bluuebunny/crossref_metadata_2025 huggingface dataset:
Reducing invasion potential of cervical cancer cells via targeted knockdown of c-Jun.
Authors: Yee Grace Pei Chien , De Souza Paul L. , Khachigian Levon M.
Published: March 2020

e22005

Background: Despite recent advent of vaccines for human papillomaviruses (HPV) in cervical cancer and increasing efforts to improve therapy, deaths still average 275,000 annually worldwide, with most women succumbing to recurrent or metastatic disease. The c-Jun oncogene is a subunit of the activating protein-1 (AP-1) transcription factor and is strongly expressed in cervical cancer, regulating the expression of HPV16 and 18 genes. AP-1 plays a major role in cell growth, migration and apoptosis in many cell types. This study examined the role of c-Jun in modulating HeLa proliferation, migration, apoptosis and invasion as well as susceptibility to cisplatin. Methods: Transient knockdown and over-expression of c-Jun was performed in HeLa cervical cancer cells using Dharmacon c-Jun siRNA and Origene Jun expression vector. c-Jun and downstream gene/protein expression was confirmed by western blot and real-time PCR and cells subject to proliferation, in vitro wound and matrigel dual-chamber transwell assays. Flow cytometry was used for analysis of cell cycle and apoptosis. Results: c-Jun protein and mRNA levels were reduced by c-Jun siRNA. c-Jun silencing inhibited cervical cancer cell proliferation. Significantly, c-Jun suppression dramatically reduced HeLa migration and invasion and targeted down-regulation of cyclooxygenase-2 (Cox2), interleukin-6 (IL-6), metalloproteinases (MMP)-1, -9 and -13 as well as HPV18 E6 and E7, genes highly expressed in cervical cancer and associated with metastatic growth. Direct siRNA knockdown of Cox2 in HeLa also reduced MMP1 and MMP9 expression suggesting an intermediary link. In HeLa cells over-expressing c-Jun, cell proliferation was not significantly increased but cell invasiveness was markedly enhanced in parallel with enhanced MMP-1 expression. Modulation of c-Jun expression did not interfere with susceptibility of HeLa cells to apoptosis in the presence of cisplatin. Conclusions: Reduced invasion potential of HeLa cells after c-Jun knockdown suggests a potential target in treatment of metastatic and recurrent cervical cancer. Data suggest a mechanism involving down-regulation of Cox2 and MMP-1 and -9 expression.
Tunneling nanotubes and intercellular communication: Differences between platinum-resistant and platinum-sensitive ovarian cancer.
Authors: Dickson Elizabeth Louise , Thayanithy Venugopal , Vogel Rachel Isaksson , Argenta Peter , Geller Melissa Ann , Subramanian Subbaya , Lou Emil
Published: March 2020

e22007

Background: Preliminary evidence suggests that cell-to-cell communication may be responsible for the development of chemotherapy resistance in ovarian cancer. We propose tunneling nanotubes (TnTs) – long, thin actin-based cell extensions – as novel candidates to explain direct communication between treatment-refractory malignant ovarian cells. The purpose of this study was to investigate TnT formation between ovarian cancer cells in vitro. Methods: Using platinum-sensitive (A2780) and resistant (C200 and SKOV3, as well as ES2) ovarian cancer cell lines, we tested various conditions to assess factors affecting TnT formation. Scratch assays were utilized as a 2-dimensional simulation of ovarian cancer invasion. To assess TnTs as a conduit for transmission of therapeutic drugs between connected cells, we used doxorubicin, which auto-fluoresces in cell culture. Results: We determined that a hyperglycemic, low-serum, acidic medium stimulated TnT formation between all ovarian cancer cells studied, and more significantly, formed direct connections between A2780 to both C200 and SKOV3 cell lines. Conversely, Everolimus or Metformin decreased TnT formation in all cell lines with continuous exposure up to 96 hours; most prominently for the platinum-sensitive cell line. Time-lapse microscopy was used to assess chronologic formation of TnTs at the advancing front of the scratch wound. Cell proliferation assays were performed and confirmed the decrease in TnTs was not due to decreased cell proliferation. We directly observed fluorescing doxorubicin within the TnTs, suggesting TnTs act as a transport mechanism for cellular communication. Conclusions: TnT formation is stimulated in conditions of cellular stress similar to those experienced in vivo and results in direct connections between cells. Our data suggests that these conduits are a potential means of cellular exchange between platinum-sensitive and resistant ovarian cancer cells. Using currently available agents to target TnTs and disrupt this communication provides a novel approach to understanding and treating the problem of platinum resistance in ovarian cancer.
Use of tumor necrosis factor receptor 2 (TNFR2)/p75 and TNF-α as an antitumor strategy.
Authors: Bae Sanggyu , Sasi Sharath , Song Jin , Perepletchikov Aleksandr , Schneider Douglas , Goukassian David
Published: March 2020

e22010

Background: TNF-α has two receptors, TNFR1/p55 and TNFR2/p75, with diverse signaling roles: p55 - predominantly cytotoxic and p75 - prosurvival and proangiogenic. Compared to WT and p55KO/p75KO, growth of tumors in p75 knockout (KO) mice was decreased >50% in Lewis lung carcinoma (LLC) and B16 melanomas (Sasi et al., Oncogene, 2012). This was associated with increased apoptosis and decreased capillary density in tumos. We hypothesized that selective blocking of p75 receptor in tumors may sensitize them to TNF induced apoptosis. Methods: p75 receptor was knockdown (KD) in LLCs using shRNA. LLC and p75KD LLCs were implanted into WT mice flanks. On day 8 post-inoculation, mouse recombinant (mr) TNF (12.5 ng/g/injection) or saline was injected x2 daily for 6 days. Tumor volumes (tV) were measured daily and tumor weights (tW) on day 16. Tumors, tubular bones and spleens were examined by 2 pathologists blindfolded to treatments. Results: Mouse weights remianed stable throughout the study in all groups. There was no difference in tV or tW between LLC minus(-) TNF and p75KD LLC-TNF tumors on day 16 (2 d days after last injection). Compared to 3 control groups, p75KD LLC+TNF showed >2-5-fold decreases in tV (p<0.001, n=8) and tW (p<0.0001, n=8). There was NO difference in tV or tW (p=ns) end of study vs before injections in p75KD LLC+TNF group whereas in 3 control groups tV and tW were increased 2.7-, 4.5- and 3.2-fold (p<0.01, p<0.0002 and p<0.0001). Pathology revealed that 1/3 of tumors in p75KD LLC+TNF group were nearly 100% necrotic. Bone marrow and spleens did not show significant TNF-induced systemic toxic effect. There was an inflammatory response in the BM and spleens of mice with necrotic tumors in +TNF group, reflected by shift of myeloid/erythroid radio in the BM and moderate increases in lymphoid follicles in the spleen. Conclusions: Our results suggest that blocking p75 expression in tumor tissue combined with administration of small exogenous doses of TNF significantly inhibits aggressive LLC tumor growth and induces nearly 100% tumor necrosis in 1/3 of the experimental group in six days, with no detectible systemic toxicity. This may represent new and effective therapy against lung neoplasms and potentially solid tumors of other origin.
Prognostic factors and survival of patients with brain metastasis (BM) from breast cancer (BC) who underwent craniotomy.
Authors: Leone Jose Pablo , Cunningham Diana E. , Lee Adrian , Bhargava Rohit , Hamilton Ronald L. , Wiener Erik , Okada Hideho , Brufsky Adam
Published: March 2020

e22017

Background: BC is the second most frequent cause of BM after lung cancer, with metastases occurring in 10-16% of all patients. BM in patients with BC is a catastrophic event that results in poor prognosis. Identification of prognostic factors associated with breast cancer brain metastases (BCBM) could help to identify patients at risk. The aim of this study was to assess clinical characteristics, prognostic factors and survival of patients with BCBM who had craniotomy and resection in a series of patients treated with modern multimodality therapy. Methods: We analyzed 42 patients with BCBM who underwent resection. Patients were diagnosed with BC between April 1994 and May 2010. Cox proportional hazards regression was selected to describe factors associated with time to BM, survival from the date of first recurrence, and overall survival (OS). Results: Median age was 51 years (range 24-74). Median follow-up was 4.2 years (range 0.6-18.5). The mean time to BM from primary diagnosis was 49 months (range 0-206.22). Patients had a median of 2 BM with a median size of 3.25 cm. The proportion of the biological subtypes of BC was ER+/HER2- 25%, ER+/HER2+ 15%, ER-/HER2+ 30% and ER-/HER2- 30%. Brain radiotherapy was given to 28 patients, of which 10 had stereotactic radiosurgery, 7 whole brain radiation, and 11 both. Median OS from the date of primary diagnosis was 5.74 years. Median survival after diagnosis of BM was 1.33 years. In multivariate Cox regression analyses, stage was the only factor associated with shorter time to the development of BM (P=0.059), whereas age was the only factor associated with survival from the date of recurrence (P=0.027) and with OS (P=0.037). Controlling for age and stage, neither the biological subtype of cancer, the radiation modality nor the site of first recurrence showed any impact on survival. Conclusions: Stage at primary diagnosis correlated with shorter time to the development of BM, while age at diagnosis was associated with shorter survival in BCBM. None of the other clinical factors had influence on survival.
Sensitive detection of tumor nucleic acids in plasma by mutation-enriched next generation sequencing.
Authors: Marziali Andre
Published: March 2020

e22041

Background: Next Generation DNA Sequencing (NGS) is becoming the new standard for mutational profiling of tumour tissue, due to its flexibility, speed, and decreasing cost. While generally exceptional in performance, NGS suffers from a sequencing error rate of 0.1% – 1%, largely due to amplification-induced artifacts in its workflow. While this does not constitute a significant problem in application of NGS to sequencing of tumour tissue, it makes NGS impractical as a method to search for low abundance mutation signatures in plasma samples. Numerous publications have shown the presence of tumour signatures in the cell-free DNA (cfDNA) circulating in plasma, but concordance between the tumour signature and the plasma signature has been limited. This is likely due to limitations in the detection technologies used to search for cfDNA in plasma. To maximize concordance between plasma and tissue, it will be essential that sensitivities reaching 0.01% and below (as little as a single tumour mutant allele per sample) be achieved, and ideally that multiple mutational hot spots be analysed to maximize the chance of detection. Current technologies are incapable of such sensitivity over a large number of mutation loci. Methods: We have developed a novel electrophoretic method that can enrich nucleic acid samples over 1,000,000-fold for up to 100 somatic mutations, enabling reliable profiling of samples containing as little as 0.01% mutant. By enriching nucleic acid samples for specific targets prior to amplification and sequencing, we enable the use of NGS in plasma-based mutation detection and profiling. Results: We present technical and clinical data demonstrating highly sensitive multiplexed mutation detection in plasma and tissue samples, demonstrating 0.01% sensitivity over 45 somatic mutations per sample. Conclusions: We have demonstrated a novel somatic mutation enrichment methodology that allows DNA sequencing to work beyond its usual limit of detection to accurately profile solid tumours by detecting their mutation signature in plasma, even when the tumour DNA is present in plasma at abundances below 0.01%.
Prognostic value of CDH2 and CDT2 expression levels in the peripheral blood (PB) specimens of patients with Ewing family of tumors (EFTs).
Authors: Przybyl Joanna , Debiec-Rychter Maria , Switaj Tomasz , Kosela Hanna Melania , Falkowski Slawomir , Wiater Katarzyna , Lugowska Iwona , Szumera-Cieckiewicz Anna , Ptaszynski Konrad , Siedlecki Janusz A. , Rutkowski Piotr
Published: March 2020

e22046

Background: EFTs are characterized by oncogenic EWSR1-FLI1 fusion gene, which can be detected in 85-90% of tumors and in 20-70% of peripheral blood specimens. It has been previously reported that downregulation of CDH2 (encoding N-cadherin) and overexpression of CDT2 [also known as DTL, encoding denticleless homolog (Drosophila)]in EFTs tumors are associated with poor prognosis. The aim of the study was to evaluate the expression levels of these markers in the circulating tumor cells and assess their utility as prognostic markers in EFTs. Methods: 10mL of PB was collected from 24 untreated adult EFTs patients at the diagnosis (period: 2009-2011, median age 30 years). 9 blood specimens from healthy individuals, and 5 untreated frozen EFTs tumor samples were used as controls. 8 EFTs patients presented metastatic disease (M1) at the diagnosis and 5 patients died during the follow-up period. Median follow-up was 22 months. Quantitative reverse transcription PCR (qRT-PCR) for CDH2 and CDT2 expression were performed in triplicate using the TaqMan Gene Expression Assays (Applied Biosystems). Mean expression level of 2 reference genes PSMC4 and EIF2B1 served as endogenous control. The fold change was calculated using ddCt method with pooled healthy individuals as calibrator. Results: At least 2-fold decrease (range 2.11-16.35) of CDH2 expression level in PB has been observed in 79% (n=19) of EFTs patients (p=0.04) as compared to healthy control. In general, CDH2 expression level in PB of EFTs patients was 3.08-fold lower compared with healthy individuals (p=0.07). All of the patients who had M1 at diagnosis (n=8) and who died during the follow-up (n=5) had CDH2 gene underexpression. CDT2 overexpression (FC range 3.28-6.97) has been observed in 17% (n=4) of EFTs patients (p=0.01). Three of these patients had M1 at diagnosis and two of these patients died during the follow-up. PB specimens presenting CDT2 overexpression clustered together with the tumor specimens. Conclusions: Abnormalities in the expression of CDH2 and CDT2 genes in PB specimens are associated with aggressive clinical course of EFTs. Diagnostic and prognostic significance of these biomarkers warrants further studies.
A potential role for myosin light chain kinase in lung adenocarcinoma tumorigenesis.
Authors: Borowicz Stanley John , Tauler Jordi , Rizzo Alicia , Garcia Joe G. N.
Published: March 2020

e22051

Background: Treatment of non-small cell lung cancer remains a clinical challenge despite the migration from cytotoxic chemotherapies to targeted agents. Myosin light chain kinase (MLCK) is a protein involved in phosphorylation of regulatory chains of myosin, thereby regulating the contraction of myosin II. The 210 KDa non-muscle MLCK isoform (nmMLCK), expressed in endothelial cells, regulates cell shape and motility, and increases EGFR-mediated proliferation of hepatocytes. Furthermore, inhibitors of MLCK such as ML-7 and ML-9 have been shown to decrease invasiveness of pancreatic prostate cancer cell lines. Clinically, levels of MLCK gene (MYLK) mRNA correlate with increased risk of disease recurrence and development of distant metastasis in lung cancer. These data suggest a potential role for MLCK in lung cancer tumorigenesis and metastasis. Methods: H-23 and H-441 human lung adenocarcinoma cell lines were treated for 12hrs with MLCK chemical inhibitor, ML-7 (20uM), and harvested at 24hr, 48hr, and 72hr for MTS Assay. Cell proliferation was assessed with MTS Assay per manufacturer protocol (Promega). H-23 cells were transiently transfected with a FLAG-tagged (WT) nmMLCK overexpression vector using Fugene Transfection Kit per manufacturer protocol (Promega). Results: While no significant difference in proliferation was noted in A-549 cells, both H-23 and H-441 cells showed a decrease in cell proliferation with inhibition of MLCK. H-23 cells also showed an increase in proliferation when transiently transfected with an MLCK-overexpression vector. Conclusions: Inhibition of endogenous nmMLCK decreases H-23 and H-441 proliferation. Conversely, an increase in nmMLCK expression in vitro increases cell proliferation in H23 cells. These results suggest that MLCK/MYLK may participate in regulation of lung adenocarcinoma cell proliferation.
Triggering receptor expressed on myeloid cells-2 (TREM-2) elicited by lung cancer cells to facilitate tumor immune evasion.
Authors: Yao Yinan , Li Hequan , Wang Yuehong , Zhou Jianying
Published: March 2020

e22054

Background: More recent evidences have argued that negative regulatory mechanism that serve to impede ongoing immune responses, which gain in interest as a complementary strategy for cancer therapy. Triggering receptor expressed on myeloid cells-2 (TREM-2) restrains phagocytosis of both dendritic cells (DCs) and macrophages(MΦs), and reduces release of inflammatory cytokines through DAP12-mediated inhibitory signal, prompting us to investigate the role of TREM-2 involved in immune dysfunction among tumor-bearing host. Methods: In lung cancer patients, monocytes of peripheral blood were analyzed by flow cytometry and MΦs around tumour cells in lung tissue were detected by immunehistochemistry. Then we prepared conditional medium containing supernatant of 3LL cells mimicing tumor microenvironment for DC and MΦ derivation, and established an orthotopic lung cancer-bearing mice model to explore the further mechanism. Results: It was showed that TREM-2 expression on monocytes was up-regulated in lung cancer patients compared with that of healthy controls, and that TREM-2 levels of macrophages had a positive correlation with TNM stage. Moreover, reduction of tumor burden, by operation or chemotherapy, led to obvious decline of TREM-2 expression. The percentage of TREM-2+DC in the murine lung signiﬁcantly increased after tumor-bearing, and such DCs preformed lower MHCII-Ia, CD86 expression, and IL-12 production, but higher IL-10 secretion. In vitro, more TREM-2+DCs and TREM-2+MΦs were harvested from ‘conditional’ medium instead of ‘normal’ medium. Also, TREM-2+DCs rather than TREM-2- DCs markedly inhibited proliferation of T cells, which could be partially abolished by anti-TREM-2 mAb. Conclusions: TREM-2 acts as a negative immune regulator in promoting lung cancer progress. Our study provides new mechanism for tumor immune escape and inhibitory checkpoint for immunotherapy.
Metronomic chemotherapy following the maximum tolerated dose as a multitarget antitumor therapy affecting angiogenesis, tumor dissemination, and cancer stem cells.
Authors: Vives Marta , Ginestà Mireia M. , Gracova Kristina , Graupera Mariona , Casanovas Oriol , Capella Gabriel , Serrano Teresa , Vinyals Francesc , Saez Berta Laquente
Published: March 2020

e22057

Background: Metronomic chemotherapy has been suggested as a maintenance administration strategy after the Maximum Tolerated Dose (MTD) treatment in a multi-targeted chemo-switch schedule (C-S). We report the effectiveness of this chemo-switch schedule in mice models of human pancreatic and ovarian cancer. Methods: In pancreas cancer models, Gemcitabine (G) was administered on four different schedules: metronomic (METG), Maximum Tolerated Dose (MTDG), chemo-switch schedule (C-SG, MTDG dosing followed by the METG) and anti-angiogenic (MTDG followed by the administration of monoclonal anti-VEGF receptor antibody DC101). In ovarian cancer model, animals were treated following a Cyclophosphamide chemo-switch schedule (C-S CTX). Results: In all these models C-S schedule had the most favorable effect, reaching at least 80% of tumor growth inhibition in absence of increased toxicity. Moreover, in the pancreas cancer model while peritoneal metastases were observed in control and MTD groups, no dissemination was observed in the MET and C-S groups. C-S treatment caused a decrease in angiogenesis and its effect on tumor growth was similar to obtained by the MTD followed by anti-angiogenic DC101 treatment. At the molecular level, C-S treatment combined an increase in thrombospondin-1 expression with a decrease in number of CD133+ cancer cells and triple positive CD133+/CD44+/CD24+ cancer stem cells. Conclusions: These findings provide confirmation that the chemo-switch schedule is a challenging clinical strategy with demonstrated inhibitory effects on tumor dissemination, angiogenesis and cancer stem cells.
Discordance of the ER- and HER2-status on disseminated tumor cells (DTCs) compared to the primary tumor (PT) in patients with early breast cancer (BC).
Authors: Jaeger Bernadette Anna Sophia , Finkenzeller Charlotte , Bock Carolin , Majunke Leonie , Jueckstock Julia Kathrin , Andergassen Ulrich , Neugebauer Julia Katharina , Pestka Aurelia , Friedl Thomas W. P. , Jeschke Udo , Janni Wolfgang , Doisneau-Sixou Sophie F , Rack Brigitte Kathrin
Published: March 2020

e22059

Background: The goal of this study was to evaluate both ER- and HER2-status on DTCs in the bone marrow of patients with early BC and to compare these with the corresponding PT. In case of discordant expression, DTCs might help in properly selecting targeted therapy according to their phenotype. Methods: Bone marrow (BM) samples were obtained from early BC patients at the time of first breast surgery (years 2006 to 2009). Two cytospins with 106 BM cells were prepared after Ficoll enrichment for mononuclear cells. We were able to determine the presence of DTCs (cytokeratin (CK)+cells) in a total of 58 patients. Simultaneously ER- and HER2-expressions were analyzed by immunocytochemistry using a triple fluorescence staining method. Briefly, antibodies (AB) directed against human ER (secondly labeled with Cy3, red), HER2 (DyLight488, blue) and CK (Coumarin-AMCA, green) were used. The manual analysis was conducted using a computerized fluorescence microscope (Axioskop, Zeiss, Germany). Criteria for CK-positivity and HER2-overexpression were the ring-like appearance of the respective membrane-staining and for ER-expression a nuclear staining. Data on ER- and HER2-status of the PT were available for all 58 and 54 of the patients respectively. Results: The median number of DTCs detected was 12 (range 1-95; total number of DTCs 1132). 41 (71%) of the patients had at least one ER+ DTC, 25 (43%) at least one HER2+ DTC, 15 (26%) at least one ER+/HER2+ DTC and 13 (22%) had DTCs neither expressing ER nor HER2. The concordance rate between ER-status on DTCs and PT was 74%. 6 (43%) patients with ER- PT had ER+ DTCs. The concordance rate between HER2-status on DTCs and PT was 52% and 22 (46%) patients with HER2- PT had HER2+DTCs. Seven out of 10 patients with a triple-negative PT had at least one DTC positive for ER, HER2, or both. Conclusions: Our study confirms that the ER- and/or HER2-status on DTCs might differ compared to the PT. This discordance, which we demonstrated at the protein level, could be especially important for patients with a triple-negative PT and ER+ or HER2+ DTCs, since they might respond favorably to an endocrine or HER2-targeted therapy.
Epidermal growth factor mutation in non-small cell lung cancer: Venezuelan experience.
Authors: Oblitas Alca George , Fuentes Maria Belen , Rodriguez Juan Jose Jose , Garcia Cristina , Perez Maria Angelina , Kubicek Karen , Nunez Patricia , Pichelbauer Ernestina
Published: March 2020

e22060

Background: Lung cancer represents the most common cause of death related to cancer worldwide. The current treatment strategy includes the determinations of the EGFR somatic mutations. Currently in Venezuela the frequency and the type of EGFR mutation in NSCLC patients is unknown and due to its importance in the decision of treatment, it was decided to execute this study with the objective of knowing our population’s characteristics with respect to the stage and mutational type of EGFR. Methods: In the period between November of 2010 until March of 2012, the samples of 395 patients with NSCLC diagnostic all over the country were obtained, these samples were evaluated to determine the EGFR mutation, confidence intervals of 95% were calculated based on the binomial distribution. Results: From the total of 395 patients, 80 presented an inadequate sample, 19 had no registry, 296 patients remained for the analysis. It was obtained an EGFR mutation rate of 11.5%, that was more common seen in male gender (56%), younger than 65 years (66%), ex smokers (59%), with Adenocarcinoma histology (97%). About the mutation type of the EGFR, the specific mutation of the exon 21 was found in 59% of the positive cases, and deletion of exon 19 in 38%. Conclusions: The EGFR mutation is present within the Venezuelan population and shows a particular regional demographic behavior.
The effect of cholesterol on cancer growth and metastasis: Experimental study with LDL receptor knockout mice.
Authors: Pu Daniel , Cui Yuqi , Liu Zhenzhen , Parthasarathy Sampath , Bai Xuefeng , Liu Zhengou
Published: March 2020

e22071

Background: Cholesterol is an essential component for normal cell growth. However, epidemiological studies show cholesterol levels may be associated with cancer incidence. The Multiple Risk Factor Intervention Trial reported a significant inverse relationship between cholesterol levels and lung cancer. The effects of cholesterol on cancer growth and metastasis have not been established. The aim of the present study was to determine if cholesterol alters cancer growth or metastasis. Methods: The study used 48 wild-type mice and 18 LDL receptor knockout mice (LDLR-/-). Mice were randomized into 3 groups: Group I (wild-type mice fed normal chow, n=23); Group II (wild-type mice fed high lipid chow, n=25); Group III (LDLR-/- mice fed high lipid chow, n=18). Human melanoma cells were injected subcutaneously for the assessment of growth and intravenously for metastasis. Mice were followed for 4 weeks. At the end of the study, mice were weighed and autopsy was performed. Melanoma weight, metastasis, and blood cholesterol levels were evaluated. Data was analyzed using One-way ANOVA and Chi-square. Results: LDL-cholesterol level was significantly higher in Group III than Group I (1103±472 vs.28±6; p<0.01). There was no significant difference in melanoma weight (2.74g ±1.77, 2.98g±1.98 vs. 2.18g±1.59, P=0.56) among the three subcutaneous injection groups. Lung metastasis was observed in all intravenous injection groups (11/11 vs. 10/10 vs. 8/8). There was no difference in melanoma weight or metastasis between male and female mice (P>0.05). There was no difference in net weight gain among the subcutaneous injection groups (4.12g±2.50 vs. 2.64g±2.79 vs. 3.66g±1.96, P=0.30). However, among the intravenous injection groups, weight gain was observed in Group II (2.78g±2.67) and Group III (2.36g±1.73) and weight loss in Group I (-0.32g±1.34, P<0.003). Conclusions: Cholesterol has no effect on melanoma growth or metastasis in mice. A high cholesterol diet may reduce weight loss in animals with metastatic melanoma. Further study is warranted to determine whether similar results exist in other types of cancers and to evaluate a possible beneficial effect of high lipid diets on patients with metastatic cancers.
Creating clinical target validation groups via quality assured transfer of robust clinical pharmacodynamic (PD) assays from the NCI: Clinical implementation of a PAR immunoassay in tumor biopsies.
Authors: Parchment Ralph E. , Kinders Robert J. , Ji Jiuping Jay , Srivastava Apurva K. , Ferry-Galow Katherine V. , Tomaszewski Joseph E. , Doroshow James H.
Published: March 2020

e22080

Background: Early phase clinical trials of investigational agents benefit from laboratory assays that quantify intended mechanism of action on molecular target (1° PD effects), desired changes in mechanistic biomarkers (2° PD effects), and 3° PD effects on cell survival. Robust PD assay results are valuable for informing go/no‐go decisions about continued development of new agents and for identifying combinations of targeted agents that cover the multiple molecular defects underlying many malignancies. Methods: Readying PD assays for clinical use involves validating analytical performance, identifying and qualifying critical reagents, demonstrating fit‐for‐purpose for the clinical protocol, and finalizing companion SOPs specifying specimen handling and processing. Because clinical PD questions often demand assay performance that meets or even exceeds clinical diagnostic assay standards, but key assay reagents are usually R and D‐rather than GMP‐grade, stringent reagent Quality Control is critical for preventing assay failures due to lot-to-lot variability. Results: The poly(ADP-ribose) (PAR) Immunoassay was the first qualified assay developed by NCI for implementation in early phase clinical trials. Development of stringent production and internal Quality Control specifications for accepting/rejecting new lots of critical reagents and a defined Quality Assurance Plan allowed a network of users to achieve consistent results and quality using R&D-grade source materials. Conclusions: The NCI’s Division of Cancer Treatment and Diagnosis is developing a clinical PD assay portfolio capable of quantifying 1°, 2°, and 3° PD effects. Proven clinical assays are formally transferred from the NCI to requesting sites in academia and industry via laboratory‐based certification and training, centralized access to SOPs, assistance with assay transfer, and participation in the assay’s Quality Assurance Plan. Funded by NCI Contract No HHSN261200800001E.
Routine molecular profiling of solid tumors in a community cancer center's genomic pathology laboratory.
Authors: Gunn Shelly , Reveles Xavier , Smith Lon S. , Rodriguez Gladys Isabel , Patnaik Amita , Beeram Muralidhar , Papadopoulos Kyriakos P. , Rasco Drew Warren , Tolcher Anthony W.
Published: March 2020

e22088

Background: Selection of targeted therapeutics for solid tumors depends upon detection of actionable genomic alterations in archival tissue. High-throughput molecular profiling at the community level would allow local pathologists to participate with oncologists in the delivery of personalized cancer medicine. Methods: Between March and December 2012, molecular profiling was performed on FFPE tumor tissue sent to Oncopath (our in-house CAP/CLIA genomic pathology laboratory) from 330 patients with diverse types of metastatic and non-resectable solid tumors. Tumor-targeted DNA was analyzed for genome-wide copy number alterations by array-based comparative hybridization (CGH), and mutation analysis (MA) using multiplex PCR with mass spectrometric genotyping (mass spec), or single gene allele-specific PCR (PCR). Results for 96 actionable genes analyzed by CGH, and MA were correlated with clinically or pre-clinically available targeted therapeutics. Actionable biomarkers for FDA approved therapies (e.g. BRAF V600E/vemurafenib) and prospective biomarkers for Phase I/II trials (e.g. CCND1 gene amplifications and CDKN2A gene deletions/CDK inhibitors) were reported for each patient. Results: Of 330 cases, 216 had at least one and as high as seven actionable biomarkers. The remaining 114 cases had either no tumor specific changes (n=17) or non-actionable oncogenic changes (n=97). The most commonly analyzed tumors were breast, brain, lung, colorectal, pancreatic, head & neck, and ovarian. Rare tumor types with positive biomarkers included an HRAS Q61R positive adenoid cystic carcinoma and a BRAF V600E positive pleomorphic xanthroastrocyoma. Tumor DNA was stored for 100% of patients and all cases were reported synoptically listing actionable biomarkers with therapeutic recommendations. Conclusions: Molecular profiling in a community cancer center facilitates biomarker detection for rational direction of patients to targeted therapeutics. New technologies for detection of actionable biomarkers will continue to be implemented in our center to improve the likelihood of effective treatment for each individual patient’s tumor.
Association of myeloid-derived suppressor cells (MDSCs) in peripheral blood with characteristics in breast cancer patients.
Authors: Shen Peng , Wang Aijuan , Ke Wei , He Mengye , Yu Lanfang , Huang Sui
Published: March 2020

e22094

Background: The immune therapy of human cancer is a promising therapeutic approach. Myeloid-derived suppressor cells (MDSCs) have been confirmed as an indispensable component of immune system and associated with tumor induced immune suppression. Thus, MDSCs play an important role in immune therapy. Although a lot of research work has been done, currently there exists limited data about MDSCs status in cancer patients. The aim of this study was to determine whether MDSCs levels in peripheral blood were different in healthy volunteers, benign breast tumor patients and breast cancer patients, and to investigate the relation between MDSCs and several characteristics in breast cancer patients. Methods: Peripheral blood cells (PBC) were collected from 36 normal healthy volunteers, 52 patients with breast cancer and 62 patients with benign breast tumor. Peripheral blood samples of breast cancer patients were obtained before treatment. Circulating MDSCs (identified as Lin-/Lo HLA-DR-CD33+) were measured by flow cytometry. Statistical methods included two sample t tests and one way ANOVA. Results: The percentage of MDSCs was higher in breast cancer than benign breast tumor patients and healthy volunteers (2.017% vs 1.578% vs1.594%; p=0.0002), and the stage III/IV breast cancer patients had more MDSCs accumulation than those in stage I/II (2.92% vs 2.155%; P=0.0806). The levels of MDSCs in peripheral blood showed a significant correlation with HER-2/neu expression in breast cancer patients, but had no correlation with incidence age, ER status, PR status and P53 expression respectively. The percentage of MDSCs was lower in HER-2/neu over expression breast cancer patients than HER-2/neu negative patients (1.715% vs 2.882%, GP=0.0012). Conclusions: MDSCs seemed to have positive correlation with clinical stage and negative correlation with HER-2/neu expression in breast cancer patients . Further research will contribute to the better understanding of the connection between MDSCs and breast cancer, to develop better approaches to breast cancer therapy.
A combined EpCAM and MCAM circulating tumor cell (CTC) CellSearch enrichment to improve CTC capture rate in stage II/III breast cancer: A Dutch Breast Cancer Trialists' Group (BOOG) side study.
Authors: Onstenk Wendy , Kraan Jaco , Charehbili Ayoub , Mostert Bianca , Smit Vincent T. H. B. M. , Kroep Judith R. , Nortier J. W. R. , Liefers Gerrit-Jan , van de Ven Saskia , Heijns Joan Barbara , Kessels Lonneke , Van Laarhoven Hanneke W. M. , Bos Monique M.E.M. , Van De Velde Cornelis J. H. , Gratama Jan-Willem , Sieuwerts Anieta M. , Martens John W. M. , Foekens John A. , Sleijfer Stefan
Published: March 2020

e22106

Background: Using the CellSearch System (Veridex, USA), ≥1 CTC/7.5 mL of peripheral blood is found in ~20% of primary breast cancer (BC) patients. However, epithelial cell adhesion molecule (EpCAM)-negative CTCs may be missed. We previously showed that a subset of BC cell lines lacks EpCAM, but does express melanoma cell adhesion molecule (MCAM) and that MCAM-positive CTCs (M-CTCs) do occur in metastatic breast cancer patients. In a side-study of the NEOZOTAC trial, a randomized study comparing neoadjuvant chemotherapy (NAC) with or without zoledronic acid in stage II/III BC, we investigated whether combined enrichment with EpCAM and MCAM increases the CTC detection rate. Besides, we hypothesized that CTC counts would correlate with pathological complete response (pCR) to NAC. Additionally, as exploratory study, circulated endothelial cells (CECs) were measured. Methods: At baseline and after the 1st chemotherapy cycle, blood was drawn from all patients in the NEOZOTAC trial who gave additional informed consent. The CellSearch System was used to count EpCAM-positive CTCs (E-CTCs) and M-CTCs. For the latter, the anti-EpCAM-ferrofluid was replaced by an anti-MCAM-ferrofluid (both Veridex). CECs were measured using flow cytometry. Results: At baseline, blood was received from 73 patients; ≥1 E-CTCs and M-CTCs were detected in 14/73 (19%, median 1) and 8/68 (12%, median 1), respectively. Combining both counts, ≥1 CTCs were detected in 17/68 (25%) of patients (p=0.01). After 1 chemotherapy cycle, E-CTCs, M-CTCs and overall CTCs were detected in 9/47 (19%), 7/45 (16%), and 13/44 (30%; p<0.01) patients. Median CEC count was 43 at baseline and 142 after cycle 1 (p<0.001). No differences in CTC and CEC count were found between the treatment arms. Baseline CTC counts, nor changes in counts, were associated with pCR. Conclusions: Combined enrichment of CTCs with EpCAM and MCAM significantly increased CTC detection rates in stage II/III BC with about 10%. CTC counts were not associated with pCR to NAC. Associations with disease free survival will follow when available.
Update on pilot study on antitumor efficacy of intravenously applied synergistic combinations of diflunisal, PAS, and aspirin in patients with advanced solid tumors.
Authors: Drevs Joachim , D'Urso Susanne , Dayton Martin , Martinez Lukas , Mueller-Huebenthal Jonas , Kamijo Izumi , Kreutz Werner
Published: March 2020

e22114

Background: Due to chronic hypoxia cancer cells are growing in a more acidic environment compared to physiological tissue. Recent preclinical in vitro and in vivo data have shown direct pH-sensitive anti-tumor activity by pore formation and apoptosis when synergistically acting combinations of Diflunisal, Paraaminosalicylic acid (PAS) and Aspirin were applied. Therefore, a pilot study was performed to evaluate its clinical relevance and its update is presented. Methods: A total of 65 patients (n=65) with advanced solid tumors and the lack of standard treatments were treated with a protocol consisting of 3-5 weeks with 2 daily i.v. infusions of Diflunisal and Aspirin for 4 days per week after they had signed an informed consent form. Response evaluation was assessed by PET/CT differentiating in metabolic and metric (RECIST) response and tumor marker where available. Results: Out of 65 patients treated 8 patients suffered from colorectal cancer, 17 from breast cancer, 6 from ovarian cancer, 3 from lung cancer, 3 from gastric cancer, 3 from glioblastoma, 3 from CUP, 1 from hemangioendothelioma, 1 from pleuramesothelioma, 1 from non-Hodgkin lymphoma, 2 from pancreatic cancer, 1 from choledochus cancer, 3 from prostate cancer, 5 from sarcomas, 2 from head and neck cancer, 1 from melanoma, 1 from cervical cancer, and 1 from thyroid cancer, respectively. Side effects related to the therapy have been fatigue grade I (30%), nausea grade I (13,3%), tinnitus (25%), hypertension (2,5%), dyspnea (3,3%) and burning sensation in tumor areas (65%). 33 patients were evaluable for metric response, 20 for metabolic and 19 for tumor marker response assessment. For tumor marker follow up 11 % had a CR, 53 % a PR, 26 % a SD of > 3 month and 11 % a PD. For metric follow up 3 % had a CR, 33 % a PR, 39 % a SD of > 3 month and 24 % a PD. For metabolic follow up 10 % had a CR, 35 % a PR, 35 % a SD of > 3 month and 20 % a PD. Conclusions: This continuing pilot study confirms a direct pH-sensitive synergistic anti-tumor activity of intravenous Diflunisal, PAS and Aspirin in patients with advanced solid tumors. Therefore, a further evaluation in a controlled clinical phase II study should be performed.
Molecular analysis in breast cancer subtypes and correlation with pathologic complete response (pCR) to neoadjuvant chemotherapy.
Authors: Gonzalez Cao Maria , Costa Carlota , Molina-Vila Miguel Angel , Cusido Maria Teresa , Viteri Ramirez Santiago , Baulies Sonia , Martinez-Bueno Alejandro , Fabregas Rafael , Tresserra Francesc , Sansano Irene , Ara Carmen , Amselen David , Ubeda Belen , Simo-Perdigo Marc , Gasco Amaya , Rodriguez Ignacio , Rosell Rafael
Published: March 2020

e22119

Background: Although it is know that pCR following neoadjuvant chemotherapy is more frequent in some subtypes of breast cancer such as Triple Negative (TN) or erb2 tumors, the predictive role of gene expression and mutation status is not well defined in this setting. Methods: We analyzed samples from 41 patients (p) prospectively treated with neoadjuvant chemotherapy (sequential AC followed by weekly TXL, or inverse sequence, plus trastuzumab for erb2 positive p). Pathologic response (PR) was classified according to Miller-Payne and RCB criteria. Radiologic evaluation was performed by ultrasonography, dynamic MR and PET-TAC after each chemotherapy sequence. We performed expression analysis of AXL, BRCA1, RAP80, BIM, EZH2, ROR1, FGFR1, PTPN12, YAP, GAS6, beta-TRCP, HIF1 alpha and ZNF217 by RT-PCR, and mutational status of p53 and PI3K genes in pretreatment biopsies. Statistical analysis was performed using Mann-Whitney U and Pearson’s chi-squared tests. Results: pCR was detected in 5 p (3TN, 2 erb2) of 25 p (9 luminal A, 5 luminal B, 6 erb2 and 5 TN) evaluated for PR at time of submitting this abstract. TN tumors had lower levels of RAP80 (p=.0013), PTPN12 (p=.003), beta TRCP (p=.001), ZNF217 (p=.014) and YAP (p=.097). Luminal B tumors had low levels of YAP and the highest levels of FGFR1 (p=.09) and ZNF217 (p=.014). Luminal A tumors had high levels of beta-TRCP (p=.003). We found no differences in BRCA1, AXL, BIM, EZH2, ROR1, GAS6 and HIF1 levels by breast cancer subtype. P with low levels of FGFR1 (p=.087), HIF1alpha (p=.07) or EZH2 (p=.005) had higher probability of pCR. No pCR was observed in p with higher levels of AXL, EZH2, RAP80, GAS6, beta TRCP, HIF alpha. Four p had p53 mutations (1 luminal B, 1 erb2 and 2 TN) and 4 p had PI3K mutations (2 luminal A, 1 erb2, 1 luminal B). There was no correlation between p53 status and PR. P with PI3K mutations did not achieve pCR vs 46% of p with wild type PI3K (p=.23). Conclusions: Gene expression profile varies by breast cancer subtype. Chemosensitivity could be higher in tumors with lower levels of FGFR1, HIF1alpha or EZH2. Further results will be presented.
Overcoming cell size escape from tumor treating fields using a varying frequency treatment paradigm in vitro.
Authors: Schneiderman Rosa S. , Giladi Moshe , Porat Yaara , Munster Mijal , Weinberg Uri , Kirson Eilon David , Palti Yoram
Published: March 2020

e22134

Background: TTFields Therapy at 200 kHz received FDA approval for the treatment of patients with recurrent glioblastoma (GBM) based on the results of a phase III clinical trial. Radiological responses were observed in 14% of the treated patients. The lack of radiological response in the remaining patients suggests that GBM cells may escape the effect of TTFields over time. The goals of the present study were to identify possible routes by which cancer cells can escape the antimitotic effect of TTFields and to explore ways to overcome such escape mechanisms. Methods: Measurements of cell size before and after 72 hours TTFields application revealed a significant volume increase in 10 cancer cell lines (17% - 101%). A2780 ovarian cancer cultures (cell volume 2.0+0.2 pL) exhibit maximal inhibition by TTFields at 200 kHz. Twenty four hour application of 200 kHz TTFields led to a 70+29% increase in average cell volume (p<0.05). FACS analysis showed that TTFields application to A2780 cells led to a 44% increase in the G2 population due to the antimitotic effect of TTFields. However, the increase in the G2 proportion is not sufficient to explain the increase in cell volume seen after TTFields application. Furthermore, after an additional 24 hours of treatment, the fraction of cells in the G2 population returned to its original percentage (30%), while the increase in cell volume remained high (67+26%). Results: Previous publications demonstrated an inverse relationship exists between cell size and optimal TTFields frequency. Thus an increase in cell size may allow cells to avoid the maximal TTFields effect. To attempt to overcome such cell size escape we decreased the frequency of the TTFields from 200 to 150 kHz after 24 hours treatment to coincide with the increase in cell volume. We found that varying the frequency from 200 to 150 kHz increased the inhibitory effect of TTFields compared to continuous treatment at 200Khz (37+10% and 57+16% decrease in cell count, respectively; p=0.043). Conclusions: This is the first evidence of an escape mechanism from the antimitotic effect of TTFields. The results presented provide a simple way to restore treatment efficacy by varying TTFields frequency in parallel to the change in cell volume.
BIM deletion polymorphism and the clinical outcome of patients with advanced NSCLC who received first-line platinum-based chemotherapy: Preliminary results.
Authors: Zhang Jie , Zheng Di
Published: March 2020

e22137

Background: The up-regulation of the proapoptotic polypeptide BIM was acquired during the cancer cell apoptosis induced either by cytotoxic agents or tyrosine kinase inhibitors (TKIs). Germline polymorphism of BIM deletion was associated with shorter progression free survival (PFS) in patients with EGFR-mutated lung cancer treated with EGFR-TKIs. The aim of this study is to investigate the correlation between the germline polymorphism of BIM deletion and the clinical outcome of patients with advanced NSCLC who received first-line platinum based chemotherapy. Methods: Since June of 2012, 140 consecutive chemo-naive advanced NSCLC patients who completed two cycles of first-line platinum based chemotherapy evaluable for response assessment were eligible for inclusion in Shanghai Pulmonary Hospital. Patients with stable disease were excluded in order to eliminate the indolent disease effect. Each patient’s genomic DNA was extracted from peripheral blood sample, then the genotype of the BIM deletion was performed using a single PCR reaction with the primers 5’-AATACCACAGAGGCCCACAG-3’ and 5’-GCCTGAAGGTGCTGAGAAAG-3’. Results: There are 49 patients who responded to chemotherapy (RE group) while 32 patients developed progressive disease (PD group). The clinical characteristics at baseline and chemotherapy regimens received were balanced between the two groups except for more stage IV patients noted in PD group (57 vs 58 years old of median age, 59% vs 62.5% of male, 63% vs 100% of stage IV in RE group and PD group respectively, and the predominant regimen with gemcitabine plus cisplatin in both groups). BIM deletion polymorphism were detected in 4 of RE group (4/49, 8.2%) and 8 of PD group (8/32, 25%) respectively, (odds ratio=0.267, 95% CI=0.073-0.977, P=0.046). Conclusions: The germline polymorphism of BIM deletion may contribute to the intrinsic resistance to the chemotherapy in advanced NSCLC.