De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.

BACKGROUND: Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. RESULTS: We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. CONCLUSIONS: We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

SMARCA5 reprograms AKR1B1-mediated fructose metabolism to control leukemogenesis.

A significant variation in chromatin accessibility is an epigenetic feature of leukemia. The cause of this variation in leukemia, however, remains elusive. Here, we identify SMARCA5, a core ATPase of the imitation switch (ISWI) chromatin remodeling complex, as being responsible for aberrant chromatin accessibility in leukemia cells. We find that SMARCA5 is required to maintain aberrant chromatin accessibility for leukemogenesis and then promotes transcriptional activation of AKR1B1, an aldo/keto reductase, by recruiting transcription co-activator DDX5 and transcription factor SP1. Higher levels of AKR1B1 are associated with a poor prognosis in leukemia patients and promote leukemogenesis by reprogramming fructose metabolism. Moreover, pharmacological inhibition of AKR1B1 has been shown to have significant therapeutic effects in leukemia mice and leukemia patient cells. Thus, our findings link the aberrant chromatin state mediated by SMARCA5 to AKR1B1-mediated endogenous fructose metabolism reprogramming and shed light on the essential role of AKR1B1 in leukemogenesis, which may provide therapeutic strategies for leukemia.

Threonine dehydrogenase regulates neutrophil homeostasis but not H3K4me3 levels in zebrafish.

l-threonine dehydrogenase (Tdh) is an enzyme that links threonine metabolism to epigenetic modifications and mitochondria biogenesis. In vitro studies show that it is critical for the regulation of trimethylation of histone H3 lysine 4 (H3K4me3) levels and cell fate determination of mouse embryonic stem cells (mESCs). However, whether Tdh regulates a developmental process in vivo and, if it does, whether it also primarily regulates H3K4me3 levels in this process as it does in mESCs, remains elusive. Here, we revealed that, in zebrafish hematopoiesis, tdh is preferentially expressed in neutrophils. Knockout of tdh causes a decrease in neutrophil number and slightly suppresses their acute injury-induced migration, but, unlike the mESCs, the level of H3K4me3 is not evidently reduced in neutrophils sorted from the kidney marrow of adult tdh-null zebrafish. These phenotypes are dependent on the enzymatic activity of Tdh. Importantly, a soluble supplement of nutrients that are able to fuel the acetyl-CoA pool, such as pyruvate, glucose and branched-chain amino acids, is sufficient to rescue the reduction in neutrophils caused by tdh deletion. In summary, our study presents evidence for the functional requirement of Tdh-mediated threonine metabolism in a developmental process in vivo. It also provides an animal model for investigating the nutritional regulation of myelopoiesis and immune response, as well as a useful tool for high-throughput drug/nutrition screening.

ROBO1 deficiency impairs HSPC homeostasis and erythropoiesis via CDC42 and predicts poor prognosis in MDS.

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic neoplasms originating from hematopoietic stem progenitor cells (HSPCs). We previously identified frequent roundabout guidance receptor 1 (ROBO1) mutations in patients with MDS, while the exact role of ROBO1 in hematopoiesis remains poorly delineated. Here, we report that ROBO1 deficiency confers MDS-like disease with anemia and multilineage dysplasia in mice and predicts poor prognosis in patients with MDS. More specifically, Robo1 deficiency impairs HSPC homeostasis and disrupts HSPC pool, especially the reduction of megakaryocyte erythroid progenitors, which causes a blockage in the early stages of erythropoiesis in mice. Mechanistically, transcriptional profiling indicates that Cdc42, a member of the Rho-guanosine triphosphatase family, acts as a downstream target gene for Robo1 in HSPCs. Overexpression of Cdc42 partially restores the self-renewal and erythropoiesis of HSPCs in Robo1-deficient mice. Collectively, our result implicates the essential role of ROBO1 in maintaining HSPC homeostasis and erythropoiesis via CDC42.

The non-cell-autonomous function of ID1 promotes AML progression via ANGPTL7 from the microenvironment.

The bone marrow microenvironment (BMM) can regulate leukemia stem cells (LSCs) via secreted factors. Increasing evidence suggests that dissecting the mechanisms by which the BMM maintains LSCs may lead to the development of effective therapies for the eradication of leukemia. Inhibitor of DNA binding 1 (ID1), a key transcriptional regulator in LSCs, previously identified by us, controls cytokine production in the BMM, but the role of ID1 in acute myeloid leukemia (AML) BMM remains obscure. Here, we report that ID1 is highly expressed in the BMM of patients with AML, especially in BM mesenchymal stem cells, and that the high expression of ID1 in the AML BMM is induced by BMP6, secreted from AML cells. Knocking out ID1 in mesenchymal cells significantly suppresses the proliferation of cocultured AML cells. Loss of Id1 in the BMM results in impaired AML progression in AML mouse models. Mechanistically, we found that Id1 deficiency significantly reduces SP1 protein levels in mesenchymal cells cocultured with AML cells. Using ID1-interactome analysis, we found that ID1 interacts with RNF4, an E3 ubiquitin ligase, and causes a decrease in SP1 ubiquitination. Disrupting the ID1-RNF4 interaction via truncation in mesenchymal cells significantly reduces SP1 protein levels and delays AML cell proliferation. We identify that the target of Sp1, Angptl7, is the primary differentially expression protein factor in Id1-deficient BM supernatant fluid to regulate AML progression in mice. Our study highlights the critical role of ID1 in the AML BMM and aids the development of therapeutic strategies for AML.

Identification of patients with locally advanced rectal cancer eligible for neoadjuvant chemotherapy alone: Results of a retrospective study.

BACKGROUND AND OBJECTIVES: Neoadjuvant chemotherapy (nCT) appears in a few clinical studies as an alternative to neoadjuvant chemoradiation (nCRT) in selected patients with locally advanced rectal cancer (LARC). We aimed to compare the clinical outcomes of nCT with or without nCRT in patients with LARC and to identify patients who may be suitable for nCT alone. MATERIALS AND METHODS: A total of 155 patients with LARC who received neoadjuvant treatment (NT) were retrospectively analysed from January 2016 to June 2021. The patients were divided into two groups: nCRT (n = 101) and nCT (n = 54). More patients with locally advanced disease (cT4, cN+ and magnetic resonance imaging-detected mesorectal fascia [mrMRF] positive [+]) were found in the nCRT group. Patients in the nCRT group received a dose of 50 Gy/25 Fx irradiation with concurrent capecitabine, and the median number of nCT cycles was two. In the nCT group, the median number of cycles was four. RESULTS: The median follow-up duration was 30 months. The pathologic complete response (pCR) rate in the nCRT group was significantly higher than that in the nCT group (17.5% vs. 5.6%, p = 0.047). A significant difference was observed in the locoregional recurrence rate (LRR); 6.9% in the nCRT group and 16.7% in the nCT group (p = 0.011). Among patients with initial mrMRF (+) status, the LRR in the nCRT group was significantly lower than that in the nCT group (6.1% vs. 20%, p = 0.007), but not in patients with initial mrMRF negative (-) (10.5% in each group, p = 0.647). Compared with the nCT group, a lower LRR was observed in patients in the nCRT group with initial mrMRF (+) converted to mrMRF (-) after NT (5.3% vs. 23%, p = 0.009). No significant difference was observed between the two groups regarding acute toxicity and overall and progression-free survivals. Multivariate analysis showed that nCRT and ypN stage were independent prognostic factors for the development of LRR. CONCLUSION: Patients with initial mrMRF (-) may be suitable for nCT alone. However, patients with initial mrMRF (+) converted to mrMRF (-) after nCT are still at high risk of LRR, and radiotherapy is recommended. Prospective studies are required to confirm these findings.

Inhibition of USP1 reverses the chemotherapy resistance through destabilization of MAX in the relapsed/refractory B-cell lymphoma.

The patients with relapsed and refractory diffuse large B-cell lymphoma (DLBCL) have poor prognosis, and a novel and effective therapeutic strategy for these patients is urgently needed. Although ubiquitin-specific protease 1 (USP1) plays a key role in cancer, the carcinogenic effect of USP1 in B-cell lymphoma remains elusive. Here we found that USP1 is highly expressed in DLBCL patients, and high expression of USP1 predicts poor prognosis. Knocking down USP1 or a specific inhibitor of USP1, pimozide, induced cell growth inhibition, cell cycle arrest and autophagy in DLBCL cells. Targeting USP1 by shRNA or pimozide significantly reduced tumor burden of a mouse model established with engraftment of rituximab/chemotherapy resistant DLBCL cells. Pimozide significantly retarded the growth of lymphoma in a DLBCL patient-derived xenograft (PDX) model. USP1 directly interacted with MAX, a MYC binding protein, and maintained the stability of MAX through deubiquitination, which promoted the transcription of MYC target genes. Moreover, pimozide showed a synergetic effect with etoposide, a chemotherapy drug, in cell and mouse models of rituximab/chemotherapy resistant DLBCL. Our study highlights the critical role of USP1 in the rituximab/chemotherapy resistance of DLBCL through deubiquitylating MAX, and provides a novel therapeutic strategy for rituximab/chemotherapy resistant DLBCL.

Evaluation of systemic inflammatory and nutritional indexes in locally advanced gastric cancer treated with adjuvant chemoradiotherapy after D2 dissection.

Background: Many studies have shown that the peripheral blood inflammatory index and nutritional index, such as the platelet lymphocyte ratio (PLR), neutrophil lymphocyte ratio (NLR), lymphocyte monocyte ratio (LMR), systemic inflammation response index (SIRI), pan-immune-inflammation value (PIV), systemic immune-inflammation index (SII), and prognostic nutrition index (PNI), are independent prognostic factors for tumors. The present study aimed to investigate the prognostic role of these peripheral blood indexes before treatment in locally advanced gastric cancer (LAGC) treated with adjuvant chemoradiotherapy after D2 dissection. Methods: A total of 89 patients with LAGC who underwent D2 gastrectomy and adjuvant chemoradiotherapy at our hospital from 2010-2018 were eligible. Systemic inflammatory indicators before treatment were evaluated. Receiver operating characteristic curve (ROC), Kaplan-Meier analysis, and Cox regression were utilized for prognosis evaluation. Results: The median follow-up time was 29.1 (4.1-115.8) months. The overall survival at 3 years (OS) and the disease-free survival (DFS) were 78.9% and 59.1%, respectively. According to the ROC curve for 3-year DFS, the best cut-off values of pre-treatment NLR, PLR, LMR, SII, SIRI, PIV and PNI were 1.7, 109.3, 2.9, 369.2, 0.58, 218.7, and 48, respectively. Multivariate Cox regression analysis showed that NLR was an independent prognostic factor for DFS (HR 2.991, 95%CI 1.085-8.248, P = 0.034). Kaplan-Meier analysis showed that a higher NLR (>1.70) was significantly associated with a poorer OS (3-year OS: 68.8% vs 92.9%, P = 0.045) and DFS (3-year DFS: 47.5% vs 80.9%, P = 0.005). In terms of the free locoregional recurrence rate (LRR), the prognosis of patients with high NLR was also significantly worse than those with low NLR (70.2% vs 96.0%, P = 0.017). Paraaortic lymph nodes were the most common site of LRR (7/14 patients). The seven cases of paraaortic lymph node metastasis occurred in patients with high NLR. Conclusions: In our retrospective analysis, we found that pretreatment NLR could serve as a prognostic factor for survival in LAGC treated with adjuvant chemoradiotherapy after D2 dissection, especially for the prediction of LRR and paraaortic lymph node metastasis. Prospective studies are needed to confirm our findings.

Targeting UHRF1-SAP30-MXD4 axis for leukemia initiating cell eradication in myeloid leukemia.

Aberrant self-renewal of leukemia initiation cells (LICs) drives aggressive acute myeloid leukemia (AML). Here, we report that UHRF1, an epigenetic regulator that recruits DNMT1 to methylate DNA, is highly expressed in AML and predicts poor prognosis. UHRF1 is required for myeloid leukemogenesis by maintaining self-renewal of LICs. Mechanistically, UHRF1 directly interacts with Sin3A-associated protein 30 (SAP30) through two critical amino acids, G572 and F573 in its SRA domain, to repress gene expression. Depletion of UHRF1 or SAP30 derepresses an important target gene, MXD4, which encodes a MYC antagonist, and leads to suppression of leukemogenesis. Further knockdown of MXD4 can rescue the leukemogenesis by activating the MYC pathway. Lastly, we identified a UHRF1 inhibitor, UF146, and demonstrated its significant therapeutic efficacy in the myeloid leukemia PDX model. Taken together, our study reveals the mechanisms for altered epigenetic programs in AML and provides a promising targeted therapeutic strategy against AML.

Influence of Adjuvant Radiotherapy Timing on Survival Outcomes in High-Risk Patients Receiving Neoadjuvant Treatments.

Purpose: To determine the relationship between time to radiotherapy (TTR) and survival outcomes in breast cancer (BC) patients treated with neoadjuvant treatments (NATs). Methods: Continuous non-metastatic BC patients receiving NAT and adjuvant radiotherapy (RT) from 2009 to 2016 were retrospectively reviewed. A multivariable Cox model with restricted cubic splines (RCSs) was used to determine the panoramic relationship between TTR and survival outcomes. Multivariable analysis was used to control for confounding factors between the groups of TTR. Results: A total of 315 patients were included. The RCS modeling demonstrated a non-linear relationship between TTR and survival outcomes. The lowest risk for distant metastasis-free survival (DMFS) and recurrence-free survival (RFS) was observed at the TTR of 12 weeks, and the lowest risk of BC-specific survival (BCSS) at 10 weeks. TTR was accordingly transformed into categorical variables as ≤10, 11-20, and >20 weeks. Multivariable analysis revealed that the TTR of ≤10 weeks was an independent prognostic factor for worse DMFS (HR = 2.294, 95% CI 1.079-4.881) and RFS (HR = 2.126, 95% CI 1.038-4.356) compared with the TTR of 10-20 weeks, while the is no difference in DMFS, RFS, and BCSS between TTR >20 weeks and TTR of 10-20 weeks. Conclusion: There exists a non-linear relationship between TTR after surgery and survival outcomes in patients treated with NAT. Early initiation of RT following surgery does not seem to be associated with a better therapeutic outcome. A relatively flexible recommendation of TTR could be adopted in clinical practice.

[Preparation and effect against cervical cancer invasion in vitro of harmine-loaded photosensitive liposomes].

Despite the development of HPV vaccines and screening programs, cervical cancer is still a serious threat to women's health. Early-stage cervical cancer is mainly treated by surgery. However, considering the serious complications after surgery, hyperthermia is recommended to enhance the effect of chemotherapy, retain the integrity of cervix, improve the treatment effect, which provides a therapeutic basis for the early treatment of cervical cancer. The photosensitive liposomes containing harmine and dye IR-780 were prepared by thin-film dispersion method and separated by Sephadex G-50 dextran gel column. The preparation conditions were optimized as the mass ratio of phospholipid to cholesterol membrane material being 8∶1 and that of drug to lipid being 1∶20. The results of HPLC showed that the encapsulation efficiency of harmine was 55.6%±0.18%. The prepared photosensitive liposomes were round and evenly distributed under transmission electron microscope, with the particle size of(125.2±0.62) nm determined by Marvin particle size analyzer and the Zeta potential of(-2.55±0.76) mV. Additionally, the photosensitive liposomes had the photothermal conversion efficiency, an important property of photothermal agent, of 27.1%±0.86%. The photosensitive liposomes stored at 4 ℃ showed stable encapsulation efficiency in the first 14 days without flocculation. The sulforhodamine B(SRB) assay was employed to determine the inhibitory effect of the liposomes on the proliferation of HeLa cells under near-infrared(NIR) irradiation or not, which showcased stronger inhibitory effect under NIR irradiation. The results of Transwell assay indicated that the prepared liposomes significantly inhibited the invasion and migration of HeLa cells in vitro. The findings of this study provide a basis for the treatment of cervical cancer with harmine.

Characteristics and Treatment Strategy of Isolated Calf Deep Venous Thrombosis after Fractures: A Review of Recent Literature.

Isolated calf deep venous thrombosis (ICDVT) includes thrombosis located at the far end of the popliteal vein, such as the anterior tibial vein, posterior tibial vein, fibular vein, and intramuscular vein of the soleus and gastrocnemius. This type of thrombosis has the highest incidence, accounting for approximately half of all deep vein thrombosis (DVT) cases; however, there is no consistent recommendation for ICDVT treatment across countries, and there is also no optimal management strategy. In recent years, increasing evidence has shown that ICDVT can develop into proximal DVT, even causing pulmonary embolism (PE). Therefore, some experts suggest anticoagulant therapy for this type of DVT, while others hold an opposing attitude. Therefore, the treatment strategy for this type of DVT has become a hot and difficult research topic. The purpose of this review is to summarize the characteristics of ICDVT and the effects of different treatment strategies by analyzing recent and important classical works in the literature in an attempt to provide recommendations for the treatment of this most common type of DVT in orthopaedic clinics.

Practical Model to Optimize the Strategy of Adjuvant Postmastectomy Radiotherapy in T1-2N1 Breast Cancer With Modern Systemic Therapy.

Purpose: The effect of adjuvant irradiation after mastectomy in early-stage breast cancer patients remains controversial. The present study aims to explore the clinical benefit obtained from adjuvant radiotherapy among post-mastectomy pT1-2N1 breast cancer patients who received adjuvant modern systemic therapy. Methods: Medical records of consecutive patients with pT1-2N1 breast cancer who received mastectomy in our institution between January 2009 and December 2016 were retrospectively reviewed. High-risk features consist of patient age, number of positive lymph nodes, T stage, and Ki67 index, which were developed previously at our institution using early-stage breast cancer patients after mastectomy without adjuvant radiotherapy. Differences of survival and local recurrence were compared between no-postmastectomy radiotherapy (PMRT) and PMRT group according to number of risk factors. The time-to-event curves were calculated by the Kaplan-Meier methods and compared by the log-rank test. Propensity score matching (PSM) was performed to reduce the imbalances in patient characteristics. Results: A total of 548 patients were enrolled (no-PMRT: 259 and PMRT: 289). After a median follow-up of 69 months, the 5-year rate of DFS, BCSS, and LRR in the overall cohort was 90.2%, 97.4%, and 3.6%, respectively. PMRT did not significantly improve DFS, BCSS, and LRRFS in the whole cohort. Patients were divided into low-risk (with no or one risk factor) and high-risk (with two or more risk factors) groups. According to the univariable and multivariable analysis, high-risk group (HR = 1.81, 95% CI 1.11-2.98, p = 0.02) was demonstrated as an independent risk factor for DFS. For the high-risk group, PMRT significantly improved DFS from 81.4% to 91.9% and BCSS from 95.5% to 98.6% and decreased the 5-year rate of LRR from 5.6% to 1.4%, respectively (p < 0.01, p = 0.05, and p = 0.06). However, no survival benefit from PMRT was observed in the low-risk group in terms of DFS, BCSS, and LRR (p = 0.45, p = 0.51, and p = 0.99, respectively). In multivariate analysis, PMRT remained an independent prognostic factor for DFS (HR = 0.50, 95% CI 0.24-1.00, p = 0.05) in the high-risk group. After PSM analysis, the survival benefit of PMRT was sustained in high-risk patients. Conclusion: PMRT significantly improved DFS in high-risk pT1-2N1 breast cancer patients, but not in low-risk patients. Independent validation of our scoring system is recommended.

Associated Mirror Therapy Enhances Motor Recovery of the Upper Extremity and Daily Function after Stroke: A Randomized Control Study.

Bimanual cooperation plays a vital role in functions of the upper extremity and daily activities. Based on the principle of bilateral movement, mirror therapy could provide bimanual cooperation training. However, conventional mirror therapy could not achieve the isolation of the mirror. A novel paradigm mirror therapy called associated mirror therapy (AMT) was proposed to achieve bimanual cooperation task-based mirror visual feedback isolating from the mirror. The study was aimed at exploring the feasibility and effectiveness of AMT on stroke patients. We conducted a single-blind, randomized controlled trial. Thirty-six eligible patients were equally assigned into the experimental group (EG) receiving AMT and the control group (CG) receiving bimanual training without mirroring for five days/week, lasting four weeks. The Fugl-Meyer Assessment Upper Limb subscale (FMA-UL) for upper extremity motor impairment was used as the primary outcome. The secondary outcomes were the Box and Block Test (BBT) and Functional Independence Measure (FIM) for motor and daily function. All patients participated in trials throughout without adverse events or side effects. The scores of FMA-UL and FIM improved significantly in both groups following the intervention. Compared to CG, the scores of FMA-UL and FIM were improved more significantly in EG after the intervention. The BBT scores were improved significantly for EG following the intervention, but no differences were found in the BBT scores of CG after the intervention. However, no differences in BBT scores were observed between the two groups. In summary, our study suggested that AMT was a feasible and practical approach to enhance the motor recovery of paretic arms and daily function in stroke patients. Furthermore, AMT may improve manual dexterity for poststroke rehabilitation.

Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65.

OBJECTIVE: To investigate the role of Zinc finger protein A20 in osteoarthritis (OA) by regulating NF-κB p65. METHODS: A20, MMP1, MMP13 and IL-1ß expressions in human OA cartilage samples were detected by qRT-PCR. IL-1ß-induced chondrocyte was treated with A20 lentivirus activation particle, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) with/without A20 siRNA. IL-6, TNF-α, and PGE2 levels were measured by ELISA, and NO production by Greiss reaction. Destabilization of the medial meniscus (DMM) surgery was used to construct the OA models, followed by injection of A20 adenovirus. MMP1 and MMP13 expression was measured by immunohistochemistry. The mRNA and protein expression were performed by qRT-PCR and western blotting, respectively. RESULTS: A20 was down-regulated in human OA cartilage samples, and negatively correlated with the expressions of MMP1, MMP13 and IL-1ß. The IL-1ß-induced chondrocyte manifested decreased A20 with increased NF-κB p65 activity. A20 overexpression suppressed the NF-κB p65 activity in IL-1ß-induced chondrocyte. Furthermore, PDTC decreased IL-1ß-induced chondrocyte apoptosis with the upregulated COL1A1, COL2A1, COL10A1 and ACAN, as well as the down-regulated MMP1, MMP13, COX2, iNOS, IL-6, TNF-α, NO and PGE2, which was reversed by A20 siRNA. In vivo, OA mice gained higher OARSI score and Mankin's score, exhibited up-regulations of MMP1 and MMP13, and decreased NF-κB p65 activity, which was improved after injection of A20 adenovirus. CONCLUSION: A20 was reduced in OA cartilage samples, and its overexpression, by suppressing the activity of NF-κB p65, could improve IL-1ß-induced chondrocyte degradation and apoptosis in vitro, as well as mitigate the inflammation in OA mice.

Prognostic and predictive significance of tumor infiltrating lymphocytes for ductal carcinoma in situ.

This study aims to identify the density of TILs in ductal carcinoma in situ (DCIS) in terms of prognostic significance with recurrence and the benefit of whole breast irradiation (WBI). The clinicopathological data of DCIS patients from Jan 2009 to Dec 2016 who received breast-conserving surgery (BCS) were retrospectively reviewed. Cox regression analysis was used to confirm independent prognostic factors of ipsilateral breast tumor recurrence (IBTR). Kaplan-Meier method was utilized to analyze IBTR and values of WBI. Touching-tumor-infiltrating lymphocytes (TILs) were defined by TILs touching or within one lymphocyte cell thickness from the malignant ducts' basement membrane. In total, 129 patients were enrolled in this analysis with 98 patients who received WBI. After a median follow-up of 53.0 months, there were 16 IBTR events with five invasive IBTRs. Univariate and multivariate analyses showed that touching-TILs >5 were an independent prognostic factor for higher IBTR (HR = 6.17, 95%CI 1.95-19.56, p < .01). The whole cohort was classified into two subgroups: dense group (>5 touching-TILs per duct) and sparse group (≤5 touching-TILs per duct). Dense touching-TILs were associated with unfavorable biologic characteristics. The 5-y rate of IBTR between dense and sparse group was 29.0% versus 4.5% (p < .01). For the sparse group, WBI significantly reduced the rate of 5-y-IBTR risk from 13.2% to 1.7% (p = .02), but there was no benefit of WBI in the dense group. Touching-TILs density was heterogeneous in patients with DCIS. Sparse touching-TILs were associated with better prognosis and benefit from WBI. Dense touching-TILs not only were associated with a higher risk of IBTR but also lack of benefit from WBI.

Identification of Patients With Locally Advanced Gastric Cancer Who May Benefit From Adjuvant Chemoradiotherapy After D2 dissection: A Propensity Score Matching Analysis.

BACKGROUND: One of the most controversial areas in gastrointestinal oncology is the benefit of postoperative chemoradiotherapy (CRT) over chemotherapy (CT) alone after D2 dissection of locally advanced gastric cancer (LAGC). We aimed to identify the LAGC patients who may benefit from adjuvant CRT. METHODS: We analyzed retrospectively 188 patients receiving radical gastrectomy with D2 dissection for LAGC in our hospital. Patients were divided into two balanced groups by using propensity score matching: CRT group (n = 94) received adjuvant CRT, and CT group received adjuvant CT alone. RESULTS: At a median follow-up of 27.10 months, 188 patients developed 79 first recurrence events (36 in CRT group and 43 in CT group). Our results showed that adjuvant CRT significantly decreased the risk of developing local regional recurrence (LRR) when compared to CT alone (14.9% vs. 25.5%, p = 0.044), while the estimated 3-year disease-free survival (DFS) was comparable between the CRT and CT groups (59.3% vs. 50.9%, p = 0.239). In the subgroup analysis, a significantly decreased LRR rate was also observed in LAGC patients with N1-3a stage after adjuvant CRT (p = 0.046), but not for N3b. Para-aortic lymph nodes (station No. 16) were the most frequent sites of LRR. After receiving radiotherapy, recurrence of 16 a2 region and 16 b1 region were significantly deceased (p = 0.026 and p = 0.044, respectively). Patients who received irradiation more than 4 months after surgery showed an increased risk of LRR (p = 0.022). CONCLUSIONS: This study showed that adjuvant CRT significantly reduced LRR after D2 dissection of LAGC. Early initiation of adjuvant RT with clinical target volume encompassing a2 and b1 regions of para-aortic lymph nodes is recommended for pN1-3a patients after D2 dissection.

How Does the Interval Between Completion of Adjuvant Chemotherapy and Initiation of Radiotherapy Impact Clinical Outcomes in Operable Breast Cancer Patients?

PURPOSE: The aim of this study was to evaluate the impact of time to radiotherapy (TTR) after completion of chemotherapy (CT), and TTR after surgery, in breast cancer (BC) patients. PATIENTS AND METHODS: Continuous breast cancer patients treated with surgery and CT followed by radiotherapy (RT) from 2009 through 2015 were retrospectively reviewed. Patients were categorized into four groups with respect to TTR after CT, i.e. <4, 4-8, 8-12, and >12 weeks, and TTR after surgery, i.e. <147, 147-180, 180-202, and >202 days. The Cox proportional hazards model was used to identify the independent effect of TTRs. RESULTS: Overall, 989 patients were enrolled. Patients with a TTR of >12 weeks after CT showed significantly worse breast cancer-specific survival (BCSS) and overall survival (OS) compared with those who had a TTR of <4 weeks (BCSS: hazard ratio [HR] 0.28, 95% confidence interval [CI] 0.1-0.76; OS: HR 0.33, 95% CI 0.13-0.88), 4-8 weeks (BCSS: HR 0.23, 95% CI 0.08-0.66; OS: HR 0.29, 95% CI 0.11-0.8), and 8-12 weeks (BCSS: HR 0.22, 95% CI 0.05-0.96; OS: HR 0.23, 95% CI 0.06-0.99). TTR after surgery showed no significant association with survival outcomes in the entire cohort, except in patients with hormone receptor (HR)-positive disease and those receiving mastectomy. In HR-positive tumors, a TTR after CT of >12 weeks remained an independent predictor for adverse BCSS and OS. CONCLUSION: Initiation of RT beyond 12 weeks after CT might compromise survival outcomes. Efforts should be made to avoid delaying RT, especially after completion of CT and in patients with HR-positive tumors, positive lymph nodes, and those receiving mastectomy.

setd2 knockout zebrafish is viable and fertile: differential and developmental stress-related requirements for Setd2 and histone H3K36 trimethylation in different vertebrate animals.

Setd2 is the only enzyme that catalyzes histone H3 lysine 36 trimethylation (H3K36me3) on virtually all actively transcribed protein-coding genes, and this mechanism is evolutionarily conserved from yeast to human. Despite this widespread and conserved activity, Setd2 and H3K36me3 are dispensable for normal growth of yeast but are absolutely required for mammalian embryogenesis, such as oocyte maturation and embryonic vasculogenesis in mice, raising a question of how the functional requirements of Setd2 in specific developmental stages have emerged through evolution. Here, we explored this issue by studying the essentiality and function of Setd2 in zebrafish. Surprisingly, the setd2-null zebrafish are viable and fertile. They show Mendelian birth ratio and normal embryogenesis without vascular defect as seen in mice; however, they have a small body size phenotype attributed to insufficient energy metabolism and protein synthesis, which is reversable in a nutrition-dependent manner. Unlike the sterile Setd2-null mice, the setd2-null zebrafish can produce functional sperms and oocytes. Nonetheless, related to the requirement of maternal Setd2 for oocyte maturation in mice, the second generation of setd2-null zebrafish that carry no maternal setd2 show decreased survival rate and a developmental delay at maternal-to-zygotic transition. Taken together, these results indicate that, while the phenotypes of the setd2-null zebrafish and mice are apparently different, they are matched in parallel as the underlying mechanisms are evolutionarily conserved. Thus, the differential requirements of Setd2 may reflect distinct viability thresholds that associate with intrinsic and/or extrinsic stresses experienced by the organism through development, and these epigenetic regulatory mechanisms may serve as a reserved source supporting the evolution of life from simplicity to complexity.

SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS.

SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.