Tumor Size Progression Research Articles

Preclinical studies of toxicity and anti-cholangiocarcinoma activity of the standardized capsule formulation of Atractylodes lancea (Thunb.) DC

BackgroundCholangiocarcinoma (CCA), the adenocarcinoma of the biliary duct, is commonly reported in Asia, with the highest incidence in northeastern Thailand. Chemotherapy of CCA has been limited by the lack of effective chemotherapeutic drugs. A series of previous in vitro and in vivo studies support further research and development of Atractylodes lancea (Thunb.) DC. (AL) as a potential candidate for treating CCA as a crude ethanolic extract. In the present study, we evaluated the toxicity and anti-CCA activity of the CMC (Chemistry, Manufacturing, and Control) capsule formulation of the ethanolic rhizome extract of AL (CMC-AL) in animals.MethodsMajor steps included acute, subchronic and chronic toxicity testing in Wistar rats and anti-CCA activity in a CCA-xenografted nude mouse model. The safety of CMC-AL was determined based on the maximum tolerated dose (MTD) and no-observed-adverse-effect level (NOAEL) according to the OECD guideline. The anti-CCA activity of CMC-AL in nude mice was evaluated after transplantation of CL-6 cells to evaluate inhibitory effects on tumor size progression and metastasis and survival time prolongation. Safety assessments included hematology, biochemistry parameters and histopathological examination. Lung metastasis was investigated using VEGF ELISA kit.ResultsAll evaluations confirmed satisfactory pharmaceutical properties of oral formulation and safety profile of the CMC-AL with no overt toxicity up to the MTD and NOAEL of 5,000 and 3,000 mg/kg body weight, respectively. CMC-AL exhibited potent anti-CCA efficacy with regard to inhibitory activity on tumor progression and lung metastasis.ConclusionsCMC-AL is safe and should be further investigated in a clinical trial as a potential therapy for CCA patients.

Targeting β-catenin using XAV939 nanoparticle promotes immunogenic cell death and suppresses conjunctival melanoma progression

Many tumors dysregulate Wnt/β-catenin pathway to promote stem-cell-like phenotype, tumorigenesis, immunosuppression, and resistance to targeted cancer immunotherapies. Therefore, targeting this pathway is a promising therapeutic approach to suppress tumor progression and elicit robust anti-tumor immunity. In this study, using a nanoparticle formulation for XAV939 (XAV-Np), a tankyrase inhibitor that promotes β-catenin degradation, we investigated the effect of β-catenin inhibition on melanoma cell viability, migration, and tumor progression using a mouse model of conjunctival melanoma. XAV-Nps were uniform and displayed near-spherical morphology with size stability for upto 5 days. We show that XAV-Np treatment of mouse melanoma cells significantly suppresses cell viability, tumor cell migration, and tumor spheroid formation compared to control nanoparticle (Con-Np) or free XAV939-treated groups. Further, we demonstrate that XAV-Np promotes immunogenic cell death (ICD) of tumor cells with a significant extracellular release or expression of ICD molecules, including high mobility group box 1 protein (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP). Finally, we show that local intra-tumoral delivery of XAV-Nps during conjunctival melanoma progression significantly suppresses tumor size and conjunctival melanoma progression compared to Con-Nps-treated animals. Collectively, our data suggest that selective inhibition of β-catenin in tumor cells using nanoparticle-based targeted delivery represents a novel approach to suppress tumor progression through increased tumor cell ICD.

Correlation Based on the WHO Grading with Tumor Control and Clinical Outcome Following Gamma Knife Radiosurgery in Meningiomas.

The aim of this study was to study the effect of Gamma Knife (GK) on meningiomas based on the World Health Organization's (WHO) grading system in terms of tumor control and final clinical outcome. This retrospective study included clinicoradiological and GK characteristics of patients who had undergone GK for meningiomas at our institute from April 1997 until December 2009. Of 440 patients, 235 underwent secondary GK for residual/recurrent lesion and 205 received primary GK. Of the 137 patients whose biopsy slide could be reviewed, 111 patients had grade I, 16 had grade II, and 10 had grade III meningiomas. Good tumor control rates were seen in 96.3% of grade I meningioma patients, 62.5% of 16 grade II, and 10% of grade III meningioma patients at median follow-up of 40 months. Age, sex, Simpson's grade of excision, and increasing peripheral dose of GK did not affect the response to radiosurgery (P > 0.05). Multivariate analysis showed that high-grade tumor and radiotherapy prior to GK were important negative predictors for progression of tumor size after GK radiosurgery (GKRS) (P < 0.05). In patients with WHO grade I meningioma, radiation therapy prior to GKRS and repeat surgery were predictors for poorer outcome. In WHO grades II and III meningiomas, no factors affected tumor control except the histology itself.

Malignant Peripheral Nerve Sheath Scalp Tumor: A Short-Term Institutional Experience with Literature Review.

Malignant peripheral nerve sheath tumor (MPNST) of the scalp is rare. These lesions are associated with neurofibromatosis type 1 (NF1), but patients had been reported without NF1 also. We tried to analyze the difference between the clinical course and outcome of the patient with MPNST having stigmata of NF1 and without it. We included five patients treated over 3 years between July 2018 and July 2021 with diffuse scalp MPNST. Two of these five patients with MPNST of the scalp had neurocutaneous stigmata of NF1. Three were female and two males with an average age of 38.40 ± 18.48 years-the youngest with NF1 being a 19-year-old female. We found dull aching pain as the most typical complaint in all patients and a repeated episode of generalized seizure in one patient. In these cases, two patients with NF1 have highly vascular tumors and attained large sizes greater than 30 cm. These two cases required preoperative digital subtraction angiography (DSA) and embolization with n-butyl acrylate. Total excision of the tumor was done in all patients with radiotherapy. Metastases within 1 year were noted in two patients with NF1, and one of these two succumbed to her illness. The rest of the three patients without NF1 are under follow-up with no evidence of disease with a maximum follow-up of 2 years. Large MPNST (size > 20 cm) are rare and reported to have been associated with and without NF1. Patients with scalp MPNST with NF1 can achieve larger size with fast progression of tumor size and higher chances of recurrence and metastases.

EV-miRome-wide profiling uncovers miR-320c for detecting metastatic colorectal cancer and monitoring the therapeutic response

PurposeMolecular composition of circulating small extracellular vesicles (EVs) does not merely reflect the cells of origin, but also is enriched in specific biomolecules directly associated with the cellular transformation. However, while most of the currently identified EV-miRs are only geared towards one-dimensional disease detection, their application for long-term tracking and treatment response monitoring has been largely elusive.MethodsWe established and optimized a rapid, sensitive and robust liquid biopsy sampling method, and further used small RNA sequencing to comprehensively catalogue EV-miRomes in association with the progression and outcome of metastatic colorectal cancer (mCRC).ResultsBy cross-comparison of EV-miRomes (n = 290) from multi-stage and longitudinal cohorts, we uncovered a 15-EV-miR signature with dual detection and long-term monitoring of tumor size progression for mCRC. From this panel, EV-miR-320c was uncovered as a strong clinical marker – aside from its diagnostic power and a therapeutic monitoring performance superior to carcinoembryonic antigen (CEA), its high expression has also been linked to lower overall survival and a greater likelihood of disease recurrence. Further, integrative analyses of tissue transcriptomic and liquid biopsy implicated this 15-EV-miR signature in programming the mesenchymal–epithelial transition (MET) for distant localization of the metastasized cells and also in creating a tumor-favoring metastatic niche.ConclusionOur clinically-oriented delineation of the mCRC-associated circulating EV-miRomes systematically revealed the functional significance of these liquid biopsy markers and further strengthen their translational potential in mCRC therapeutic monitoring.Graphical abstract

Exposure\u2013response analyses of cabozantinib in patients with metastatic renal cell cancer

AimIn the registration trial, cabozantinib exposure ≥ 750 ng/mL correlated to improved tumor size reduction, response rate and progression free survival (PFS) in patients with metastatic renal cell cancer (mRCC). Because patients in routine care often differ from patients in clinical trials, we explored the cabozantinib exposure–response relationship in patients with mRCC treated in routine care.MethodsCabozantinib trough concentrations (Cmin) were collected and average exposure was calculated per individual. Exposure–response analyses were performed using the earlier identified target of Cmin > 750 ng/mL and median Cmin. In addition, the effect of dose reductions on response was explored. PFS was used as measure of response.ResultsIn total, 59 patients were included:10% were classified as favourable, 61% as intermediate and 29% as poor IMDC risk group, respectively. Median number of prior treatment lines was 2 (0–5). Starting dose was 60 mg in 46%, 40 mg in 42% and 20 mg in 12% of patients. Dose reductions were needed in 58% of patients. Median Cmin was 572 ng/mL (IQR: 496–701). Only 17% of patients had an average Cmin ≥ 750 ng/mL. Median PFS was 52 weeks (95% CI: 40–64). No improved PFS was observed for patients with Cmin ≥ 750 ng/mL or ≥ 572 ng/ml. A longer PFS was observed for patients with a dose reduction vs. those without (65 vs. 31 weeks, p = .001). After incorporating known covariates (IMDC risk group and prior treatment lines (< 2 vs. ≥ 2)) in the multivariable analysis, the need for dose reduction remained significantly associated with improved PFS (HR 0.32, 95% CI:0.14–0.70, p = .004).ConclusionIn these explorative analyses, no clear relationship between increased cabozantinib exposure and improved PFS was observed. Average cabozantinib exposure was below the previously proposed target in 83% of patients. Future studies should focus on validating the cabozantinib exposure required for long term efficacy.

Increased Plasma Soluble PD-1 Concentration Correlates with Disease Progression in Patients with Cancer Treated with Anti-PD-1 Antibodies.

Immune checkpoint inhibitors (ICIs) confer remarkable therapeutic benefits to patients with various cancers. However, many patients are non-responders or develop resistance following an initial response to ICIs. There are no reliable biomarkers to predict the therapeutic effect of ICIs. Therefore, this study investigated the clinical implications of plasma levels of soluble anti-programmed death-1 (sPD-1) in patients with cancer treated with ICIs. In total, 22 patients (13 with non-small-cell lung carcinoma, 8 with gastric cancer, and 1 with bladder cancer) were evaluated for sPD-1 concentration using enzyme-linked immunosorbent assays for diagnostic and anti-PD-1 antibody analyses. sPD-1 levels were low before the administration of anti-PD-1 antibodies. After two and four cycles of anti-PD-1 antibody therapy, sPD-1 levels significantly increased compared with pretreatment levels (p = 0.0348 vs. 0.0232). We observed an increased rate of change in plasma sPD-1 concentrations after two and four cycles of anti-PD-1 antibody therapy that significantly correlated with tumor size progression (p = 0.024). sPD-1 may be involved in resistance to anti-PD-1 antibody therapy, suggesting that changes in sPD-1 levels can identify primary ICI non-responders early in treatment. Detailed analysis of each cancer type revealed the potential of sPD-1 as a predictive biomarker of response to ICI treatment in patients with cancer.

Plasma D-Dimer and Fibrinogen Levels Correlates with Tumor Size and Disease Progression in Nigerian Breast Cancer Patients

Fourty-five breast cancer patients and 50 apparently healthy sex-matched controls from the University of Ilorin Teaching Hospital were enrolled in this study. Plasma D-dimer and fibrinogen were found to be significantly higher than controls; APTT was significantly shorter than the controls. D-dimer and fibrinogen were also significantly positively correlated with ECOG, disease stage, lymph node involvement, and tumor size. On multivariate analysis, D-dimer and fibrinogen were found to be independently related to lymph node involvement. This study shows that plasma D-dimer and fibrinogen levels are elevated in breast cancer patients, and both are markers of disease progression.

Prolonged Time from Diagnosis to Breast-Conserving Surgery is Associated with Upstaging in Hormone Receptor-Positive Invasive Ductal Breast Carcinoma

BackgroundTime to surgery (TTS) has been suggested to have an association with mortality in early-stage breast cancer.ObjectiveThis study aims to determine the association between TTS and preoperative disease progression in tumor size or nodal status among women diagnosed with clinical T1N0M0 ductal breast cancer.MethodsWomen diagnosed with clinical T1N0M0 ductal breast cancer who had breast-conserving surgery as their first definitive treatment between 2010 and 2016 (n = 90,405) were analyzed using the National Cancer Database. Separate multivariable logistic regression models for hormone receptor (HR)-positive and HR-negative patients, adjusted for clinical and demographic variables, were used to assess the relationship between TTS and upstaging of tumor size (T-upstaging) or nodal status (N-upstaging).ResultsT-upstaging occurred in 6.76% of HR-positive patients and 11.00% of HR-negative patients, while N-upstaging occurred in 12.69% and 10.75% of HR-positive and HR-negative patients, respectively. Among HR-positive patients, odds of T-upstaging were higher for 61–90 days TTS (odds ratio [OR] 1.18, 95% confidence interval [CI] 1.05–1.34) and ≥91 days TTS (OR 1.47, 95% CI 1.17–1.84) compared with ≤30 days TTS, and odds of N- upstaging were higher for ≥91 days TTS (OR 1.35, 95% CI 1.13–1.62). No association between TTS and either T- or N-upstaging was found among HR-negative patients. Other clinical and demographic variables, including grade, tumor location, and race/ethnicity, were associated with both T- and N-upstaging.ConclusionTTS ≥61 and ≥91 days was a significant predictor of T- and N-upstaging, respectively, in HR-positive patients; however, TTS was not associated with upstaging in HR-negative breast cancer. Delays in surgery may contribute to measurable disease progression in T1N0M0 ductal breast cancer.

Breast Cancer Care in a COVID-19 Pandemic Epicenter.

Most breast cancer patients are initially treated with surgery, with elective timing Moderate delays in timing do not impact surgical outcome, as they are not significantly associated with a change in tumor size nor significant disease progression 1 The oncology literature in fact supports sometimes delaying surgery with neoadjuvant endocrine therapy or chemotherapy William Wycherley wrote: “Necessity, mother of invention ” [ ]in response to social distancing, surgeons are learning how to delicately balance risk and benefit in new, meaningful ways

Triple Immunotherapy Overcomes Immune Evasion by Tumor in a Melanoma Mouse Model.

Background: Melanoma is a malignancy with increasing incidence that underlies most skin cancer-related deaths. Advanced melanoma patients still have poor prognosis despite recently developed immunotherapies. This study devises a triple immunotherapy to treat melanoma in a mouse model. The combination includes anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, Monophosphoryl-lipid-A (MPLA), and an Indolamine-Dioxygenase-1 (IDO1) inhibitor. The aim of the study is, first, to rule out any major toxic effects related to this therapy and, second, to assess its antitumor effects.Methods: Cancer-free C57BL/6 mice were randomized into control groups and groups receiving single, dual, or triple therapies of the defined treatments. Clinical signs, weight gain, and histological sections from their main organs were assessed. Then, melanoma-bearing mice were segregated into similar groups, monitored for survival, and their tumor size was measured repeatedly. Finally, flow cytometry was used to analyze immune cell populations in the tumor masses including CD4+, CD8+, and regulatory T cells in addition to natural killer cells.Results: No adverse effects were detected in any of the treated groups. Survival analysis indicated that the groups receiving dual or triple therapies had prolonged survival compared to the controls. However, the group receiving triple therapy was the only group to show statistically significant increase in survival compared to the controls. Tumor size progression paralleled the survival outcome. The group receiving the triple therapy showed statistically significant smaller tumor sizes compared to all the other groups throughout the whole monitoring period. Flow cytometry used to analyze immune cell populations in the tumor mass indicated that the triple immune therapy was capable of significantly enhancing the natural killer cell counts as well as the CD3+CD4+/Treg and CD3+CD8+/Treg ratios possibly enhancing the anti-tumorigenic environment.Conclusions: Generated data rule out any major adverse events pertaining to the triple immunotherapy and reveal its enhanced effectiveness in thwarting melanoma progression over all other tested treatments.

Efficacy of somatostatin analog (SSA) monotherapy for well-differentiated grade 3 (G3) gastroenteropancreatic neuroendocrine tumors (NETs).

617 Background: Currently, there is no published data on the efficacy of SSAs for well-differentiated G3 NETs. Randomized trials have demonstrated a progression-free survival (PFS) benefit and limited tumor response for lower grade 1-2 NETs, but the optimal systemic therapy for metastatic/unresectable G3 NETs is unknown. We sought to evaluate the efficacy of SSAs in G3 NETs. Methods: We performed a retrospective analysis of Mayo Clinic patients treated with SSAs for metastatic/unresectable G3 NETs, querying data from 1992 - present. Inclusion criteria were: centrally reviewed pathology confirming well-differentiated morphology, G3 based on WHO classification, SSA monotherapy, and radiological data available to assess response. Patients who had prior lines of treatment were included as long they subsequently were treated with single-agent SSA. Poorly-differentiated tumors were excluded. The primary endpoint was PFS. Best overall response was determined by radiographic regression, stabilization, or progression of tumor size. Results: Ninety patient records were reviewed, with 14 meeting inclusion criteria (diagnosed 2014 – 2018). Median Ki-67 proliferative index was 25%. Two patients (14%) had a partial response to SSA therapy, five (36%) had stable disease, and seven (50%) had progressive disease. The estimated median PFS was 4.4 months (95% CI 2.9 – 24). Of the 7 patients with stable disease or partial response, the median time to progression was 8.7 months. Three patients had stable disease for greater than 9 months (24, 29 and 42 months, respectively). Overall survival was not estimable. There was no association of Ki-67 index with PFS based on a proportional hazards model. Conclusions: This is the first report on the efficacy of SSAs for G3 NETs. Although half of the patients in our series had at least stable disease, the PFS was modest at only 4.4 months. Given their favorable side-effect profile compared to cytotoxic chemotherapy, SSAs may present an attractive option to be further explored in a prospective fashion. We are presently updating this data by reassessing response using RECIST criteria.

Abstract A073: Using prebaseline and on-treatment tumor assessments to compare time to tumor size progression of hormonal therapy + sapanisertib vs an extrapolated hormonal therapy alone from a single-arm phase 2 study

Abstract Introduction: This is a single-arm phase 2 trial testing the efficacy of the investigational dual TORC 1 and 2 inhibitor, sapanisertib (TAK-228), and hormonal therapy (HT) in estrogen receptor-positive patients relapsed or refractory to hormonal therapy and everolimus (NCT02049957). We sought to estimate the potential progression-free survival (PFS) benefit of adding sapanisertib to continued HT treatment by comparing the tumor growth rate, and therefore extrapolated time-to-tumor size progression (TTTSP) in the absence of sapanisertib, to the observed TTTSP in sapanisertib-treated patients. Experimental Procedure: Using the most recent historical prebaseline scans from the subset of response evaluable patients enrolled in the study with scans available (N=40), we estimated the exponential rate of tumor size increase on continued HT alone and extrapolated that rate to a virtual TTTSP, defined per RECIST 1.1 as the time until the sum of diameters (SoD) of target lesions was predicted to exceed 120% of the baseline SoD. We applied Kaplan-Meier (KM) analysis to estimate the potential benefit of sapanisertib added to HT by comparing 3 different PFS estimators to the TTTSP distribution for HT alone: (1) the model-estimated TTTSP obtained by fitting a tumor dynamic model to the on-treatment tumor size data (N = 40), (2) the observed PFS in the same patient population for which prebaseline scans were available (N = 40), and (3) the observed PFS in the entire response evaluable study population (N = 94). Results: Because TTTSP does not account for new lesions or death, the longest times to event were observed using the model-estimated TTTSP of HT with sapanisertib. Accordingly, the strongest separation between the various time-to-event metrics for the HT + sapanisertib arm and the model-based HT-only TTTSP ‘arm’ was observed using the model-estimated TTTSP of HT + sapanisertib (HR = 0.16, 95% CI: [0.07,0.36]). Smaller but statistically significant separation between observed HT + sapanisertib PFS and model-based HT-only TTTSP KM curves was observed in the subset of patients for whom prebaseline scans were available (HR = 0.40, 95% CI: [0.25,0.7]). A similar separation was seen using the full set of 94 evaluable patients in the active arm, irrespective of whether they provided a prebaseline scan (HR = 0.46, 95% CI: [0.3,0.75]), confirming that PFS of the analysis subset was reflective of the larger population. Conclusion: Under the assumption that HT-only patients would have PFS events at least as quickly as the model-based TTTSP, given that progression by new lesions and/or death are not captured by TTTSP, the observed HT + sapanisertib PFS to model-based HT-only TTTSP hazard ratio in the response-evaluable patient subset with prebaseline scans may provide a conservative estimate of what would be observed in a head-to-head HT +/- sapanisertib study in this patient population. Our analysis suggests that using such a modeling approach may aid program decision making by estimating 2 “arms” of time-to-event data from a single-arm study that captures each patient’s most recent prebaseline scan. It predicts that sapanisertib adds benefit to HT in terms of longer times to RECIST progression/TTTSP (median: 5.5 months vs 1.6 months). Citation Format: Dean Bottino, Yaming Hang, Rachel Neuwirth, Chirag Patel, Farhad Sedarati, Douglas V Faller, Annette Schmid. Using prebaseline and on-treatment tumor assessments to compare time to tumor size progression of hormonal therapy + sapanisertib vs an extrapolated hormonal therapy alone from a single-arm phase 2 study [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A073. doi:10.1158/1535-7163.TARG-19-A073

In vivo longitudinal imaging of RNA interference-induced endocrine therapy resistance in breast cancer.

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor-positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence-labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)-regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox-induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor-bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen-treated control group. Longitudinal imaging was performed by a home-built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper-measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e-7) correlated with the tumor size progression for the mice with shRNA-induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.

Undetected pituitary adenoma in a patient with retinitis pigmentosa

BackgroundRetinitis pigmentosa (RP) is one of the most severe hereditary retinal disorders with a worldwide prevalence reaching 1 in every 3000–5000 people and a total of almost one million affected individuals. RP is heterogeneous in its clinical presentations but typically presents as progressive visual dysfunction, including nyctalopia in adolescence, restricted peripheral vision (tunnel vision) in young adults, and loss of central vision at an advanced age.Case descriptionHerein, we want to report a case of RP who presented with gradual worsening of vision and headache, and further evaluation revealed a concomitant non-functional pituitary macroadenoma. Ophthalmologic evaluation revealed a little chance for him to regain his vision, so the patient refused to undergo endoscopic surgical resection. However, he is still under clinic-radiologic follow-up, to be evaluated for progression in tumor size and obstructive hydrocephalus.ConclusionPresenting with similar symptoms of tunnel vision, the simultaneous occurrence of these two diseases in a patient may delay the diagnosis of the latter, leading to its progression.

Abstract 1242: Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype

Abstract Currently there are few successful targeted therapies for non-small cell lung cancer (NSCLC). Targeting mutations in EGFR, BRAF, ALK etc. provide treatment options only for a defined subset of adenocarcinoma patients leaving majority of adenocarcinoma and squamous carcinoma patients without any options. Activating KRAS mutations are found in 15-30% of all NSCLC patients; however, existing treatment options for KRAS-mutant cancer are not effective in the subset with concomitant loss of LKB1 (kinase that phosphorylates AMPK). Several studies demonstrate the importance of mitochondrial metabolism and oxidative phosphorylation (OXPHOS) in lung tumorigenesis. Components of OXPHOS complexes and markers of mitochondrial biogenesis are highly predictive of reduced overall survival in NSCLC patients. A study using genetically engineered mouse models (GEMMs) for lung cancer (KrasLSLG12D/+Trp53-/- and KrasLSL-G12D/+Lkb1-/-) showed that along with glucose, lactate contributes to the TCA cycle. This highlights the importance of OXPHOS in these models. Also, heme is a prosthetic group for several of these OXPHOS proteins and is involved in oxygen transport and utilization. Hence, we aim to investigate unique therapeutic targets by examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype. We used LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc)) GEMM model for our study. Briefly, the mice were infected with AdenoCre virus, and sacrificed at different intervals in the progression of the tumors. For histological and immunohistochemical analysis, lung tissues were formalin fixed, paraffin embedded and sectioned (5µm thick). H&amp;E staining showed consistent progression in tumor size over time. We will examine the time-course expression of OXPHOS, heme pathway proteins, and hemoproteins to determine the extent to which this lung tumor subtype depends on heme and respiratory proteins for its progression. Citation Format: Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh. Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1242.

RREB1-induced upregulation of the lncRNA AGAP2-AS1 regulates the proliferation and migration of pancreatic cancer partly through suppressing ANKRD1 and ANGPTL4

Long noncoding RNAs (lncRNAs) have been reported to be involved in a variety of human diseases, including cancers. However, their mechanisms have not yet been fully elucidated. We investigated lncRNA changes that may be associated with pancreatic cancer (PC) by analyzing published microarray data, and identified AGAP2-AS1 as a relatively overexpressed lncRNA in PC tissues. qRT-PCR assays were performed to examine expression levels of AGAP2-AS1. MTT assays, colony formation assays, and EdU assays were used to determine the proliferative capacity of cells. Flow cytometry and TUNEL assays were used to study the regulation of AGAP2-AS1 in the cell cycle and apoptosis. Transwell experiments were used to study changes in cell invasion and metastasis, and a nude mouse model was established to assess the effects of AGAP2-AS1 on tumorigenesis in vivo. RNA sequencing was performed to probe AGAP2-AS1-related pathways. Subcellular fractionation and FISH assays were used to determine the distribution of AGAP2-AS1 in PC cells, and RIP and ChIP were used to determine the molecular mechanism of AGAP2-AS1-mediated regulation of potential target genes. Increased expression of AGAP2-AS1 was associated with tumor size and pathological stage progression in patients with PC. RREB1 was found to activate transcription of AGAP2-AS1 in PC cells. AGAP2-AS1 affected proliferation, apoptosis, cycle arrest, invasion, and metastasis of PC cells in vitro, and AGAP2-AS1 regulated PC proliferation in vivo. Furthermore, AGAP2-AS1 epigenetically inhibited the expression of ANKRD1 and ANGPTL4 by recruiting zeste homolog 2 (EZH2), thereby promoting PC proliferation and metastasis. In summary, our data show that RREB1-induced upregulation of AGAP2-AS1 regulates cell proliferation and migration in PC partly through suppressing ANKRD1 and ANGPTL4 by recruiting EZH2. AGAP2-AS1 represents a potential target for the diagnosis and treatment of PC in the future.

Detection of Circulating Tumor DNA in Patients With Leiomyosarcoma With Progressive Disease.

Leiomyosarcoma (LMS) is a soft tissue sarcoma characterized by multiple copy number alterations (CNAs) and without common recurrent single nucleotide variants. We evaluated the feasibility of detecting circulating tumor DNA (ctDNA) with next-generation sequencing in a cohort of patients with LMS whose tumor burden ranged from no evidence of disease to metastatic progressive disease. Cell-free DNA in plasma samples and paired genomic DNA from resected tumors were evaluated from patients with LMS by ultra-low passage whole genome sequencing (ULP-WGS). Sequencing reads were aligned to the human genome and CNAs identified in cell-free DNA and tumor DNA by ichorCNA software to determine the presence of ctDNA. Clinical data were reviewed to assess disease burden and clinicopathologic features. We identified LMS ctDNA in eleven of sixteen patients (69%) with disease progression and total tumor burden over 5 cm. Sixteen patients with stable disease or low disease burden at the time of blood draw were found to have no detectable ctDNA. Higher ctDNA fraction of total cell-free DNA was associated with increasing tumor size and disease progression. Conserved CNAs were found between primary tumors and ctDNA in each case, and recurrent CNAs were found across LMS samples. ctDNA levels declined following resection of progressive disease in one case and became detectable upon disease relapse in another individual patient. These results suggest that ctDNA, assayed by a widely available sequencing approach, may be useful as a biomarker for a subset of uterine and extrauterine LMS. Higher levels of ctDNA correlate with tumor size and disease progression. Liquid biopsies may assist in guiding treatment decisions, monitoring response to systemic therapy, surveying for disease recurrence and differentiating benign and malignant smooth muscle tumors.

Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein

Recombinant strains of Lactococcus lactis NZ9000 that express native and codon-optimized E6 protein (fused to the SPusp45 secretion signal) were successfully constructed by using the nisin-controlled gene expression (NICE) system. Expression of the recombinant strains was evaluated by Western blot analysis. Female mice of strain C57BL/6 were immunized orally with recombinant lactococci expressing inducible E6 oncoprotein and the antigen-specific antibody production (IgA and IgG) and cytokines were measured by ELISA and ELISPOT assay, respectively. Our outcomes indicate that the HPV-16 E6 specific IL-2- and IFN-γ-secreting lymphocytes in the antigen-stimulated intestinal mucosal lymphocytes, splenocytes and vaginal lymphocytes were significantly higher than the control groups. We showed that L. lactis having codon-optimized E6 oncogene had better inhibitory effect on tumor growth, better treatment effects on progression of tumor size, and better survival rate in comparison with L. lactis having native E6 oncogene, (P < 0.0001).In conclusion, the rE6 protein displayed by L. lactis can induce humoral and cellular immunity. Taken together, these preclinical results represent a promising step towards the development of recombinant L. lactis as a live oral vector vaccine to treat the HPV-16 associated with cervical cancer.

Abstract 3872: Quantitative measurement of immune-modulatory mediators within tumors of freely moving mice utilizing in vivo microdialysis

Abstract Monitoring changes in biochemical elements within tumors is crucial to understanding cancer biology and to helping with the development of novel therapies. Yet to date, experimental techniques enabling sensitive and quantitative measurement of the levels of small molecules contributing to tumor development have been limited in preclinical oncology models. In the current set of experiments, we implemented in vivo microdialysis to measure signaling molecules and oncometabolites in the tumor microenvironment of freely moving rodents. To this aim, a small dialysis probe is implanted in syngeneic tumors in mice. Probes are perfused with artificial dialysate fluid and dialysate containing molecules from the tumor microenvironment is collected at regular intervals. Levels of biochemicals in the dialysate are then quantified by liquid chromatography-mass spectrometry (LC-MS), a robust and sensitive quantitative technique. We report here proof-of-concept results supporting the use of in vivo microdialysis coupled to LC-MS in cancer research. First, we tested whether implantation of dialysis probe affects in vivo tumor growth. To do so, tumor volume from probe-implanted and non-implanted MC38 allografts in mice was monitored daily for up to 10 days following probe implantation. We found that the growth curve from probe-bearing MC38 tumors was similar to probe-free tumors, revealing that probe implantation did not influence tumor size progression. Next, the levels of metabolites that are known to be involved in tumor development were measured in tumor allografts of freely-moving mice using in vivo microdialysis. Relevant levels of arginine, ornithine and putrescine were quantified by LC-MS. Levels of key molecules of the adenosine signaling and tryptophan/kynurenine pathway metabolites in MC38 tumors were also quantified. To better understand the homogeneity of this particular allograft model, we measured the concentration of the above-mentioned molecules in different regions of a tumor. To this end, two separate probes were implanted in the center and the peripheral area of the same 300 mm3 MC38 allograft. We found that levels of measured molecules were similar in dialysates collected from both probe locations, suggesting homogenous levels of biochemicals throughout the microenvironment of these non-necrotic tumors. Our proposed experimental approach allows for a quantitative and sensitive measurement of key biochemical mediators of tumor progression. In vivo microdialysis in murine tumor models may be used to elucidate the mechanisms by which therapies, such as chemotherapy and immune checkpoint inhibitors, modulate the tumor microenvironment. Our efforts in preclinical cancer research has the potential to bring new insights on the mechanisms underlying cancer development and help the discovery of next-generation therapies for cancer Citation Format: Nadege Morisot, Julien Roeser, Hajo Schiewe, Holden Brown Janssens, Marieke van der Hart, Arash Rassoulpour. Quantitative measurement of immune-modulatory mediators within tumors of freely moving mice utilizing in vivo microdialysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3872.