Ablative 5-Fraction Stereotactic MRI-Guided Adaptive Radiotherapy for Oligometastatic Pancreatic Adenocarcinoma.

INTRODUCTION: Metastatic pancreatic ductal adenocarcinoma (PDAC) carries a poor prognosis and significant morbidity from local tumor progression. We investigated outcomes among oligometastatic PDAC patients treated with stereotactic magnetic resonance image-guided ablative radiotherapy (SMART) to primary disease. METHODS: We performed a retrospective multi-institutional analysis of oligometastatic PDAC at diagnosis or with metachronous oligoprogression during induction chemotherapy treated with primary tumor SMART. Outcomes of interest included overall survival (OS), progression-free survival (PFS), freedom from locoregional failure (FFLRF), and freedom from distant failure (FFDF). Acute and late toxicity were reported and in exploratory analyses patients were stratified by the number of metastases, SMART indication, and addition of metastasis-directed therapy. RESULTS: From 2019 to 2021, 22 patients with oligometastatic PDAC (range: 1-6 metastases) received SMART to the primary tumor with a median follow-up of 11.2 months from SMART. Nineteen patients had de novo synchronous metastatic disease and three had metachronous oligoprogression. Metastasis location most commonly was liver only (40.9%), multiple organs (27.3%), lungs only (13.6%), or abdominal/pelvic nodes (13.6%). All patients received either FOLFIRINOX (64%) or gemcitabine/nab-paclitaxel (36%) followed by SMART (median 50 Gy, 5 fractions) for local control (77%), pain control (14%), or local progression (9%). Additionally, 41% of patients received other metastasis-directed treatments. The median OS from diagnosis and SMART was 23.9 months and 11.6 months, respectively. Calculated from SMART, the median PFS was 2.4 months with 91% of patients having distant progression, and 1-year local control was 68. Two patients (9%) experienced grade 3 toxicities, gastric outlet obstruction, and gastrointestinal bleed without grade 4 or 5 toxicity. CONCLUSION: There was minimal morbidity of local disease progression after SMART in this cohort of oligometastatic PDAC. As systemic therapy options improve, additional strategies to identify patients who may derive benefits from local consolidation or metastasis-directed therapy are needed.

Neoadjuvant chemotherapy and stereotactic body radiation therapy for borderline resectable pancreas adenocarcinoma: influence of vascular margin status and type of chemotherapy.

BACKGROUND: The influence of chemotherapy type and vascular margin status after sequential chemotherapy and stereotactic body radiation therapy (SBRT) for borderline resectable pancreatic cancer (BRPC) is unknown. METHODS: A retrospective review was performed on BRPC patients treated with chemotherapy and 5-fraction SBRT from 2009 to 2021. Surgical outcomes and SBRT-related toxicity were reported. Clinical outcomes were estimated by Kaplan-Meier with log rank comparisons. RESULTS: A total of 303 patients received neoadjuvant chemotherapy and SBRT to a median dose of 40 Gy prescribed to the tumor-vessel interface and median dose of 32.4 Gyto 95% of the gross tumor volume. One hundred and sixty-nine patients (56%) were resected and benefited from improved median OS (41.1 vs 15.5 months, P < 0.001). Close/positive vascular margins were not associated with worse OS or FFLRF. Type of neoadjuvant chemotherapy did not influence OS for resected patients, but FOLFIRINOX was associated with improved median OS in unresected patients (18.2 vs 13.1 months, P = 0.001). CONCLUSION: For BRPC, the effect of a positive or close vascular margin may be mitigated by neoadjuvant therapy. Shorter duration neoadjuvant chemotherapy as well as the optimal biological effective dose of radiotherapy should be prospectively explored.

Cancer outcomes are independent of preoperative CA 19-9 in anatomically resectable pancreatic ductal adenocarcinoma: A retrospective cohort analysis.

BACKGROUND AND OBJECTIVES: Current guidelines recommend neoadjuvant therapy for pancreatic ductal adenocarcinoma (PDAC) patients with anatomically resectable tumors but elevated CA 19-9. However, this recommendation is based on data from anatomically resectable and borderline resectable PDAC patients. Therefore, we analyzed the association of preoperative CA 19-9 with oncologic outcomes in a cohort of anatomically resectable PDAC patients. METHODS: A single-institution PDAC database from 2007 to 2015 included patients who underwent guideline-based staging and were anatomically resectable. Patients with bilirubin above 1.5 after decompression, nonsecretors of CA 19-9, and borderline resectable patients were excluded. Statistical analysis included frequency testing and regression modeling for recurrence and survival. RESULTS: One hundred forty-four PDAC patients were identified; 16 (11.1%) had elevated preoperative CA 19-9 ≥ 1000. A CA 19-9 level ≥1000 was not associated with demographic, clinical, or pathological factors. After adjustment for potential confounders, CA 19-9 levels (continuous, median, 500 U/mL, or 1000 U/mL cut-offs) were not associated with recurrence or overall survival (OS). CONCLUSIONS: Although guidelines recommend CA 19-9 to determine the management of anatomically resectable PDAC patients, CA 19-9 was not associated with recurrence or OS in this cohort. Our findings do not suggest that CA 19-9 alone should determine the PDAC treatment strategy.

Role of Arabidopsis AtPI4Kγ3, a type II phosphoinositide 4-kinase, in abiotic stress responses and floral transition.

Phosphoinositides (PIs) are essential metabolites which are generated by various lipid kinases and rapidly respond to a variety of environmental stimuli in eukaryotes. One of the precursors of important regulatory PIs, phosphatidylinositol (PtdIn) 4-phosphate, is synthesized by PtdIns 4-kinases (PI4K). Despite its wide distribution in eukaryotes, its role in plants remains largely unknown. Here, we show that the activity of AtPI4Kγ3 gene, an Arabidopsis (Arabidopsis thaliana) type II PtdIn 4-kinase, is regulated by DNA demethylation and some abiotic stresses. AtPI4Kγ3 is targeted to the nucleus and selectively bounds to a few PtdIns. It possessed autophosphorylation activity but unexpectedly had no detectable lipid kinase activity. Overexpression of AtPI4Kγ3 revealed enhanced tolerance to high salinity or ABA along with inducible expression of a host of stress-responsive genes and an optimal accumulation of reactive oxygen species. Furthermore, overexpressed AtPI4Kγ3 augmented the salt tolerance of bzip60 mutants. The ubiquitin-like domain of AtPI4Kγ3 is demonstrated to be essential for salt stress tolerance. Besides, AtPI4Kγ3-overexpressed plants showed a late-flowering phenotype, which was caused by the regulation of some flowering pathway integrators. In all, our results indicate that AtPI4Kγ3 is necessary for reinforcement of plant response to abiotic stresses and delay of the floral transition.

Management of melanoma brain metastases.

Relapses in the brain remain a major obstacle to cure in many patients with advanced melanoma. At present, the management of melanoma brain metastases continues to rely heavily on surgical and radiotherapeutic interventions, which have become safer and more effective with modern imaging, surgery and radiation technologies. Additionally, novel targeted and immunotherapeutic agents, shown to generate meaningful intracranial response and survival benefit in patients with melanoma brain metastases when compared with historical controls, expand systemic treatment options for this subset of patients. These systemic therapies become particularly important when intracranial disease burden precludes neuro- or radio-surgery. Considerable multidisciplinary research effort is ongoing to improve outcomes for melanoma patients with brain metastases, a key challenge in the management of advanced melanoma.

Substance P induces M2-type macrophages after spinal cord injury.

The potential benefits or the tissue-damaging effects of inflammatory response after central nervous system injuries have long been disputed. Recent studies have noted that substance P (SP), a neuropeptide, plays an important role in the wound-healing process by recruiting bone marrow stem cells to the injured tissue. In this study, we examined whether SP can enhance recovery from spinal cord injury (SCI) in Sprague-Dawley rats through its known function of stem cell mobilization and/or through the modulation of inflammation. We examined proinflammatory and anti-inflammatory cytokines and markers for macrophage subtypes. SP treatment modulated the SCI microenvironment toward a more anti-inflammatory and reparative one by inducing interleukin-10 and M2 macrophages and suppressing inducible nitric oxide synthase and tumor necrosis factor-α. This modulation was achieved at 1 day much earlier than SP-stimulated bone marrow stem cells' mobilization. Early intervention of the devastating inflammatory response by SP treatment caused the lesion cavity to become filled with robust axonal outgrowth that overlaid the M2 macrophages at 2 weeks--all of which culminated in tissue sparing and improvement in functional recovery from the SCI. SP is therefore a potential anti-inflammatory modulator for the treatment of injury-induced inflammatory central nervous system disorders.

Lidocaine suppresses the increased extracellular signal-regulated kinase/cyclic AMP response element-binding protein pathway and pro-inflammatory cytokines in a neuropathic pain model of rats.

BACKGROUND AND OBJECTIVE: Rats which have undergone spinal nerve ligation (SNL) display increases in the expression of extracellular signal-regulated kinase (ERK 1/2) and cyclic AMP response element-binding (CREB) protein. The present study was designed to determine whether lidocaine has a beneficial effect on the treatment of neuropathic pain by analysing related proteins. METHODS: Twenty-four male Sprague­Dawley rats were randomly allocated to three groups (eight per group): shamoperated (control) group, a neuropathic pain and normal saline group (NP+NS), a neuropathic pain and lidocaine group (NP+Lido, 2mgkg(-1) h(-1)). Anaesthetised rats received left L5 and L6 SNL. The mechanical withdrawal threshold test was performed 7 days after SNL and for 7 days with the pump implanted (saline or lidocaine). At post-implanted pump day 7, their brains and spinal cords were harvested. ERK 1/2, CREB proteins and mRNA amounts of pro-inflammatory cytokines (tumour necrosis factor a, intercellular adhesion molecule 1, monocyte chemo-attractive protein 1 and macrophage inflammatory protein 2) were assessed by immunoblotting or reverse transcriptase-PCR on samples collected from the three groups. RESULTS: Lidocaine increased the mechanical withdrawal threshold of a neuropathic rats. In only spinal tissues, ERK 1/2 and CREB proteins in the NP+Lido group was significantly reduced to 39%, and 48% in comparison with the NP+NS group. The NP+Lido group showed a significant reduction in mRNA amounts of pro-inflammatory cytokines compared with the NP+NS group (P<0.05). CONCLUSION: These results suggest that lidocaine therapy may be effective in treating neuropathic pain after spinal nerve injury, and that these effects may occur via suppression of ERK 1/2 and CREB signalling proteins and anti-inflammatory effects.

Hydrogen peroxide causes mitochondrial DNA damage in corneal epithelial cells.

PURPOSE: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas. METHODS: Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced. RESULTS: In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia. CONCLUSIONS: Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.

Hypersensitive response to oxidative stress in keratoconus corneal fibroblasts.

PURPOSE: It is unclear whether the oxidative damage found in keratoconus (KC) corneas results from innate defects of corneal fibroblasts or is due to excessive environmental challenges encountered by the patient with KC. The purpose of this study was to explore whether KC cells have inherent, exaggerated hypersensitivity to oxidative stressors. METHODS: Normal and KC corneal stromal fibroblasts were incubated in neutral or low-pH conditions, with or without hydrogen peroxide (H(2)O(2)). Reactive oxygen/nitrogen species (ROS/RNS) production was measured with 2',7'-dichlorodihydrofluorescein diacetate dye. Caspase-9 and -12 activities were measured by fluorochrome inhibitor to caspase (FLICA) assays. Long-extension polymerase chain reaction (LX-PCR) was used to amplify mtDNA. RNA was extracted, full-length cDNA synthesized, and PCR performed for mitochondria-encoded genes. Mitochondrial membrane potential (DeltaPsim) was measured by a cationic dye assay. RESULTS: In neutral pH conditions, KC fibroblasts had increased ROS production (P = 0.047), higher RNA levels for cytochrome c oxidase (complex IV) subunit II (P < 0.05), and decreased cathepsin K RNA (P = 0.04) compared with levels in normal cultures. In low-pH conditions, KC fibroblasts had decreased DeltaPsim (P = 0.015) and increased activation of caspase-9 (P = 0.013) and -12 (P = 0.01) compared with normal cultures. Changes in DeltaPsim were independent of cathepsin inhibition. The combination of low-pH+H(2)O(2) treatment degraded intact mtDNA and decreased the mtDNA-to-nuclear DNA ratio. CONCLUSIONS: Cultured KC fibroblasts have an inherent, hypersensitive response to oxidative stressors that involves mitochondrial dysfunction and mtDNA damage. KC fibroblast hypersensitivity may play a role in the development and progression of keratoconus.

Transduced human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult.

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1-HSP27 against cell death and ischemic insults. When PEP-1-HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1-HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1-HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1-HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.

7-Ketocholesterol activates caspases-3/7, -8, and -12 in human microvascular endothelial cells in vitro.

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 microM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.

Evaluation of in vitro effects of bevacizumab (Avastin) on retinal pigment epithelial, neurosensory retinal, and microvascular endothelial cells.

PURPOSE: To evaluate the short-term in vitro safety of bevacizumab (Avastin) in human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28), and human microvascular endothelial (HMVECad) cells. METHODS: ARPE-19 and R28 cells were treated with 0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, and 1 mg/mL of bevacizumab for 2, 6, and 24 hours. HMVECad cells were treated with 5 ng/mL of vascular endothelial growth factor (VEGF) and 0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, and 1 mg/mL of either bevacizumab for 2, 6, and 24 hours or a nonspecific human purified immunoglobulin (IgG) for 24 hours. Cell viability was measured using trypan blue dye exclusion assay. RESULTS: The cell viabilities of ARPE-19 cells, R28 cells, and HMVECad cells treated with bevacizumab were not significantly different (P > 0.05) from that of untreated controls. There was no significant difference (P > 0.05) between viabilities of HMVECad cells treated with bevacizumab and IgG. CONCLUSION: This study suggests that bevacizumab, at concentrations at or above the dose normally used in clinical practice, is not toxic to human retinal pigment epithelial, rat neurosensory retinal, or human microvascular endothelial cells in vitro. This report is consistent with the recent report of lack of toxicity of intravitreal bevacizumab in rabbits as well as the lack of apparent toxicity in clinical use.

Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts.

PURPOSE: To determine whether keratoconus (KC) corneal fibroblast cultures have increased reactive oxygen species (ROS) production and are more susceptible to stress-related challenges. METHODS: Normal (n = 9) and KC (n = 10) stromal fibroblast cultures were incubated in either neutral- or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic (green) dye was used to measure the mitochondrial membrane potential (delta psi m). RESULTS: KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P < 0.001 and 1.8-fold, P < 0.001, respectively) and catalase activity (P < 0.01) with higher concentrations of H2O2 compared with normal cultures (P = 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P < 0.02), showed higher caspase-3 activity (7.5-fold, P < 0.02) and decreased delta psi m (2.6-fold, P < 0.04), and had decreased cell viability (37%, P < 0.005 vs. 20%, P < 0.27) compared with normal fibroblasts. CONCLUSIONS: Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H2O2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas (increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC.

Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity.

PURPOSE: Keratoconus is a non-inflammatory thinning disorder of the corneal stroma. Recently, we showed that these corneas contain inducible nitric oxide synthase and an accumulation of nitrotyrosine, representing oxidative damage from peroxynitrite. Previously, we suggested that keratoconus corneas and their cell cultures have alterations in a gelatinase system with increased matrix metalloproteinase-2 (MMP-2) activity and decreased tissue inhibitor of metalloproteinase-1 (TIMP-1). This study examines whether a peroxynitrite donor (3-morpholinosydomine N-ethylcarbamide, SIN-1) or nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP) could alter TIMP-1 and/or MMP-2 in vitro. METHODS: Normal stromal fibroblasts were cultured in the presence or absence of either SIN-1 or SNAP for varying times. These cultures were analyzed by western and northern blot analyses, gelatin zymography, and a quantitative gelatinase/MMP assay. RESULTS: In vitro, SIN-1 treatment led to protein nitration, increased RNA levels of TIMP-1 and MMP-2, and loss of TIMP-1 immunostaining, but did not diminish gelatinase activity. SNAP treatment led to activation of MMP-2 and significantly increased gelatinase/MMP activity, without a change in TIMP-1 levels. CONCLUSIONS: Our data show that peroxynitrite or nitric oxide can decrease TIMP-1 and increase gelatinase activity, respectively. This demonstrates a relationship between elements of oxidative stress and tissue degradation in human corneal fibroblasts. This effect may play a significant role in the stromal thinning that occurs in keratoconus.