Laparoscopic versus open radical hysterectomy in FIGO 2018 early-stage cervical adenocarcinoma: Long-term survival outcomes after propensity score matching.

OBJECTIVE: To compare the long-term survival outcomes of laparoscopic radical hysterectomy (LRH) and open radical hysterectomy (ORH) in International Federation of Gynecology and Obstetrics (FIGO) 2018 early-stage cervical adenocarcinoma. METHODS: Based on the clinical diagnosis and treatment for cervical cancer in mainland China (Four C) database, the medical records of 1098 patients with FIGO 2018 early-stage cervical adenocarcinoma were retrospectively reviewed. Long-term and short-term survival outcomes of the two groups were compared using a multivariate Cox regression model and the log-rank method in the whole study population and after propensity score matching. RESULTS: There was no difference in disease-free survival (hazard ratio [HR] 0.921, 95% confidence interval [CI]: 0.532-1.595, p = 0.770) and overall survival (HR 1.168, 95% CI: 0.526-2.592, p = 0.702) between LRH (n = 468) and ORH (n = 468) in the risk-adjusted analysis. LRH resulted in significantly lower estimated blood loss (342.7 vs. 157.5 mL, p < 0.001) and shorter postoperative anal exhaust time (2.8 vs. 2.5 days, p < 0.001) in risk-adjusted analysis. The overall rates of intraoperative complications (2.4% vs. 4.3%, p = 0.100) and postoperative complications (7.5% vs. 6.2%, p = 0.437) showed no significant difference between the two groups. However, the LRH group had a significantly higher incidence of ureter injury (0.4% vs. 2.4%, p = 0.012) and great vessel injury (0.0% vs. 0.9%, p = 0.045) compared to the other group. No statistical variation in the site of recurrence was observed between the two groups (p = 0.613). CONCLUSIONS: LRH has comparable survival outcomes with ORH and was associated with earlier recovery in FIGO 2018 early-stage adenocarcinoma of the uterine cervix. However, the LRH group had higher risk of ureter injury and great vessel injury.

Genomic profiling identifies genes and pathways dysregulated by HEY1-NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis.

Mesenchymal chondrosarcoma is a rare, high-grade, primitive mesenchymal tumor. It accounts for around 2-10% of all chondrosarcomas and mainly affects adolescents and young adults. We previously described the HEY1-NCOA2 as a recurrent gene fusion in mesenchymal chondrosarcoma, an important breakthrough for characterizing this disease; however, little study had been done to characterize the fusion protein functionally, in large part due to a lack of suitable models for evaluating the impact of HEY1-NCOA2 expression in the appropriate cellular context. We used iPSC-derived mesenchymal stem cells (iPSC-MSCs), which can differentiate into chondrocytes, and generated stable transduced iPSC-MSCs with inducible expression of HEY1-NCOA2 fusion protein, wildtype HEY1 or wildtype NCOA2. We next comprehensively analyzed both the DNA binding properties and transcriptional impact of HEY1-NCOA2 expression by integrating genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and expression profiling (RNA-seq). We demonstrated that HEY1-NCOA2 fusion protein preferentially binds to promoter regions of canonical HEY1 targets, resulting in transactivation of HEY1 targets, and significantly enhances cell proliferation. Intriguingly, we identified that both PDGFB and PDGFRA were directly targeted and upregulated by HEY1-NCOA2; and the fusion protein, but not wildtype HEY1 or NCOA2, dramatically increased the level of phospho-AKT (Ser473). Our findings provide a rationale for exploring PDGF/PI3K/AKT inhibition in treating mesenchymal chondrosarcoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Uterine corpus invasion in cervical cancer: a multicenter retrospective case-control study.

OBJECTIVE: To determine the accuracy of uterine corpus invasion (UCI) diagnosis in patients with cervical cancer and identity risk factors for UCI and depth of invasion. METHODS: Clinical data of patients with cervical cancer who underwent hysterectomy between 2004 and 2016 were retrospectively reviewed. UCI was assessed on uterine pathology. Independent risk factors for UCI and depth of invasion were identified using binary and ordinal logistic regression models, respectively. RESULTS: A total of 2,212 patients with cervical cancer from 11 medical institutions in China were included in this study. Of these, 497 patients had cervical cancer and UCI, and 1,715 patients had cervical cancer and no UCI, according to the original pathology reports. Retrospective review of the original pathology reports revealed a missed diagnosis of UCI in 54 (10.5%) patients and a misdiagnosis in 36 (2.1%) patients. Therefore, 515 patients with cervical cancer and UCI (160 patients with endometrial invasion, 176 patients with myometrial invasion < 50%, and 179 patients with myometrial invasion ≥ 50%), and 1697 patients with cervical cancer without UCI were included in the analysis. Older age, advanced stage, tumor size, adenocarcinoma, parametrial involvement, resection margin involvement, and lymph node metastasis were independent risk factors for UCI. These risk factors, except resection margin involvement, were independently associated with depth of UCI. CONCLUSIONS: UCI may be missed or misdiagnosed in patients with cervical cancer on postoperative pathological examination. Older age, advanced stage, tumor size, adenocarcinoma, parametrial involvement, resection margin involvement, and lymph node metastasis were independent risk factors for UCI and depth of UCI, with the exception of resection margin involvement.

Lipoblastomas presenting in older children and adults: analysis of 22 cases with identification of novel PLAG1 fusion partners.

Lipoblastomas are benign neoplasms of embryonal white fat that typically present in the first 3 years of life and show a lobular arrangement of maturing adipocytes with variable degrees of myxoid change. We systematically studied the clinicopathologic and genetic features of lipoblastomas arising in older children and adults. Cases with a diagnosis of lipoblastoma or maturing lipoblastoma in patients >3 years of age were retrieved from our archives. Immunostaining for CD34 and desmin and molecular studies (FISH, RNA sequencing) were performed. Twenty-two cases (8F; 14M) were identified in patients ranging from 4 to 44 years of age (median 10 years). Sites included extremity (n = 15), head and neck (n = 4), and trunk (n = 3) with tumor sizes varying from 1.6 to 17.5 cm (median 5). Only three tumors had histologic features of "conventional" lipoblastoma. The majority of tumors (n = 14) were composed of variably sized lobules of mature adipose tissue partitioned by thin fibrous septa ("maturing"). The remaining five cases consisted predominantly of bland spindled to plump ovoid cells embedded in a fibrous stroma, with a vaguely plexiform arrangement of small myxoid and adipocytic nodules ("fibroblastic"). CD34 was diffusely positive in all cases tested (21/21), while desmin immunoreactivity was identified in 12 of 21 cases (diffuse = 7, focal = 5). PLAG1 rearrangements were identified in 13 tumors in the entire cohort (59%), including all 5 fibroblastic tumors. RNA sequencing detected eight PLAG1 fusion partners, of which two were known (CHCHD7 and COL3A1) and six were novel (SRSF3, HNRNPC, PCMTD1, YWHAZ, CTDSP2, and PPP2R2A). Twelve cases had follow-up (1-107 months; median 21 months), and no recurrences were reported. Lipoblastomas may occur in older children and adults and may be difficult to recognize due to their predominantly adipocytic or fibrous appearance. Awareness that lipoblastomas may occur in older patients, careful evaluation for foci showing more typical morphologic features, ancillary immunohistochemistry for CD34 and desmin, and molecular genetic studies to identify PLAG1 rearrangements are the keys to recognizing these tumors.

Rph1 coordinates transcription of ribosomal protein genes and ribosomal RNAs to control cell growth under nutrient stress conditions.

Coordinated regulation of ribosomal RNA (rRNA) synthesis and ribosomal protein gene (RPG) transcription by eukaryotic RNA polymerases (RNAP) is a key requirement for growth control. Although evidence for balance between RNPI-dependent 35S rRNA production and RNAPII-mediated RPG transcription have been described, the molecular basis is still obscure. Here, we found that Rph1 modulates the transcription status of both rRNAs and RPGs in yeast. We show that Rph1 widely associates with RNAPI and RNAPII-transcribed genes. Deletion of RPH1 remarkably alleviates cell slow growth caused by TORC1 inhibition via derepression of rRNA and RPG transcription under nutrient stress conditions. Mechanistically, Rim15 kinase phosphorylates Rph1 upon rapamycin treatment. Phosphorylation-mimetic mutant of Rph1 exhibited more resistance to rapamycin treatment, decreased association with ribosome-related genes, and faster cell growth compared to the wild-type, indicating that Rph1 dissociation from chromatin ensures cell survival upon nutrient stress. Our results uncover the role of Rph1 in coordination of RNA polymerases-mediated transcription to control cell growth under nutrient stress conditions.

The Paf1 complex transcriptionally regulates the mitochondrial-anchored protein Atg32 leading to activation of mitophagy.

Mitophagy is a critical process that safeguards mitochondrial quality control in order to maintain proper cellular homeostasis. Although the mitochondrial-anchored receptor Atg32-mediated cargo-recognition system has been well characterized to be essential for this process, the signaling pathway modulating its expression as a contribution of governing the mitophagy process remains largely unknown. Here, bioinformatics analyses of epigenetic or transcriptional regulators modulating gene expression allow us to identify the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) as a transcriptional repressor of ATG genes. We show that Paf1C suppresses glucose starvation-induced autophagy, but does not affect nitrogen starvation- or rapamycin-induced autophagy. Moreover, we show that Paf1C specifically regulates mitophagy through modulating ATG32 expression. Deletion of the genes encoding two core subunits of Paf1C, Paf1 and Ctr9, increases ATG32 and ATG11 expression and facilitates mitophagy activity. Although Paf1C is required for many histone modifications and gene activation, we show that Paf1C regulates mitophagy independent of its positive regulatory role in other processes. More importantly, we also demonstrate the mitophagic role of PAF1C in mammals. Overall, we conclude that Paf1C maintains mitophagy at a low level through binding the promoter of the ATG32 gene in glucose-rich conditions. Dissociation of Paf1C from ATG32 leads to the increased expression of this gene, and mitophagy induction upon glucose starvation. Thus, we uncover a new role of Paf1C in modulating the mitophagy process at the transcriptional level. ABBREVIATIONS: AMPK: AMP-activated protein kinase; ATP5F1A: ATP synthase F1 subunit alpha; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chlorophenylhydrazone; DFP: chelator deferiprone; GFP: green fluorescent protein; H2B-Ub1: H2B monoubiquitination; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; KD: kinase dead; OPTN, optineurin; Paf1: polymerase-associated factor 1; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RT-qPCR: real-time quantitative PCR; SD-N: synthetic dropout without nitrogen base; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; YPD: yeast extract peptone dextrose; YPL: yeast extract peptone lactate.

Effects of preoperative radiotherapy or chemoradiotherapy on postoperative pathological outcome of cervical cancer--from the large database of 46,313 cases of cervical cancer in China.

OBJECTIVE: To investigate the effect of preoperative radiotherapy or chemoradiotherapy combined with radical surgery on pathological outcomes in cervical cancer patients. METHODS: Based on a large Chinese cervical cancer database of clinical diagnosis and treatment (C4 Project), the postoperative pathological outcomes of patients who received preoperative radiotherapy or chemoradiotherapy followed by open surgery (PR group) or surgery alone (SD group) were compared. RESULTS: Among the strictly selected patients, the incidence of lymph node metastasis in the PR group (n = 574) was higher than that in the SD group (231 VS 9; P < 0.001), while the incidence of vascular space invasion was lower than that in the SD group (72 VS 2041; P < 0.001). The logistic regression analysis showed that preoperative radiotherapy was a protective factor for parametrial involvement, positive surgical margins, deep cervical stromal invasion, and vascular space invasion (P < 0.05). The median number of resected lymph nodes in both groups was 18. After 1:1 case matching, the incidence of deep cervical stromal invasion and vascular space invasion was reduced by preoperative radiotherapy (292 vs 376, P < 0.001; 60 vs 106, P < 0.001). Logistic regression analysis indicated that preoperative radiotherapy was a protective factor for deep cervical stromal invasion and vascular space invasion (P < 0.05). The median numbers of resected lymph nodes in the two groups were 18 and 19, separately. CONCLUSION: Preoperative radiotherapy can reduce both the incidence of deep cervical stromal invasion and vascular space invasion, but it cannot reduce lymph node positivity, parametrial involvement and positive surgical margins.

Whole-transcriptome sequencing identifies a distinct subtype of acute lymphoblastic leukemia with predominant genomic abnormalities of EP300 and CREBBP.

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300- and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.

Effect of vitamin D receptor knockout on cornea epithelium wound healing and tight junctions.

PURPOSE: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas. METHODS: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas. Mice were on a normal or high lactose, Ca(2+), and PO4(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine corneal epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. RESULTS: Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. CONCLUSIONS: Vitamin D receptor knockout affects mouse corneal epithelium wound healing and tight junction integrity.

Enhancement of vitamin D metabolites in the eye following vitamin D3 supplementation and UV-B irradiation.

PURPOSE: This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure. METHODS: Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy. RESULTS: 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3. CONCLUSIONS: There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

Vitamin D enhances corneal epithelial barrier function.

PURPOSE: The purpose of this study was to determine whether 25-hydroxyvitamin D(3) (25(OH)D(3)) and/or its active metabolite, 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), can enhance corneal epithelial barrier function. The authors also determined if corneas contain mRNA for the vitamin D receptor (VDR) and 1α-hydroxylase, the enzyme required to convert 25(OH)D(3) to 1,25(OH)(2)D(3), and measured vitamin D metabolite concentrations in aqueous and vitreous humor. METHODS: RT-PCR was used to examine mouse, rabbit, and human corneal epithelial VDR and 1α-hydroxylase mRNA. Vitamin D metabolites were measured using a selective vitamin D derivatizing agent and mass spectroscopy. Barrier function experiments were performed by measuring inulin permeability (IP) and/or transepithelial resistance (TER) in control, 25(OH)D(3)-, and 1,25(OH)(2)D(3)-treated human and rabbit corneal epithelial monolayers cultured on permeable inserts. Ca(2+) was removed, then reintroduced to the culture medium while IP and TER readings were taken. Occludin levels were examined using Western blotting. RESULTS: All corneal samples were positive for both VDR and 1α-hydroxylase mRNA. All vitamin D metabolites except for unhydroxylated vitamin D(3) were detected in aqueous and vitreous humor. Epithelial cells showed increased TER, decreased IP, and increased occludin levels when cultured with 25(OH)D(3) and 1,25(OH)(2)D(3). CONCLUSIONS: We conclude that corneas contain mRNA for VDR and 1α-hydroxylase as well as significant vitamin D concentrations. 25(OH)D(3) and its active metabolite 1,25(OH)(2)D(3), both enhance corneal epithelial barrier function.

Lysophosphatidic acid-activated Cl- current activity in human systemic sclerosis skin fibroblasts.

OBJECTIVES: SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl- current (IClLPA) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls. METHODS: Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure IClLPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. RESULTS: Fibroblasts cultured from SSc skin show significantly increased IClLPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. CONCLUSIONS: Elevated IClLPA activity is a hallmark of SSc skin fibroblasts. Blocking IClLPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.

ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation.

To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl(-) currents (I(Cl,LPA) and I(Cl,VRAC), respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I(Cl,LPA) and I(Cl,VRAC) currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased I(Cl,LPA) and I(Cl,VRAC) activity in the presence of transforming growth factor-beta(1) (TGF-beta(1)) compared with controls, whereas ClC-3 overexpression resulted in increased I(Cl,LPA) activity in the absence of TGF-beta(1). ClC-3 knockdown also resulted in a reduction of alpha-smooth muscle actin (alpha-SMA) protein levels in the presence of TGF-beta(1), whereas ClC-3 overexpression increased alpha-SMA protein expression in the absence of TGF-beta(1). In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the I(Cl,LPA) current activity, and participates in the fibroblast-to-myofibroblast transition.

Chloride channel activity in human lung fibroblasts and myofibroblasts.

It is well established that transforming growth factor (TGF)-beta stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (I(Cl-LPA)) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of I(Cl-LPA) on fibroblast-to-myofibroblast differentiation. We recorded I(Cl-LPA) using the amphotericin perforated-patch technique. We activated I(Cl-LPA) using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-alpha-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-beta (myofibroblasts) had significantly elevated alpha-SMA levels and I(Cl-LPA) current density compared with control fibroblasts. I(Cl-LPA) activation was blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 microM). DIDS and NPPB, in a dose-dependent manner, significantly reduced alpha-SMA levels in HLF stimulated with TGF-beta. These results demonstrate the receptor-mediated activation of I(Cl-LPA) by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal I(Cl-LPA) activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.