounting for 23.8% of the patients. Of the 63 patients with molecular data available, 23.8% harbored TP53 MT and 10% with SF3B1 MT. TP53 status did not impact OS (MT 86.4 vs. wild-type (WT) 73.3 months; P = .72) but those with SF3B1 mutations had a significantly shorter mOS compared to WT (23.9 vs. 83.5 months; P = .001). Multivariate analysis confirmed lenalidomide response and SF3B1 mutations are independently associated with outcomes.ConclusionOur findings indicate many del(5q) MDS patients will benefit from lenalidomide but survival after its failure is limited. Mutations known to have prognostic impact in MDS at large may not have the same implications in the del(5q) subset.     

E2F1-mediated GINS2 transcriptional activation promotes tumor progression through PI3K/AKT/mTOR pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has high morbidity and mortality rates. It is therefore imperative to study the underlying mechanism of HCC to identify potential prognostic biomarkers and therapeutic targets. Recently, GINS2 has been identified to be a cancer-promoting gene in different cancer types. Nevertheless, the exact mechanism of GINS2 in HCC remains to be elucidated. To systematically explore the significance of GINS2, we first assessed the relative expression of GINS2 in pan-cancers based on data obtained from the HCCDB, TIMER, and TCGA databases. Then, we explored the clinical significance of GINS2 in HCC through Kaplan-Meier method as well as univariate and multivariate cox regression analysis. Additionally, functional enrichment analysis of GINS2 was done through GO, KEGG, PPI network, and immune cell infiltration analyses. Functional experiments were also conducted to investigate the biological significance of GINS2 in HCC cell lines. Our research revealed that GINS2 is involved in HCC progression and highlighted its potential value as a crucial diagnostic and therapeutic target for HCC.     

B7-H1 and B7-H3 are independent predictors of poor prognosis in patients with non-small cell lung cancer

B7-H1 and B7-H3, two members of the B7 family that are thought to regulate T-cell activation, are expressed in human non-small cell lung cancer (NSCLC). However, their prognostic significance is poorly understood. In the present study we reported that B7-H1 and B7-H3 were expressed in 96/128 (72.7%) and 89/128 (69.5%) samples, respectively. B7-H1 and B7-H3 expression and the number of infiltrating T-cell intracellular antigen-1+ and interferon-γ+ cells in NSCLC tissues were significantly higher than those in the adjacent tissues (p<0.01). High B7-H1 or B7-H3 expression was associated with lymph node metastasis and TNM stage (p<0.05, respectively). Sex, TNM stage, B7-H1, B7-H3, and T-cell intracellular antigen-1 expression remained significant prognostic factors after adjusting for other prognostic factors in a multivariate Cox proportional hazards regression model. In vitro studies revealed that knockdown of B7-H3 on tumor cells enhanced T-cell growth and interferon-γ secretion when stimulated by anti-CD3 and anti-CD28 monoclonal antibodies. Interferon-γ reduced CXCR4 expression on cancer cells and inhibited the CXCL12-induced cell migration. B7-H1 and B7-H3 are independent predictors of poorer survival in patients with NSCLC. Interference of the signal pathways of these negative regulatory molecules might be a new strategy for treating NSCLC.     

AEG -1和 PI3K 在食管鳞癌组织中的表达及与临床病理学特征和预后的关系

目的：观察 AEG -1和 PI3K 在食管鳞癌组织及癌旁组织中的表达差异，了解其与食管鳞癌患者临床病理学特征及相互之间的关系。方法：免疫组化法检测80例食管鳞癌患者手术标本和62例癌旁食管组织中 AEG -1和 PI3K 的表达，分析其与临床病理学特征及生存率的关系。结果：AEG -1和 PI3K 在食管鳞癌组织中的表达显著高于癌旁食管组织；与癌组织浸润深度、TNM 分期、患者的预后有显著相关性。结论：AEG -1可能是食管鳞癌的癌基因之一，有可能是通过活化食管鳞癌组织中的 PI3K/ Akt 通路从而增强癌细胞的增殖、迁移、侵袭能力。     

朗格汉斯细胞组织细胞增生症中 BRAF V600E 和 MAP2K1基因突变的分析及其临床意义

目的：探讨我国朗格汉斯细胞组织细胞增生症（Langerhans cell histiocytosis,LCH）中 BRAF V600E 和MAP2K1基因突变发生状况及其临床意义。方法：随机选取35例 LCH 组织标本,采用桑格测序法检测其中BRAF V600E 和 MAP2K1基因突变状况,免疫组化法检测 BRAF V600E 蛋白的表达。分析 BRAF V600E、MAP2K1基因突变与 LCH 临床基本资料（年龄、性别、单／多系统）的关系。结果：在35例 LCH 患者中,男女比例为1．7∶1,82．9％侵及骨组织,97．1％是单系统 LCH（single system LCH,SS －LCH）,2．9％是多系统 LCH （multi －system LCH,MS －LCH）。桑格测序法检测 BRAF V600E 基因突变率为17．1％,MAP2K1基因突变率为14．3％,MAP2K1与 BRAF V600E 基因突变有互异性;免疫组化法检测 BRAF V600E 阳性表达率为28．6％,涵盖了桑格测序法测得的突变病例。BRAF V600E 和 MAP2 K1基因突变更多出现在未成年组（35．7％和28．6％）,其中 BRAF V600E 突变在未成年人组与成人组间有显著性差异（P ＝0．028）;BRAF V600E 和MAP2K1基因突变对生存的影响无统计学差异（P ＞0．05）。结论：我国 LCH 患者大部分都是 SS －LCH,主要侵及的部位是骨组织,且预后良好,5年生存率为97．1％。桑格法所测的 BRAF V600E 和 MAP2K1基因突变率均低于西方报道,两者存在互异性,分别为17．1％和14．3％。所有 MAP2K1基因突变都是点突变,没有框内缺失突变,发现一个新的突变位点：c．112 G ＞A p．E38K;BRAF V600E 和 MAP2K1基因突变主要发生于未成年组中,提示各年龄层中 LCH 的发病机理可能不同,可能 RAS ／RAF ／MEK／ERK 通路在未成年人 LCH 中发挥更重要的作用;另外这两种突变对 LCH 的生存无影响。     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

Targeting the PI3K/AKT/mTOR pathway in biliary tract cancers: A review of current evidences and future perspectives

Biliary tract cancers (BTCs) are a group of invasive neoplasms, with increasing incidence and dismal prognosis. In advanced disease, the standard of care is represented by first-line chemotherapy with cisplatin and gemcitabine. In subsequent lines, no clear recommendations are currently available, highlighting the need for novel therapeutic approaches.The PI3K/AKT/mTOR pathway is a core regulator of cell metabolism, growth and survival, and is involved in BTCs carcinogenesis and progression. Mutations, gene copy number alterations and aberrant protein phosphorylation of PI3K, AKT, mTOR and PTEN have been thoroughly described in BTCs and correlate with poor survival outcomes.Several pre-clinical evidences state the efficacy of PI3K/AKT/mTOR pathway inhibitors in BTCs, both in vitro and in vivo. In the clinical setting, initial studies with rapamycin analogs have shown interesting activity with an acceptable toxicity profile. Novel strategies evaluating AKT and PI3K inhibitors have risen serious safety concerns, pointing out the need for improved patient selection and increased target specificity for the clinical development of these agents, both alone and in combination with chemotherapy.This review extensively describes the role of the PI3K/AKT/mTOR pathway in BTCs and examines the rationale of its targeting in these tumors, with particular focus on clinical activity, toxicities and perspectives on further development of PI3K/AKT/mTOR pathway inhibitors.