Longer Doubling Time Research Articles

Pilot Study of Donor-Engrafted Clonal Hematopoiesis Evolution and Clinical Outcomes in Allogeneic Hematopoietic Cell Transplantation Recipients Using a National Registry

Improved treatment options, such as reduced-intensity conditioning (RIC), enable older patients to receive potentially curative allogeneic hematopoietic cell transplantation (HCT). This progress has led to increased use of older HLA-matched sibling donors. An unintended potential risk associated with older donors is transplantation of donor cells with clonal hematopoiesis (CH) into patients. We aimed to determine the prevalence of CH in older HLA-matched sibling donors pretransplantation and to assess the clinical impact of donor-engrafted CH on HCT outcomes. This was an observational study using donor peripheral blood samples from the Center for International Blood and Marrow Transplant Research repository, linked with corresponding recipient outcomes. To explore engraftment efficiency and evolution of CH mutations following HCT, recipient follow-up samples available through the Bone Marrow Transplant Clinical Trials Network (Protocol 1202) were included. Older donors and patients (both ≥55 years) receiving first RIC HCT for myeloid malignancies were eligible. DNA from archived donor blood samples was used for targeted deep sequencing to identify CH. The associations between donor CH status and recipient outcomes, including acute graft-versus-host disease (aGVHD), chronic GVHD (cGVHD), overall survival, relapse, nonrelapse mortality, disease-free survival, composite GVHD-free and relapse-free survival, and cGVHD-free and relapse-free survival, were analyzed. A total of 299 donors were successfully sequenced to detect CH. At a variant allele frequency (VAF) ≥2%, there were 44 CH mutations in 13.7% (41 of 299) of HLA-matched sibling donors. CH mostly involved DNMT3A (n=27; 61.4%) and TET2 (n= 9; 20.5%). Post-HCT samples from 13 recipients were also sequenced, of whom 7 had CH+ donors. All of the donor CH mutations (n=7/7; 100%) were detected in recipients at day 56 or day 90 post-HCT. Overall, mutation VAFs remained relatively constant up to day 90 post-HCT (median change, .005; range, -.008 to .024). Doubling time analysis of recipient day 56 and day 90 data showed that donor-engrafted CH mutations initially expand then decrease to a stable VAF; germline mutations had longer doubling times than CH mutations. The cumulative incidence of grade II-IV aGVHD at day 100 was higher in HCT recipients with CH+ donors (37.5% versus 25.1%); however, the risk for aGVHD by donor CH status did not reach statistical significance (hazard ratio, 1.35; 95% confidence interval, .61 to 3.01; P=.47). There were no statistically significant differences in the cumulative incidence of cGVHD or any secondary outcomes by donor CH status. In subset analysis, the incidence of cGVHD was lower in recipients of grafts from DNMT3A CH+ donors versus donors without DNMT3A CH (34.4% versus 57%; P=.035). Donor cell leukemia was not reported in any donor-recipient pairs. CH in older HLA-matched sibling donors is relatively common and successfully engrafts and persists in recipients. In a homogenous population (myeloid malignancies, older donors and recipients, RICr, non-cyclophosphamide-containing GVHD prophylaxis), we did not detect a difference in cGVHD risk or other secondary outcomes by donor CH status. Subgroup analyses suggest potential differential effects by clinical characteristics and CH mutations. Larger prospective studies are needed to robustly determine which subsets of patients and CH mutations elicit meaningful impacts on clinical outcomes.

Risk Factor Analysis for Second Treatment-Free Remission Failure from the Canadian TKI Discontinuation (TRAD) Trial in CML Patients: Treatment-Free Remission Accomplished By Dasatinib

Background The Canadian TKI discontinuation (TRAD) trial (NCT02268370) has evaluated whether Dasatinib (DA) rechallenge can sustain a second treatment-free remission (TFR2) after failure of Imatinib (IM) discontinuation (DISC) for the first TFR (TFR1) attempt. We previously reported that: 1) The 12-month molecular relapse-free survival (mRFS) rate for TFR1 is 58.0%; 2) Re-challenge with DA following TFR1 failure after IM DISC restores deep molecular response (MR) quickly; 3) 12 months DA rechallenge does not significantly improve TFR2 rate (EHA 2022). The clinical risk factors associated with TRF2 failure are evaluated in this study. Patients and Methods This prospective study (BMS CA180-543, NCT#02268370) had 3 phases: 1) IM discontinuation for TFR1, 2) DA rechallenge, and 3) DA discontinuation for TFR2. Key inclusion criteria included: 1) CML in chronic phase, 2) minimum duration of IM therapy 3 years, 3) minimum duration of MR4.5 or deeper over 2 years. Molecular relapse was defined as an increase BCR::ABL qPCR >MR4 on 2 consecutive occasions, or a single increase in BCR::ABL qPCR >MR3. DA treatment was started at 100mg daily after molecular relapse was confirmed, and continued for at least 12 months after achieving ≥MR4 until TFR2 is attempted. Molecular relapse-free survival (RFS) after DA discontinuation was calculated and analyzed according to the risk factors. Results With a median follow-up duration of 27.5 months (range 1.8-51), in the TFR2 phase, 35 pts stopped DA for a TFR2. Only 3 of 35 pts (8.6%), maintained the MR at last follow-up, the remaining 32 lost the MR within a median 3.65 months. The actuarial mRFS rate at 6 and 12 months was 22.9% (95% CI, 10.8-37.6%) and 10.0% (2.7-23.1%). Potential risk factors for TFR2 from three different phases were evaluated, and 5 were found to be associated with TFR2 failure in univariate analysis (table 1): 1. In the DA rechallenge phase, failure to achieve MMR in less than 1 month with DA rechallenge increased the risk of TFR2 failure, in comparison to those who achieved MMR in less than a month (HR 4.656, p=0.0002). 2. Prior to DA DISC phase, any detectable BCR::ABL1 qPCR level between MR4 to MR5.4 showed an increased risk of TFR2 failure, compared to undetectable BCR::ABL1 qPCR level below MR5.5 (HR 4.04, p=0.0001). 3. Among the variables during the IM DISC phase: 4. Molecular relapse pattern: in comparison to the loss of MR4 only, those who lost MMR had an increased risk of TFR2 failure (HR 4.163, p= 0.001). 5. Time to loss of MR within 2.8 months after IM DISC increased the risk of TFR2 failure, in comparison who lost MR beyond 2.8 months (HR 4.717, p=0.0002). 6. Shorter doubling time (DT) at 2 months below 12.75 days and above 0 increased the risk of TFR2 failure, compared to DT at 2 months above 12.75 days or below 0 (HR 654, p=0.002) Multivariate analysis was performed to identify independent risk factors, identifying 3 clinical factors predictive of TFR2 failure: 1) Failure to achieve MMR in less than 1 month following DA rechallenge (p=0.007, HR 9.542 [1.819-50]); 2) DT below 12.75 days at 2 months after IM DISC (p=0.015, HR 4.368 [1.334-14.30]); and 3) any detectable BCR::ABL1 transcript level between MR4 and MR5.4 before DA DISC (p=0.047, HR 2.759 [1.014-7.513]). A risk score model was generated incorporating these 3 clinical risk factors. A score of 1 was assigned to each one of these risk factors. The group with a score of 0 vs 1-3 was labeled as low vs high-risk, respectively. The low-risk group (n=7) showed 85.7% and 34.3% of TFR2 rate at 6 and 12 months, while the high-risk group (n=24) showed 7.4% and 3.7% of TFR2 rate at 6 and 12 months, (p=0.0003; HR 6.518 [2.219-19.95] for high-risk group vs low risk; Figure 1). Conclusion As we have previously reported, DA treatment rarely achieves a sustained TFR2 after failing the first IM DISC attempt. However, our current result suggests that TFR2 attempt can be successful in a selected group of patients if: 1) Rapid reduction of initial molecular kinetics is achieved within 1 month of DA rechallenge, 2) an undetectable BCR::ABL1 qPCR level is reached before TFR2 attempt 3) longer doubling time at 2 months after IM DISC is noted. The proposed risk model can be used as a predictive tool for TFR2 failure. Further studies to improve TFR2 rate should investigate the addition of new therapies and consider the use of innovative tools with higher precision and sensitivity to quantify the MR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PD54-06 ACTIVE OBSERVATION OF BIOCHEMICAL RECURRENCE WITHOUT TREATMENT FOLLOWING RADICAL PROSTATECTOMY: LONG TERM ANALYSIS OF OUTCOMES

You have accessJournal of UrologyCME1 May 2022PD54-06 ACTIVE OBSERVATION OF BIOCHEMICAL RECURRENCE WITHOUT TREATMENT FOLLOWING RADICAL PROSTATECTOMY: LONG TERM ANALYSIS OF OUTCOMES Erica Huang, Linda My Huynh, Adam Gordon, Ryan Chandhoke, Blanca Morales, Douglas Skarecky, Joshua Tran, and Thomas Ahlering Erica HuangErica Huang More articles by this author , Linda My HuynhLinda My Huynh More articles by this author , Adam GordonAdam Gordon More articles by this author , Ryan ChandhokeRyan Chandhoke More articles by this author , Blanca MoralesBlanca Morales More articles by this author , Douglas SkareckyDouglas Skarecky More articles by this author , Joshua TranJoshua Tran More articles by this author , and Thomas AhleringThomas Ahlering More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002631.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Biochemical recurrence (BCR) following radical prostatectomy (RP) is an unreliable predictor of metastatic progression or prostate cancer death, potentially resulting in overtreatment. Little has been published on identification and observation of men without treatment via radiation (RT) and/or androgen deprivation (ADT). The present study seeks to characterize findings of men managed using PSA doubling time (DT) kinetics to direct active observation (AO). METHODS: A retrospective cohort analysis of 1864 men undergoing RP between June 2002 and September 2019 was conducted. BCR (PSA >0.2 ng/dl, x2) patients were assessed for treatment intervention (RT and/or ADT) versus observation with DT kinetics. Initial risk was determined as a DT <12 month. Further assessment was based on DT change over time. Patients with rapidly decreasing DT were treated; increasing or slowly decreasing DT were observed without treatment. RESULTS: Median follow-up was 7.3 years (IQR 3.9-10.7). 407/1864 (21.8%) experienced BCR. 136/407 (33.4%) were observed without RT and/or ADT, at 0% PCSM and average current DT 26.0±19.9 months. Patient with lower pGGG’s had longer DT’s (p=0.02). 93/136 (68.4%) patients had increasing DT’s at last follow-up, while patients with decreasing DT’s (37/136, 27.2%) were characterized by long DT’s (mean 19.36 mos, calculated from first 4 PSA’s post-RP) and slowly decreasing DT’s (Table 1).Presently, 69 (50%) have been actively observed for 7.3-18 years, with no PCSM. All PCSM’s occurred in higher risk patients who were treated (Figure 1, p <0.001). CONCLUSIONS: This suggests that a significant portion (33.4%) of men develop a benign BCR post-RARP without need for RT and/or ADT, with 0% PCSM (median 7.3 years). These men are characterized by long DT’s and increasing or slowly decreasing DT kinetics. Source of Funding: N/A © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e917 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Erica Huang More articles by this author Linda My Huynh More articles by this author Adam Gordon More articles by this author Ryan Chandhoke More articles by this author Blanca Morales More articles by this author Douglas Skarecky More articles by this author Joshua Tran More articles by this author Thomas Ahlering More articles by this author Expand All Advertisement PDF DownloadLoading ...

Characterization of SOX2, OCT4 and NANOG in Ovarian Cancer Tumor-Initiating Cells.

Simple SummarySOX2, OCT4, and NANOG are enriched in ovarian cancer spheroids and correlate with tumor-initiating cell markers, CD117 and ALDH/CD133. SOX2, relative to OCT4 or NANOG, is a stronger indictor of chemoresistance, tumor-initiation, and recurrent disease. Including SOX2 in evaluation of ovarian cancer TICs may improve reliability of TIC markers and our understanding of mechanisms of relapse.The identification of tumor-initiating cells (TICs) has traditionally relied on surface markers including CD133, CD44, CD117, and the aldehyde dehydrogenase (ALDH) enzyme, which have diverse expression across samples. A more reliable indication of TICs may include the expression of embryonic transcription factors that support long-term self-renewal, multipotency, and quiescence. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and may indicate TICs with high relapse potential. We evaluated a panel of eight ovarian cancer cell lines grown in standard 2-D culture or in spheroid-enriching 3-D culture, and correlated expression with growth characteristics, TIC marker expression, and chemotherapy resistance. RNA-sequencing showed that cell cycle regulation pathways involving SOX2 were elevated in 3-D conditions. HGSOC lines had longer doubling-times, greater chemoresistance, and significantly increased expression of SOX2, OCT4, and NANOG in 3-D conditions. CD117+ or ALDH+/CD133+ cells had increased SOX2, OCT4, and NANOG expression. Limiting dilution in in vivo experiments implicated SOX2, but not OCT4 or NANOG, with early tumor-initiation. An analysis of patient data suggested a stronger role for SOX2, relative to OCT4 or NANOG, for tumor relapse potential. Overall, our findings suggest that SOX2 may be a more consistent indicator of ovarian TICs that contribute to tumor repopulation following chemotherapy. Future studies evaluating SOX2 in TIC biology will increase our understanding of the mechanisms that drive ovarian cancer relapse.

Life Table Parameters of the Tomato Leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), on Three Solanaceous Host Plants

The larvae of Tuta absoluta are harmful leafminers that attacks different solanaceous plants such as tomato, potato, colour pepper, eggplant and many others. The life tables of a population are important to control pests because they give the most comprehensive description of the growth, survival and fecundity. Life tables of T. absoluta reared on tomato, potato and eggplant in the laboratory were determined and analysed using the age-stage, two-sex life tables. Developmental periods of eggs, larvae, pupae and adults, and pre-oviposition and oviposition periods of T. absoluta fed as larvae on tomato, potato or eggplant were estimated. Fecundity of a female fed on the same three hosts was 198.3, 172.2 and 133.7 eggs/female, respectively. Life table parameters such as the net reproductive rates (R0) (92.25, 76.25 and 67.82 days), the mean generation time (T0) (31.96, 36.06 and 36.94 days), the intrinsic rate of increase (rm) (0.142, 0.117 and 0.113/day) and the finite population increase (λ) (1.15, 1.12 and 1.12/day) were obtained for T. absoluta fed on tomato, potato and eggplant, respectively. The shortest doubling time (DT) was obtained with tomato leaves (4.9 ± 0.06 days), while the longest DT was obtained with eggplant leaves (6.30 ± 0.08 days). Based on the results herein, it appears that the development and reproductive potentiality of T. absoluta depends on its host plant; this pest can therefore be more dangerous to its suitable host (tomato) than other hosts.

Effect of a Point Mutation in mprF on Susceptibility to Daptomycin, Vancomycin, and Oxacillin in an MRSA Clinical Strain.

We previously reported the sequential recovery of daptomycin-nonsusceptible MRSA clinical isolates with an L431F substitution in the MprF protein. The aim of the present study is to determine the effect of this mutation by replacing the mprF gene on the chromosome of a daptomycin-susceptible progenitor strain, CGK5, to obtain CGK5mut having the L431F MprF mutation. Compared to CGK5, the daptomycin and vancomycin MICs of CGK5mut increased from 0.5 to 3 μg/ml and from 1.5 to 3 μg/ml, respectively; however, its oxacillin MIC decreased from 128 to 1 μg/ml in medium without added 2% NaCl. The expression levels of vraSR and several other cell-wall synthesis-related genes were significantly increased in CGK5mut, and the mutant also had significantly reduced negative cell membrane charge, thicker cell wall, and longer doubling time. These features were abolished in the reverse mutant carrying F431L MprF, confirming the pleiotropic effects of the L431F MprF mutation. We believe that this is the first work that shows a single MprF missense mutation can lead to not only changes in the cell membrane but also increased expression of vraSR and subsequently increased resistance to daptomycin and vancomycin while simultaneously conferring increased susceptibility to oxacillin in an isogenic MRSA strain.

Outcomes and Prognostic Factors in Men Receiving Androgen Deprivation Therapy for Prostate Cancer Recurrence after Radical Prostatectomy

We sought to determine clinicopathological factors associated with early progression in men on androgen deprivation therapy as well as cancer specific and overall survival. We also assessed whether certain prostate specific antigen thresholds at androgen deprivation therapy initiation are associated with poorer outcomes. We identified 2,418 men with rising prostate specific antigen after undergoing radical prostatectomy at a single institution between 1987 and 2007 in a prospectively maintained registry. Early progression was defined as clinical progression within 2 years of initiating androgen deprivation therapy. The primary study outcome was cancer specific and overall survival. The risk of early progression while on androgen deprivation therapy was lower for prostate specific antigen doubling time 3 to less than 9 months (OR 0.19) and less than 9 months or longer (OR 0.10, each p <0.001) prior to androgen deprivation therapy. Independent predictors of cancer specific survival were metastatic disease at androgen deprivation therapy initiation (HR 2.60), prostate specific antigen 5 to 50 ng/ml (HR 2.68) and 50 ng/ml or greater (HR 4.33), and doubling time 3 to less than 9 months (HR 0.54) and 9 months or longer (HR 0.45, all p<0.001). Independent predictors of overall survival were prostate specific antigen 5 to 50 ng/ml (HR 3.10) and 50 ng/ml or greater (HR 5.20, each p <0.001). In men in whom androgen deprivation therapy was initiated for relapse after radical prostatectomy prostate specific antigen doubling time less than 3 months and prostate specific antigen 5 ng/ml or greater were adverse prognostic factors for early progression and cancer specific survival. Prostate specific antigen 5 ng/ml or greater also predicted shorter overall survival. Longer doubling time and prostate specific antigen less than 5 ng/ml were associated with lower risk and these men may not require immediate androgen deprivation therapy.

Prognostic factors in men receiving androgen deprivation therapy (ADT) for recurrent prostate cancer: Using absolute PSA and PSA doubling time (DT) to guide timing of ADT initiation.

129 Background: ADT is the first-line treatment for men experiencing recurrence after undergoing radical therapy for prostate cancer. However, timing of ADT initiation is controversial and there are limited data on prognostic factors in patients starting ADT. Methods: We identified consecutive men who underwent radical prostatectomy (RP) for localized prostate cancer at our institution between 1987 and 2007 and who subsequently received salvage ADT. Early progression on ADT was defined as development of metastatic disease within 2yrs of initiation. The primary outcomes of interest were cancer-specific (CSS) and overall survival (OS). Results: A total of 2418 men were included. Median age at RP was 64yrs and median follow-up was 13.9yrs. 48% and 20% of men had pathologic Gleason scores of 7 and 8-10 respectively. The median PSA was 2.6ng/ml, while 385 men (16%) had metastatic disease at receipt of ADT. Overall, 1060 men (44%) developed clinical metastases, with 625 (59%) of these doing so within 2yrs of starting ADT. On multivariable analysis, longer PSA DT before ADT was associated with lower odds of early progression on ADT (DT 3-9mths, OR = 0.19; DT ≥9mths, OR = 0.10, both p &lt; 0.001). 10- and 20-year CSS were 89% and 70%, and 10- and 20-year OS were 82% and 40% respectively. Independent predictors of lower CSS included metastatic disease at time of ADT (HR = 2.60), PSA at ADT of 5-50ng/ml (HR = 2.68) and &gt; 50ng/ml (HR = 4.33, all p &lt; 0.001), while longer PSA DT was associated with higher CSS (DT 3-9mths, HR = 0.54; DT ≥9mths, HR = 0.45, both p &lt; 0.001). PSA at ADT of 5-50ng/ml (HR = 3.10) and &gt; 50ng/ml (HR = 5.20, both p &lt; 0.001) were independent predictors of OS. Conclusions: PSA DT &lt; 3mths and absolute PSA at ADT initiation of ≥ 5ng/ml are adverse prognostic indicators in men receiving salvage ADT for relapse after RP. For patients with these features, their risk of early progression and death should be part of a discussion about the timing of ADT and consideration given to more aggressive treatment strategies. Conversely, men with biochemical relapse who have longer DT and PSA &lt; 5ng/ml are at lower risk and could make an informed decision to defer ADT initiation.

Comparison of NheA toxin production and doubling time between Bacillus cereus and Bacillus thuringiensis

In this study, we compared the toxin gene expression, NheA toxin production, doubling time, and viable cell number for several strains of the food poisoning bacteria Bacillus cereus and the microbial pesticide Bacillus thuringiensis. The two B. cereus and six B. thuringiensis strains evaluated were confirmed to possess and transcribe the nheABC, hblCDA, and cytK genes using polymerase chain reaction (PCR) and reverse-transcription PCR. NheA toxin production was compared based on the absorbance at 414 nm using a Tecra BDE-VIA kit. The NheA-specific production (absorbance/viable cell number) values indicated that the two B. thuringiensis var. kurstaki isolates from microbial pesticide produced the highest amount of toxin (0.66–0.95) than other B. thuringiensis (0.14–0.45) and the B. cereus strains (0.19–0.31). However, the B. thuringiensis strains had longer doubling time (20–26 min) than the B. cereus strains (18–19 min). Interestingly, two B. thuringiensis var. kurstaki isolates produced the highest amount of NheA toxin, and their doubling times (20–22 min) were close to those of the B. cereus strains tested.

Life-table study of mango leaf cutting weevil, Deporaus marginatus Pascoe (Coleoptera: Curculionidae) feeding on four mango cultivars

Mango leaf cutting weevil, Deporaus marginatus Pascoe (Coleoptera: Curculionidae) is a devastating pest of freshly emerged mango foliage. The immature development, survivorship and adult reproduction of the weevil were investigated on four popular mango cultivars (Langra, Amrapali, Fazli and Ashwina) under the laboratory conditions and life-table parameters were estimated. The immature developmental periods ranged from 17.3 to 19.8days with female ratio of 5.5–6.2. Ault longevity ranged 43.6days for female and 66.2days for males. Effects of the host plant varieties were not significant on the immature development or sex ratio, but significantly influenced the adult performances; longevity, reproduction and also life table parameters. Among the mango varieties tested, Ashwina variety could be the most preferable with higher population growth rate while the Langra and Fazli were the lesser suitable with longer doubling time or lower reproduction. Further study could pursue the nutritional and antibiotic mechanisms which could be responsible for the different performance of the herbivore insect on the mango plants.

INFLUENCE OF INITIAL CELL CONCENTRATIONS ON THE GROWTH RATE AND BIOMASS PRODUCTIVITY OF MICROALGAE IN DOMESTIC WASTEWATER

The aim of this study was to compare the specific growth rate and biomass productivity of microalgae in domestic wastewater according to the initial cell concentration. The initial microalgae cell concentrations tested started from 103 cell/mL, 104 cell/mL, 105 cell/mL, 106 cell/mL, and 107 cell/mL under outdoor condition. The result revealed that the highest biomass productivity occurred at 106 cell/mL concentration with a value of 1.24 × 104 cell/mL/day, 0.26 day-1 of specific growth rate, and a doubling time of 2.63 days. Meanwhile, the lowest biomass productivity occurred at 103 cell/mL concentration with the lowest specific growth rate of 0.1 day-1 and the longest doubling time, which reached up to 7.14 day. As a result, the initial cell concentration of microalgae did influence the algal biomass productivity and growth rate differently. Thus, the maximum growth rate and biomass productivity were obtained at 106 cell/mL concentration which is recommended to be used in biotechnology industries and any wastewater treatment.

Abstract 4067: Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines

Abstract Background: Chemoresistance is the major obstacle for the success of cancer treatment. The advanced breast cancer (BC) is commonly pan-resistant to different anticancer drugs. BC contains a small population of cancer stem cells (CSCs). CSCs express stem cell markers (CD24low/CD44high, ALDH+, CD133+) and are resistant to a wide range of anticancer drugs. Therefore, CSCs may be responsible for the pan-chemoresistance in advanced BC. We have recently developed several acquired chemoresistant BC cell lines. In this study, we investigated CSC traits in these cell lines and examined the cross-resistance of these cell lines to a panel of anticancer drugs. Disulfiram (DS), an antialcoholism drug, shows high cytotoxicity in several different types of cancer cell lines in vitro. It also targets CSCs and reverses pan-chemoresistance. However, the clinical application of DS in cancer treatment is limited by its very short half-life in the bloodstream. In order to increase the bio-stability of DS in the bloodstream, we recently developed a pluronic micelle-encapsulated DS (PM-DS). In this study, we examined the cytotoxicity of a newly developed (PM-DS) in the resistant cell lines. The effect of PM-DS on CSC traits in the resistant BC cell lines was also examined. Cell lines and Methods: Cell lines: MDA-MB-231, MCF7 and T47D and acquired resistant BC cell lines [gemcitabine (MDA-MB-231GEM100), 5-fluouracil (MCF7FU10) and doxorubicin (T47DDOX100nM)]; Methodologies: MTT assay, Western blot, CSC markers detection (ALDH, CD24low/CD44high, CD133), embryonic stem cell markers detection (Nanog, Sox2, Oct 4). Results: The resistant cell lines are highly cross-resistant to 6 conventional anticancer drugs e.g doxorubicin, gemcitabine, docetaxel, cisplatin, 5-fluouracil and vincristine. In comparison with the parental cells, the resistant cell lines have significantly longer doubling time. Significantly higher population of resistant cells expresses high levels of CSC markers e.g. ALDH+, CD24low/CD44high, and CD133+ and embryonic stem cell markers (OCT4, Sox2 and Nanog). DS was successfully encapsulated in pluronic micelles with a high entrapment efficiency of about 90% and our in vitro release studies showed that DS was slowly released from the micelles. Our formulation PM-DS has a relatively small size of about 200nm with high drug loading content (90μgDS/1mgPM). PM-DS was highly toxic to chemoresistant cell lines. It also targeted CSC population and enhanced cytotoxicity of anticancer drugs in the resistant cell lines. Conclusions: The acquired resistant BC cell lines are highly cross-resistant to 6 different kinds of anticancer drugs. CSCs may be responsible for the cross-chemoresistance. PM-DS targets CSC population and reverses the acquired pan-chemoresistance in acquired resistant BC cell lines. Key words: Disulfiram, micelles, breast cancer, stem-like cells. Citation Format: EREBI P. TAWARI, Peng Liu, Zhipeng Wang, Vinodh Kannappan, Christopher Mcconville, Angel Armesilla, John Darling, Juan Irache, Krassimira Yoncheva, Weiguang Wang, Petar Petrov. Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2015-4067

PTH-104 Tumour doubling time of colorectal metastases in patients selected for liver resection does not influence post-operative disease-free survival

Introduction The aim of this study was to assess the potential association between the change in diameter of colorectal liver metastases between pre-operative imaging and liver resection and disease-free survival in patients who do not receive pre-operative liver-directed chemotherapy. Method Analysis of a prospectively maintained database of patients undergoing liver resection for colorectal liver metastases between 2005 and 2012 was undertaken. Change in tumour size was assessed by comparing the maximum tumour diameter at radiological diagnosis determined by imaging and the maximum tumour diameter measured at examination of the resected specimen in 157 patients. Results The median interval from first scan to surgery was 99 days and the median increase in tumour diameter in this interval was 38%, equivalent to a tumour doubling time (DT) of 47 days. Tumour DT prior to liver resection was longer in patients with T1 primary tumours (119 days) than T2–4 tumours (44 days) and shorter in patients undergoing repeat surgery for intra-hepatic recurrence (33 days) than before primary resection (49 days). The median disease-free survival of the quartile of patients with the shortest DT (1.77 years) was similar to that of the quartile with the longest DT (1.77 years) (P = 0.760) and multivariate analysis revealed no association between tumour DT and disease-free survival. Conclusion The rate of growth of colorectal liver metastases prior to surgery should not be used as a prognostic factor when counselling patients prior to resection. Disclosure of interest None Declared.

Human plasma and human platelet-rich plasma as a substitute for fetal calf serum during long-term cultivation of mesenchymal dental pulp stem cells.

Our aims were to isolate and cultivate mesenchymal dental pulp stem cells (DPSC) in various media enriched with human blood components, and subsequently to investigate their basic biological properties. DPSC were cultivated in five different media based on α MEM containing different concentrations of human plasma (HP), platelet-rich plasma (PRP), or fetal calf serum (FCS). The DPSC biological properties were examined periodically. We cultivated DPSC in the various cultivation media over 15 population doublings except for the medium supplemented with 10% HP. Our results showed that DPSC cultivated in medium supplemented with 10% PRP showed the shortest average population doubling time (DT) (28.6 ± 4.6 hours), in contrast to DPSC cultivated in 10% HP which indicated the longest DT (156.2 ± 17.8 hours); hence this part of the experiment had been cancelled in the 6th passage. DPSC cultivated in media with 2% FCS+ITS (DT 47.3 ± 10.4 hours), 2% PRP (DT 40.1 ± 5.7 hours) and 2% HP (DT 49.0 ± 15.2 hours) showed almost the same proliferative activity. DPSC's viability in the 9th passage was over 90% except for the DPSC cultivated in the 10% HP media. We proved that human blood components are suitable substitution for FCS in cultivation media for long-term DPSC cultivation.

The effects of chemical interactions and culture history on the colonization of structured habitats by competing bacterial populations

BackgroundBacterial habitats, such as soil and the gut, are structured at the micrometer scale. Important aspects of microbial life in such spatial ecosystems are migration and colonization. Here we explore the colonization of a structured ecosystem by two neutrally labeled strains of Escherichia coli. Using time-lapse microscopy we studied the colonization of one-dimensional arrays of habitat patches linked by connectors, which were invaded by the two E. coli strains from opposite sides.ResultsThe two strains colonize a habitat from opposite sides by a series of traveling waves followed by an expansion front. When population waves collide, they branch into a continuing traveling wave, a reflected wave and a stationary population. When the two strains invade the landscape from opposite sides, they remain segregated in space and often one population will displace the other from most of the habitat. However, when the strains are co-cultured before entering the habitats, they colonize the habitat together and do not separate spatially. Using physically separated, but diffusionally coupled, habitats we show that colonization waves and expansion fronts interact trough diffusible molecules, and not by direct competition for space. Furthermore, we found that colonization outcome is influenced by a culture’s history, as the culture with the longest doubling time in bulk conditions tends to take over the largest fraction of the habitat. Finally, we observed that population distributions in parallel habitats located on the same device and inoculated with cells from the same overnight culture are significantly more similar to each other than to patterns in identical habitats located on different devices inoculated with cells from different overnight cultures, even tough all cultures were started from the same −80°C frozen stock.ConclusionsWe found that the colonization of spatially structure habitats by two interacting populations can lead to the formation of complex, but reproducible, spatiotemporal patterns. Furthermore, we showed that chemical interactions between two populations cause them to remain spatially segregated while they compete for habitat space. Finally, we observed that growth properties in bulk conditions correlate with the outcome of habitat colonization. Together, our data show the crucial roles of chemical interactions between populations and a culture’s history in determining the outcome of habitat colonization.

Nicotine exposure during differentiation causes inhibition of N-myc expression

BackgroundThe ability of chemicals to disrupt neonatal development can be studied using embryonic stem cells (ESC). One such chemical is nicotine. Prenatal nicotine exposure is known to affect postnatal lung function, although the mechanisms by which it has this effect are not clear. Since fibroblasts are a critical component of the developing lung, providing structure and secreting paracrine factors that are essential to epithelialization, this study focuses on the differentiation of ESC into fibroblasts using a directed differentiation protocol.MethodsFibroblasts obtained from non-human primate ESC (nhpESC) differentiation were analyzed by immunohistochemistry, immunostaining, Affymetrix gene expression array, qPCR, and immunoblotting.ResultsResults of these analyses demonstrated that although nhpESCs differentiate into fibroblasts in the presence of nicotine and appear normal by some measures, including H&E and SMA staining, they have an altered gene expression profile. Network analysis of expression changes demonstrated an over-representation of cell-cycle related genes with downregulation of N-myc as a central regulator in the pathway. Further investigation demonstrated that cells differentiated in the presence of nicotine had decreased N-myc mRNA and protein expression and longer doubling times, a biological effect consistent with downregulation of N-myc.ConclusionsThis study is the first to use primate ESC to demonstrate that nicotine can affect cellular differentiation from pluripotency into fibroblasts, and in particular, mediate N-myc expression in differentiating ESCs. Given the crucial role of fibroblasts throughout the body, this has important implications for the effect of cigarette smoke exposure on human development not only in the lung, but in organogenesis in general.

The mechanisms of surface chemistry effects of mesoporous silicon nanoparticles on immunotoxicity and biocompatibility

Despite steadily increasing insights on the biocompatibility of PSi nanoparticles (NPs), an extensive biosafety study on the immune and red blood cells (RBCs) is still lacking. Herein, we evaluated the impact of the PSi NPs' surface chemistry on immune cells and human RBCs both in vitro and in vivo. Negatively charged hydrophilic and hydrophobic PSi NPs caused less ATP depletion and genotoxicity than the positively charged amine modified hydrophilic PSi NPs, demonstrating the main role of PSi NPs' surface charge on the immunocompatibility profile. Furthermore, cells with lower metabolic activity, longer doubling time, and shorter half-life were more sensitive to the concentration- and time-dependent toxicity in the following order: T-cells ≈ monocytes > macrophages ≈ B-cells. RBC hemolysis and imaging assay revealed a significant correlation between the surface chemistry, the amount of the PSi NPs adsorbed on the cell surface and the extent of morphological changes. The in vivo results showed that despite mild renal steatosis, glomerular degeneration, hepatic central vein dilation and white pulp shrinkage in spleen, no notable changes were observed in the serum level of biochemical and hematological factors. This study is a comprehensive demonstration of the mechanistic direct and indirect genotoxicity effects of PSi NPs, elucidating the most influencing properties for the PSi NPs' design.

The Role of Host Gender in the Pathogenesis of Cryptococcus neoformans Infections

Cryptococcus neoformans (Cn) is a pathogenic yeast and the cause of cryptococcal meningitis. Prevalence of disease between males and females is skewed, with males having an increased incidence of disease. Based on the reported gender susceptibility differences to Cn in the literature, we used clinical isolates from Botswanan HIV-infected patients to test the hypothesis that different gender environments exerted different selective pressures on Cn. When we examined this data set, we found that men had significantly higher risk of death despite having significantly higher CD4+ T lymphocyte counts upon admittance to the hospital. These observations suggested that Cn strains are uniquely adapted to different host gender environments and that the male immune response may be less efficient in controlling Cn infection. To discriminate between these possibilities, we tested whether there were phenotypic differences between strains isolated from males and females and whether there was an interaction between Cn and the host immune response. Virulence phenotypes showed that Cn isolates from females had longer doubling times and released more capsular glucoronoxylomannan (GXM). The presence of testosterone but not 17-β estradiol was associated with higher levels of GXM release for a laboratory strain and 28 clinical isolates. We also measured phagocytic efficiency, survival of Cn, and amount of killing of human macrophages by Cn after incubation with four isolates. While macrophages from females phagocytosed more Cn than macrophages from males, male macrophages had a higher fungal burden and showed increased killing by Cn. These data are consistent with the hypothesis that differential interaction between Cn and macrophages within different gender environments contribute to the increased prevalence of cryptococcosis in males. This could be related to differential expression of cryptococcal virulence genes and capsule metabolism, changes in Cn phagocytosis and increased death of Cn-infected macrophages.

A “Twist box” Code of p53 Inactivation: Twist box:p53 Interaction Promotes p53 Degradation

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates itsMDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

Abstract 2032: A novel function of WW domain binding protein 2 (WBP2) in regulating cytoskeletal function and cellular division through binding to co-chaperone BAG3

Abstract BAG3, a BAG co-chaperone family member, co-localizes with actin microfilaments and influences processes that involve actin function, including adhesion and migration. Because actin function regulates cell division, we hypothesized that the BAG3 WW-domain regulates cellular division through its cytoskeletal association. We show that BAG3 co-localizes with leading lamellipodia exclusively during G2 and mitosis phases. Co-immunoprecipitation of BAG3 and pulldown of GST-tagged BAG3 WW-domain confirmed that BAG3 WW-domain association with actin increased during G2 and mitosis relative to asynchronous cells. Deletion of the BAG3 WW-domain caused an abnormal cellular morphology, degenerate F-actin organization, longer doubling time (p&amp;lt;0.05, n=4), and three-fold increase in polynucleated cells relative to empty vector and full length-BAG3 (FL-BAG3; p&amp;lt;0.001, n=3). We confirmed polyploidy development and absence of cytokinesis in αWW-BAG3 cells by live-cell imaging, and observed mitotic delay relative to control and FL-BAG3 cells. To identify a potential regulation mechanism for BAG3-actin binding, we identified WBP2 as a novel BAG3 WW domain interacting protein in a yeast two-hybrid assay. BAG3 and WBP2 co-immunoprecipitated only in G1 and S phases, indicating cell cycle-specific BAG3-WBP2 binding. WBP2 silencing caused indiscriminate BAG3 co-immunoprecipitation with actin across all cell cycle phases, suggesting that association with WBP2 prevents BAG3-actin binding. We are the first to identify a function for WBP2, which our data show regulates BAG3 WW-domain association with actin in a cell cycle-specific manner. Because actin dysfunction has been proposed to contribute to chromosomal instability and tumor formation, the WW-domain of BAG3 is a potential target to reduce abnormal cell division. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2032. doi:1538-7445.AM2012-2032