DOI: 10.5061/dryad.ttdz08m7z
title: Reshaping the landscape of locoregional treatments for breast cancer liver metastases: A novel, intratumoral, p21-targeted percutaneous therapy increases survival in BALB/c mice inoculated with 4T1 triple negative breast cancer cells in the liver

description: Patients with disseminated metastatic disease from breast cancer are
 likely to have liver involvement in &gt;50% of cases at some point
 during disease progression. These patients have a poor prognosis; and,
 when treated with the standard of care systemic therapy they have a median
 survival of &lt;9-months. Increasing survival in breast cancer
 patients will likely require the administration of better therapies that
 are specifically targeted to treat distant metastases. One approach to
 increasing treatment efficacy for breast cancer liver metastases is
 through the application locoregional therapies. Locoregional therapies are
 an appealing interventional approach for breast cancer patients with liver
 metastases since these tumor lesions are accessible via minimally invasive
 procedures that can be administered using either ultrasound or CT imaging.
 Current locoregional therapies to treat breast cancer liver metastases are
 non-specific and have not produced significant increases in survival. The
 goal of this study was to design and test a targeted locoregional
 therapeutic intervention for breast cancer liver metastases. The lead
 candidate, a fixed-dose small-molecule drug called MBC-005, was tested in
 vitro and then the efficacy was evaluated in a BALB/c mouse liver
 metastases model. A novel formulation of N-allyl noroxymorphone
 hydrochloride incorporated into an alginate-based gel overcomes many of
 the limitations associated with the administration of small-molecule
 drugs, which include solubility, off-target toxicity, and enzymatic
 degradation. In vitro results demonstrated that MBC-005 mediated its
 anti-tumorigenic effect through a p21-dependent mechanism via a novel
 molecular pathway, in which N-allyl noroxymorphone component of MBC-005
 stimulated the opioid growth factor receptor to increase p21 expression.
 Intratumoral administration of MBC-005 increased survival 3.9-fold in mice
 and significantly decreased tumor volume 4-fold. While many cytotoxic
 therapies increase p21 expression as a response to DNA damage, MBC-005
 increased p21 expression independent cytotoxic DNA damage. MBC-005 did not
 induce off-target toxicity; and, as such, would be amenable to multiple
 rounds of administration. Nevertheless, it is notable that the positive
 effects of MBC-005 treatment on increasing survival and decreasing tumor
 volume in mice was achieved using a single dose.
description: <strong>Material Studies</strong>
 A modified USP apparatus 2 was fitted with in-situ UV fiber optic
 probes that was used to perform the dissolution evaluation. Solubility was
 assessed in 10 dissolution media (e.g., buffers with and without
 surfactants) to determine the ideal ‘sink’ conditions for dissolution. UV
 wavelengths were scanned to identify the spectra and the lambda max. The
 limit of detection and limit of quantification were identified, and a
 dissolution profile was determined. Each method was run in triplicate over
 72-hours using fiber optic dip probes. Viscosity was
 assessed using the SV-10 tuning-fork sine wave viscometer (A&D
 Weighing). Samples were prepared and mixed for 2-minutes, allowed to sit
 for 5-minutes, then briefly mixed a second time prior to testing. The
 tuning-fork sensor plates were inserted into MBC-005 and viscosity and
 temperature data were collected at 5-second intervals.
 <strong>In vitro Studies</strong>
 Primary human mammary epithelial cells (HUMEC) were used as a
 non-transformed control cell line. HUMEC’s were maintained in Leibovitz’s
 L-15 media (Lonza), without serum but supplemented with the Mammary
 Epithelial Cell Growth Kit (ATCC). The MCF7 human breast cancer tumor
 cell-line (HR+/HER2-) is derived from a metastatic adenocarcinoma that
 possesses both estrogen and progesterone receptors (e.g., hormone receptor
 positive). The BT474 human breast cancer tumor cell-line is a ductal
 carcinoma derived from a solid mass (HR+/HER2+). MCF7 and BT474 cells were
 maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10%
 fetal calf serum (FCS, v/v) and 1% penicillin–streptomycin (Cellgro,
 Corning Life Sciences) with 1% glutamine (Glutagro, Corning Life
 Sciences). All cells used in this study were obtained from ATCC.
 <strong>Cell Viability Assays</strong>
 Cells were treated with serial doses of our formulation of
 N-allyl noroxymorphone dihydrate (Mallinckrodt Pharmaceuticals)
 solubilized in acetic acid, citric acid monohydrate, and 0.9% normal
 saline (Spectrum Chemical). Viable cell number was determined with the
 methyl tetrazolium (MTT) assay. After 72-hours of treatment, 5-mg/mL of
 the MTT reagent (w/v, Sigma) was added to each well and incubated for
 2-hours, after which the cells were lysed with 500-µL of DMSO (Sigma).
 50-µL of solution was added in duplicate to a 96-well plate and MTT
 absorbance was measured at 570-nm on a Tecan Spark monochromator. The
 effects on cell proliferation were determined by normalizing treated wells
 relative to mean values from non-treated wells, as follows: Fold change in
 cell number = 100*[treated cells optical density/mean control optical
 density]. The CyQuant lactate dehydrogenase
 cytotoxicity assay (LDH; Invitrogen) was used to determine cell death
 following treatment with N-allyl noroxymorphone. Briefly, at the
 appropriate time points after treatment with N-allyl noroxymorphone, 50-µL
 of cell culture media from each experimental plate was transferred to a
 96-well plate, in duplicate. 50-µL of Reaction Mixture was aliquoted into
 each sample well and mixed by gentle tapping. The plate was incubated for
 30-minutes at 37ºC in the dark, and absorbance was immediately read at
 490-nm and 680-nm. The background measured at 680-nm was subtracted from
 the 490-nm reading to determine LDH activity. Fold change in LDH was
 determined using the following equation: LDH Fold Change = 100*[treated
 cells optical density/ control well optical density].
 <strong>Spheroid Assay</strong>
 The spheroid assay was used to determine long terms effects of
 N-allyl noroxymorphone therapy on tumor cell survival. Tumor cells in
 regular DMEM (5x10<sup>4</sup> per implant) were mixed with
 equal parts (v/v) of Matrigel (Corning Life Sciences), allowed to settle
 in an incubator for 30-minutes, scanned using an Epson 700 scanner to
 determine initial spheroid area, and then carefully covered with media and
 placed back in the cell incubator. At the conclusion of the experiment,
 cells were fixed in 70% EtOH and stained with 1% crystal violet and then
 scanned again. Area fractions were determined using FIJI (ImageJ), and the
 initial area fraction for each implant was subtracted from the area
 fraction at the conclusion of the experiment.
 <strong>The p21 Assay</strong>
 Cells were disassociated using a commercially available lysis
 buffer (Cell Signaling Technology), to which 1-mM of the protease
 inhibitor phenylmethylsulfonyl fluoride was added. Protein lysates were
 analyzed using the Pierce BCA assay to determine total protein
 concentrations (ThermoFisher). Total p21<sup>Waf1/Cip1</sup>
 protein concentrations were assayed using a commercially available
 colorimetric sandwich ELISA assay (Cell Signaling Technology). Data were
 compared to a standard curve supplied with the ELISA kit.
 <strong>Mouse Xenograft Studies</strong>
 The efficacy of MBC-005 in treating breast cancer metastasis in
 the liver was evaluated using a syngeneic mammary carcinoma model
 utilizing orthotopic implantation of 4T1-Luc2 cells into the intrahepatic
 space in 8-weeks old female BALB/c mice (N=96 total/ N=12 per group). A
 Cox Proportional Hazard Model was employed to assess the Hazard Ratio (HR)
 to determine if the following parameters contributes to animal survival:
 treatment (Control versus MBC-005), initial tumor volume, final tumor
 volume, median tumor volume, animal weight at the conclusion of the
 studies, metastases, bioluminescence analysis, and liver weight.
 The 4T1-luc2 murine mammary gland tumor
 cell-line is an implantable triple-negative breast cancer that was
 obtained from Perkin-Elmer. The cell line was maintained in RPMI-1640
 medium supplemented with 10% fetal bovine serum, 2-mM glutamine,
 100-units/ mL penicillin G sodium, 100-µg/mL streptomycin sulfate, and
 25-µg/mL gentamicin. Cell-line bioluminescent intensity was verified by
 limiting dilution before and after cell implant. For
 tumor implantation, the 4T1-luc2 tumor cells were resuspended in 50%
 Matrigel (Corning) at 4x10<sup>6</sup> cells/ mL. Prior to
 undergoing intrahepatic implantation surgery, the animals were
 administered 0.15-mg/ kg buprenorphine. Each mouse was surgically injected
 intra-hepatically into the left lateral lobe with
 2x10<sup>5</sup> cells in a 50-μL suspension under isoflurane
 anesthesia. On day 7 after the implantation of the 4T1-luc2 tumor cells,
 mice were randomized into treatment groups and all animals except those
 the no treatment control group were administered with doxorubicin (5-mg/
 kg) intravenously. Mice in the MBC-005 treatment groups were concurrently
 treated with 25-mL of MBC-005 (<strong>Table 1)</strong> via
 intra-hepatically injection into the left lateral lobe.
 The surgeries, animal monitoring, and imaging were completed at
 Charles River Laboratory Morrisville (Morrisville, NC). Animals were
 maintained in barrier housing in a clean room with 12-hours light/ dark
 cycle as part of an IACUC approved study. Animal welfare considerations
 were observed during this study, including efforts to minimize suffering
 and distress, which included subcutaneous administration of 0.15-mg/ kg of
 buprenorphine as an anesthetic immediately following surgery and as needed
 subsequently. Animals were anesthetized via inhalation using 4% isoflurane
 to reach a surgical plane of anesthesia and then maintained with 1% to 2%
 isoflurane. Following tumor implantation and treatment, animals were
 monitored daily and weighed every 2-days. Animals were euthanized when
 they were observation have lost 30% of body weight or on three consecutive
 weight measurements lost 25% of body weight. Animals were also euthanized
 when they lost 15% of body weight and, in conjunction, had impaired hind
 limb function, had hindlimb paralysis, or were moribund. Animals were also
 euthanized when the tumor volume was greater than or equal to
 1000-mm<sup>3</sup>. Animals that completed the study and
 survived to day 60 following treatment were also euthanized. Animals were
 euthanized using CO<sub>2</sub> asphyxiation followed by
 cervical dislocation, after which animals were bled via cardiac puncture
 for clinical chemistry and hematology assessments. Necropsies was
 performed for individual animal and livers were removed for histologic
 processing. <strong>Liver Weight
 Measurements</strong> Following gross necropsy,
 livers were collected. Liver weight was obtained before being fixed in 10%
 neutral buffered formalin. Liver weights were measured using a balance and
 corrected for the animal weight to produce a % liver weight.
 <strong>Bioluminescence Imaging</strong>
 Bioluminescent images were acquired on day 0 and then bi-weekly
 until the end of the study.  Luciferase activity was measured using the
 IVIS SpectrumCT (Perkin Elmer) equipped with a CCD camera, mounted on a
 light-tight specimen chamber.  On each imaging day, animals were injected
 with 0.22-µm of VivoGlo D-Luciferin substrate (150-mg/ kg; i.p. at
 10-mL/kg, split across two injections) and placed in an anesthesia
 induction chamber (2.5% to 3.5% isoflurane in oxygen). Upon sedation,
 animals were placed in a ventral position in the imaging chamber, equipped
 with stage heated at physiological temperature, for image acquisition
 starting ten minutes after luciferin injection. Flux was quantified and
 reported as 10<sup>6</sup> photons/ second (p/s). 
 <strong>Histology</strong> After
 weighing the livers, samples were placed in 10% neutral buffered formalin
 and allowed to fix for at least 72-hours. Livers were dehydrated using
 serial concentrations of ethanol (e.g., 30%, 50%, 70%, and 100%) and then
 toluene. Livers were then laid flat, embedded in paraffin, and then
 sectioned at 5-mm on a rotary microtome. Sections were then stained with
 hematoxylin and eosin. Livers were sectioned at three levels (L1, L2, and
 L3). One section was evaluated from each level, which were separated by
 645-mm (e.g., L1 was 645-mm from L2 and L2 was 645-mm from L3). Liver
 sections were imaged at 4x using a Nikon Eclipse Ni microscope and a Nikon
 DS-Fi2 digital camera. Composite images were generated using a tiling
 method, in which images were taken periodically in a grid pattern and then
 assembled into a final image using Photoshop (Adobe).
 <strong>Histomorphology</strong>
 Histomorphometric analysis was used to determine tumor volume and
 the volume of necrotic tissue in the left lobe of the liver, in which the
 tumor and MBC-005 were implanted. Volumes for the whole liver lobe were
 determined using Fiji (v1.54f) using image thresholding. Tumor volumes and
 the volume of necrotic tissue were determined using QuPath (v0.5.1-arm64)
 pathology analysis software. Volumes were estimated based on the work of
 Cruz-Orive and the Cavalieri Principle. Briefly, the area fraction
 (<em>A</em>) for a particular level (e.g.,
 <em>A<sub>L1</sub></em>) was multiplied by the
 distance (<em>d</em>) between sections located in adjacent
 levels (i.e., between L1 and L2), in which <em>d</em>=645-mm.
 The composite volume (<em>V<sub>t</sub></em>) for
 each parameter (e.g., total volume, tumor volume, of volume of necrosis)
 was calculated by summing the volumes for each level, such that:
 <em>V<sub>t</sub></em> =
 <em>V<sub>L1</sub></em> +
 <em>V<sub>L2</sub></em> +
 <em>V<sub>L3</sub></em> =
 (<em>A<sub>L1</sub></em>*645-mm) +
 (<em>A<sub>L2</sub></em>*645-mm) +
 (<em>A<sub>L3</sub></em>*5-mm). The final term
 <em>A<sub>L3</sub></em> is multiplied by 5-mm to
 account for thickness of the final section. The volume fraction
 (<em>V<sub>f</sub></em>) was calculated by
 dividing the tumor volume
 (<em>V<sub>t(tumor)</sub></em>) by the total liver
 volume (<em>V<sub>t(liver)</sub></em>). Likewise,
 the volume fraction of necrosis
 (<em>V<sub>f</sub></em>) was calculated by
 dividing the volume of necrosis
 (<em>V<sub>t(necrosis)</sub></em>) by the total
 liver volume
 (<em>V<sub>t(liver)</sub></em>).
 <strong>Statistical Analyses</strong>
 Means and standard deviations were calculated for the MTT assay
 data, the LDH assay data, and the spheroid assay data. The normalized
 dose-response data from the MTT assay were assessed using an ‘inhibitor
 concentration versus normalized dose response’ non-linear regression
 analysis. Dose-response regression lines were assessed for statistical
 differences between groups using an Extra-Sum-of-Squares F-test to
 determine if the groups were statistically different or, alternatively,
 could be modeled using a single regression line (a=0.05). LDH and the
 spheroid assay data were assessed for significant differences using either
 one-way ANOVA followed by correction for multiple comparisons with
 Dunnett’s test (a=0.05). Bioluminescent data for the
 mouse studies were analyzed using a Malthusian exponential growth model
 was developed using non-linear least-squares regression to estimate the
 doubling time for the tumor volume, which served as a surrogate for local
 tumor growth. Regression lines were assessed for statistical differences
 between groups using an Extra-Sum-of-Squares F-test to determine if the
 groups were statistically different or, alternatively, could be modeled
 using a single regression line (a=0.05).
 Histomorphometric analyses were first evaluated normality using
 the Shapiro-Wilks test. All of the groups were found to be normal or
 lognormal, and a one-way ANOVA was used to assess differences between
 treatment groups. Consistent with Glantz’s et al. Welch’s correction with
 an unpaired ‘t’ was used to identify differences between the NTC group and
 the MBC-005 treatment groups (a=0.05). A Cox
 Proportional Hazard Regression Model was used to assess survival and to
 calculate the parameter estimates (b<sub>i</sub>) and the
 hazard ratio (HR = exp(b<sub>i</sub>)) for the following
 parameters: treatment (Control versus MBC-005), initial tumor volume,
 final tumor volume, median tumor volume, fracture incidence, the presence
 of metastases, and final body weight.  All data analyses were performed
 using GraphPad Prism version 9.4.0, GraphPad Software, San Diego,
 California USA.
description: # Reshaping the landscape of locoregional treatments for breast cancer
 liver metastases: A novel, intratumoral, p21-targeted percutaneous therapy
 increases survival in BALB/c mice inoculated with 4T1 triple negative
 breast cancer cells in the liver Dataset DOI:
 [10.5061/dryad.ttdz08m7z](10.5061/dryad.ttdz08m7z) ## Description of the
 data and file structure #### File: Fig2\_Viscosity.csv Mean viscosity of
 the MBC-005 biomaterial viscosity as a function of time (seconds). #####
 Variables * Time (seconds) * Viscosity 1 (milli Pascals * seconds) *
 Viscosity 2 (milli Pascals * seconds) * Viscosity 3 (milli Pascals *
 seconds) #### File: Fig4\_Elution\_Rate.csv Data showing the rate at which
 N-allyl noroxymorphone releases from MBC-005. The variables are the
 concentration of N-allyl noroxymorphone (sample 1, 2, 3, 4) versus the
 square root of time (Sqrt Time). ##### Variables * Sqrt Time (seconds) *
 Sample 1 (milligrams) * Sample 1 (milligrams) * Sample 2 (milligrams) *
 Sample 2 (milligrams) #### File: Fig4\_Elution\_Nonlin\_fit.csv Non-linear
 regression of the N-allyl noroxymorphone data in Figure 4. ##### Variables
 * Sample 1 (milligrams) * Sample 2 (milligrams) #### File:
 Fig5\_MTT\_Cell\_Number.csv Relative change in tumor cell number (MCF7 and
 BT474) and non-tumor cell (HUMEC) number measured with the MTT assay of
 proliferation as a function of N-allyl noroxymorphone dose in milli molar
 concentrations. Since the assay measures relative change, the values are
 percentages and therefore dimensionless. ##### Variables * HUVEC Vascular
 Endothelium  * HUVEC Vascular Endothelium * HUVEC Vascular Endothelium *
 HUVEC Vascular Endothelium * HUMEC Breast Epithelium * HUMEC Breast
 Epithelium * HUMEC Breast Epithelium * HUMEC Breast Epithelium * BT474
 Breast Cancer * BT474 Breast Cancer * BT474 Breast Cancer * BT474 Breast
 Cancer #### File: Fig5\_Non\_Linear\_Fit\_MTT\_Cell\_Number.csv The
 non-linear analysis for figure 5. ##### Variables * HUVEC Vascular
 Endothelium * HUMEC Breast Epithelium * BT474 Breast Cancer #### File:
 Fig6\_LDH.csv Relative lactate dehydrogenase (i.e., cell death) values
 corrected for cell number for the MCF7 and BT474 breast cancer tumor cells
 with respect to different concentrations of N-allyl noroxyorphone (milli
 molar). #### File: Fig6\_LDH\_1way\_ANOVA\_2.csv ANOVA analysis of MCF7
 LDH values.  ##### Variables * Number of families  #### File:
 Fig6\_LDH\_1way\_ANOVA.csv ANOVA analysis of BT474 LDH values.  #####
 Variables * Number of families #### File:
 Fig7\_\_\_Spheroid\_Area\_Fraction\_BT474.csv Area fraction - a
 dimensionless value - of BT474 cells following treatment with N-allyl
 noroxymorphone using the spheroid assay which measures clonogenic growth
 following a toxic insult.  #### File:
 Fig7\_\_\_Spheroid\_Area\_Fraction\_MCF7.csv Area fraction - a
 dimensionless value - of MCF7 cells following treatment with N-allyl
 noroxymorphone using the spheroid assay which measures clonogenic growth
 following a toxic insult.  #### File:
 Fig7\_\_\_Spheroid\_Area\_Fraction\_BT474\_1way\_ANOVA.csv ANOVA analysis
 of spheroid area fraction data. ##### Variables * Number of families ####
 File: Fig7\_\_\_Spheroid\_Area\_Fraction\_MCF7\_1way\_ANOVA.csv ANOVA
 analysis of spheroid area fraction data. ##### Variables * Number of
 families #### File: Fig8\_LDH.csv Relative lactate dehydrogenase (i.e.,
 cell death) values corrected for cell number for the MCF7 and BT474 breast
 cancer tumor cells relative to the MC3 non-tumor cells. ##### Variables *
 MC3 24-hrs * MC3 72-hrs * MCF7 24-hrs * MCF7 72-hrs * BT474 24-hrs * BT474
 72-hrs #### File:
 Fig8\_LDH\_Brown-Forsythe\_and\_Welch\_ANOVA\_tests\_of\_Col\_\_Corrected\_LDH\_ZetaMAST\_.csv ANOVA analysis of LDH values.  ##### Variables * Number of families #### #### File: Fig8\_MTT.csv Relative change cell number measured with the MTT assay of proliferation for the MCF7 and BT474 breast cancer tumor cells relative to the MC3 non-tumor cells. ##### Variables * MC3 24-hrs * MC3 72-hrs * MCF7 24-hrs * MCF7 72-hrs * BT474 24-hrs * BT474 72-hrs #### File: Fig8\_MTT\_Brown-Forsythe\_and\_Welch\_ANOVA\_tests\_of\_Col\_\_Corrected\_MTT\_ZetaMAST\_.csv ANOVA analysis of MTT values.  ##### Variables * Number of families #### File: Fig9\_BT474\_p21.csv Percent change in p21 proteins values corrected by total protein for the MCF7 tumor cells using an ELISA assay (values are dimensionless) treated with 0- or 1-mM N-allyl noroxymorphone. ##### Variables * 0-mM * 1-mM #### File: Fig9\_MCF7\_p21\_Welch\_ttest.csv T-test using's Welch's correction for the p21 values. ##### Variables * Table Analyzed * 24- & 48-hr MCF7 p21 Expression #### #### File: Fig9\_MCF7\_p21.csv Percent change in p21 proteins values corrected by total protein for the BT474 tumor cells using an ELISA assay (values are dimensionless) treated with 0- or 1-mM N-allyl noroxymorphone. ##### Variables * 0-mM * 1-mM #### File: Fig9\_BT474\_p21\_Welch\_ttest.csv T-test using's Welch's correction for the p21 values. ##### Variables * Table Analyzed * 24- & 48-hr BT474 p21 Expression #### File: Fig10\_2way\_ANOVA\_of\_Col\_\_MCF7\_MTT\_Nalx\_vs\_Dox.csv Relative change cell number measured with the MTT assay of proliferation for tumor cells relative to doxorubicin. ##### Variables * Compare column means (main column effect) #### File: Fig11\_Cox\_regression\_of\_Cox\_Survival.csv Cox hazard regression for animal survival. ##### Variables * Table Analyzed * Cox Survival #### File: Fig12\_Nonlin\_fit\_of\_Bioluminescence\_(p\_\_\_s)\_Liver.csv Non-linear regression of the bioluminescence values in the liver from mice inoculated with 4T1 breast cancer cells.  ##### Variables * No Treatment: * ZetaMAST - No N-allyl noroxymorphone * ZetaMAST - 30-microg N-allyl noroxymorphone * ZetaMAST - 60-microg N-allyl noroxymorphone * ZetaMAST - 120-microg N-allyl noroxymorphone * ZetaMAST - 180-microg N-allyl noroxymorphone * ZetaMAST - 240-microg N-allyl noroxymorphone * ZetaMAST - 480-microg N-allyl noroxymorphone #### File: Fig12\_Nonlin\_fit\_of\_Bioluminescence\_(p\_\_\_s).csv Non-linear regression of the bioluminescence values in mice inoculated with 4T1 breast cancer cells.  ##### Variables * No Treatment: * ZetaMAST - No N-allyl noroxymorphone * ZetaMAST - 30-microg N-allyl noroxymorphone * ZetaMAST - 60-microg N-allyl noroxymorphone * ZetaMAST - 120-microg N-allyl noroxymorphone * ZetaMAST - 180-microg N-allyl noroxymorphone * ZetaMAST - 240-microg N-allyl noroxymorphone * ZetaMAST - 480-microg N-allyl noroxymorphone #### File: Fig13\_2way\_ANOVA\_of\_\_\_Liver\_Weights.csv Liver weights measured in milligrams. ##### Variables * Table Analyzed * % Liver Weights #### File: Fig15\_Brown-Forsythe\_and\_Welch\_ANOVA\_tests\_of\_Histology\_Necrosis\_Volume.csv Relative necrosis volume determined using histomorphology. ##### Variables * Number of families #### File: Fig15\_Brown-Forsythe\_and\_Welch\_ANOVA\_tests\_of\_Histology\_Tumor\_Volume.csv Relative tumor volume determined using histomorphology. ##### Variables * Number of families ## Code/software N/A ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A