Tumor Tissue Sections Research Articles

Detecting breast cancer using artificial intelligence: Convolutional neural network.

One of the most broadly founded approaches to envisage cancer treatment relies upon a pathologist's efficiency to visually inspect the appearances of bio-markers on the invasive tumor tissue section. Lately, deep learning techniques have radically enriched the ability of computers to identify objects in images fostering the prospect for fully automated computer-aided diagnosis. Given the noticeable role of nuclear structure in cancer detection, AI's pattern recognizing ability can expedite the diagnostic process. In this study, we propose and implement an image classification technique to identify breast cancer. We implement the convolutional neural network (CNN) on breast cancer image data set to identify invasive ductal carcinoma (IDC). The proposed CNN model after data augmentation yielded 78.4% classification accuracy. 16% of IDC (-) were predicted incorrectly (false negative) whereas 25% of IDC (+) were predicted incorrectly (false positive). The results achieved by the proposed approach have shown that it is feasible to employ a convolutional neural network particularly for breast cancer classification tasks. However, a common problem in any artificial intelligence algorithm is its dependence on the data set. Therefore, the performance of the proposed model might not be generalized.

70-Gene Signature and Tumor-Stroma Ratio Select Different Groups of Patients with Breast Cancer at Risk for Recurrence

Background: Strategies for breast cancer care are improving. However, there is a clinical need for a better selection of patients who are at risk for recurrence and benefit from treatment with chemotherapy. The 70-gene signature (70-GS) guides chemotherapy decision making based on the mRNA microarray assessment but is expensive. The tumor-stroma ratio (TSR) is a morphological and cheap biomarker based on the amount of stroma in the primary tumor. In the present study the association between the 70-GS and TSR was investigated. Materials and methods: Frozen tumor tissue sections of 102 ER+/Her2- patients were send to Agendia for 70-GS analysis in an earlier study. The results of the 70-GS were available, and hematoxylin and eosin stained sections of the 102 tumors were scored for TSR. Concordance between the TSR and the 70-GS was analyzed. No data of patient outcome is available. Results: Of the patients in the 70-GS high risk category (n=46), 23 patients (50%) were classified into the stroma-high group according to previously established thresholds. 55% (n=31) of the low-risk 70-GS patients, had a stroma-low profile. The overall concordance between the 70-GS and TSR was 53% and no association was observed (Chi-square: p=0.917). Patients with a 70-GS high-risk test result had a significantly higher tumor grade and stroma-high patients were significantly older and had larger tumors. Conclusion: In conclusion, no association between the 70-GS and TSR was found. This suggests that both methods select different groups of patients at risk for recurrence, and may have additional prognostic value.

Increased formation of reactive oxygen species during tumor growth: Ex vivo low-temperature EPR and in vivo bioluminescence analyses

Previous studies have shown that reactive oxygen species (ROS) such as superoxide or hydrogen peroxide generated at low levels can exert a tumor-promoting role via a redox-signaling mechanism. Reports also suggest that both tumorigenesis and tumor growth are associated with enhanced ROS formation. However, whether ROS levels or ROS-derived oxidative marker levels increase during tumor growth remains unknown. In this study, in vivo bioluminescence imaging with a boronate-based pro-luciferin probe was used to assess ROS formation. Additionally, probe-free cryogenic electron paramagnetic resonance was used to quantify a characteristic aconitase [3Fe4S]+ center that arises in the tumor tissue of mouse xenografts from the reaction of the native [4Fe4S]2+ cluster with superoxide. Results indicated that tumor growth is accompanied by increased ROS formation, and revealed differences in oxidant formation in the inner and outer sections of tumor tissue, respectively, demonstrating redox heterogeneity. Studies using luciferin and pro-luciferin probes enabled the assessment of tumor size, ROS formation, and bioenergetic status (e.g., ATP) in luciferase-transfected mice tumor xenografts. Probe-free ex vivo low-temperature electron paramagnetic resonance can also be translated to clinical studies.

Dual stimuli-guided lipid-based delivery system of cancer combination therapy

The combination therapy, as an emerging strategy for improved clinical efficacy of cancer therapy, may not achieve effective response owing to the lack of highly selective and efficient tumor targeting. Herein, a dual stimuli-guided chemo/magnetothermal combination therapy system based upon histamine dodecyl carbamate (HDC)-coated doxorubicin (DOX)/magnetite-loaded solid lipid nanoparticles (SLNs) was developed for enhanced anticancer effects. Taking advantage of the dual pHe-induced electrostatic and magnetic guidance, the in vitro cellular uptake of these functionalized SLNs by TRAMP-C1 cancer cells was highly enhanced, leading to remarkably increased anticancer ability. With the highly selective delivery of the therapeutics toward tumor via the dual stimuli-mediated guidance, the effective growth inhibition of tumors with the small initial size (ca 50 mm3) by only chemotherapy was observed whereas the combination therapy was essentially required to fully inhibit the growth of large tumors (200 mm3). The IHC staining of tumor tissue sections with the combination therapy against large tumors showed the appreciable increase of tumor cell apoptosis and reduction of tumor angiogenesis. The results suggest that the dual stimuli-guided combination therapy system developed herein be prominent in fully inhibiting tumor growth even with the solid tumors of large size at the onset of the treatment.

Associations of the BRAF V600E Mutation and PAQR3 Protein Expression with Papillary Thyroid Carcinoma Clinicopathological Features.

The BRAFV600E mutation is the most prevalent genetic event in patients with papillary thyroid cancer (PTC). However, no study has investigated the expression of PAQR3 in papillary thyroid tissues in relation to the BRAFV600E mutation and the clinicopathological features of PTC patients. Furthermore, the potential associations of the BRAFV600E mutation, PAQR3 expression and clinicopathological parameters in the cancerous tissues of PTC patients have not been investigated. This study was conducted on 60 patients with PTC who were treated surgically at our institution from 2017 to 2018. PCR was used to amplify DNA by the amplification refractory mutation system (ARMS) method to detect BRAFV600E gene mutations. In addition, immunohistochemical techniques were utilized to assess PAQR3 expression in tumor tissue sections. The BRAFV600E mutation was associated with lymph node metastasis (LNM, p < 0.05) but not with other clinicopathological features. Low PAQR3 expression was associated with extrathyroidal extension and LNM (χ2 = 7.143, p = 0.009; χ2 = 6.459, p = 0.014, respectively). Furthermore, a statistically significant association was observed between chronic lymphocytic thyroiditis and LNM (χ2 = 5.275, p = 0.0250). A linear relationship between the BRAFV600E mutation and PAQR3 protein expression has not been identified. These factors may be independent risk factors of extrathyroidal extension and LNM in PTC and be used to indicate the invasiveness of PTC tumors. Higher quality, multivariate analyses based on larger samples from around the world are urgently needed to further validate and revise our findings in the future.

Precision biomarker discovery powered by microscopy image fusion-assisted high spatial resolution ambient ionization mass spectrometry imaging

Mass spectrometry imaging (MSI) using the ambient ionization technique enables a direct chemical investigation of biological samples with minimal sample pretreatment. However, detailed morphological information of the sample is often lost due to its limited spatial resolution. In this study, predictive high-resolution molecular imaging was produced by the fusion of ambient ionization MSI with optical microscopy of routine hematoxylin and eosin (H&E) staining. Specifically, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) mass spectrometry were employed to visualize lipid and protein species on mice tissue sections. The resulting molecular distributions obtained by ambient ionization MSI-microscopy fusion were verified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MSI and immunohistochemistry (IHC) staining. Label-free molecular imaging with 5-μm spatial resolution can be acquired using DESI and nanoDESI, whereas the typical spatial resolution of ambient ionization MSI was ∼100 μm. In this regard, sharpened molecular histology of tissue sections was achieved, providing complementary references to the pathology. Such a multi-modal integration enables the discovery of potential tumor biomarkers. After image fusion, more than a dozen potential biomarkers on a metastatic mouse lung tissue section and Luminal B breast tumor tissue section were identified.

Hsp60 and IL-8 axis promotes apoptosis resistance in cancer

BackgroundInterleukin-8 (IL-8) and heat shock protein 60 (Hsp60) play crucial roles in cell survival and maintenance of cellular homoeostasis. However, cross talks between these two proteins are not defined.MethodsIL-8 expression in tumour tissue sections was analysed by immunohistochemistry. IL-8 expression and release in cancer cells was quantified using enzyme-linked immunosorbent assay (ELISA). Apoptosis was quantified using caspase activity and Annexin-V/PI staining.ResultsWe observed IL-8 release from cancer cells in response to histone deacetylase inhibitor, apicidin (Api), and non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, thapsigargin (TG). IL-8 release was increased upon TG-treatment. TG-induced IL-8 expression was reduced in the presence of Api in Bax-dependent manner. Increased apoptosis was associated with decreased IL-8 expression in response to combined treatment of TG and Api. TG and Api combination induced caspase-8 and caspase-9 dependent apoptosis. Hsp60 knockdown abrogated IL-8 expression induced by Api, TG, and their combination. The level of TGF-β, an upstream regulator of IL-8, was decreased upon Hsp60-silencing. Knocking down Hsp60 decreased IL-8 expression and its release in prostate cancer cell xenograft tumours in SCID mice.ConclusionThis study describes the underlying mechanism associated with apoptosis resistance mediated via Hsp60-IL-8 axis in cancer.

Increased 14C-acetate accumulation in IDH-mutated human glioblastoma: implications for detecting IDH-mutated glioblastoma with 11C-acetate PET imaging.

Recently, the potential value of isocitrate dehydrogenase (IDH) mutation as a prognostic marker in glioblastomas has been established. Glioblastomas are classified by their IDH mutation status under the 2016 WHO classification system. However, noninvasive diagnostic methods for the mutation status in glioblastoma patients have not been established so far. The purpose of this study was to evaluate the difference of acetate metabolism between in glioblastomas with wild-type IDH and in those with IDH mutation by comparing the uptake of 14C-acetate using genetically engineered glioblastoma cell lines in vitro and in vivo. We established glioblastoma cells (U251) expressing IDH1 R132H and examined the cell uptake of [1-14C]acetate. Biodistribution studies and an autoradiographic study for U251 cell tumor-bearing mice (BALB/c-nu/nu) with or without the IDH1 mutation were performed 1h after [1-14C]acetate administration. Significantly higher uptake of [1-14C]acetate was observed in U251/IDH1 R132H cells than in U251/IDH1 wild-type cells both in vitro (10.11 ± 0.94 vs. 4.26 ± 0.95%dose/mg, p = 0.0047) and in vivo (0.97 ± 0.14 vs. 0.66 ± 0.05%ID/g; p = 0.0037). Tumor-to-muscle ratios were also significantly higher in U251/IDH1 R132H tumors (3.36 ± 0.41 vs. 1.88 ± 0.59, p = 0.0030). The autoradiographic study shows the entirely higher radioactivity of the U251/IDH1 R132H tumor tissue section than that of the U251/IDH1 Wild-type tumor. In vitro and in vivo studies demonstrated that the uptake of radiolabeled acetate was significantly higher in IDH-mutated cells than in IDH-wild-type cells.

Identification of molecular markers for pre-thrombotic state: validation in the rabbits with tibia fracture or lung cancer

Background: Currently there is no reports on identifying molecular markers for pre-thrombotic state in animal experimental settings. It’s necessary to explore the potential molecular markers for pre-thrombotic state to provide evidence for the pharmacotherapy of venous thromboembolism (VTE). Methods: A total of 36 male rabbits were randomly divided into Sham, Tibia fracture, Lung cancer, Tibia fracture and lung cancer group. Pro-thrombin time (PT), thrombin time (TT), activated partial pro-thrombin time (APTT), fibrinogen (FIB), D-dimer (D-D) and anti-thrombin III (AT-III) at baseline, 1, 7, 14 and 21 days after operation were detected. Immuno-histochemical staining of tumor tissue sections was performed to observe the expression of CD31, VEGF and vWF. Western blot and qPCR were used to detect the expression levels of ANG and VEGF gene in tumor tissues. Furthermore, we analyzed the tumor tissue with HE staining, immunochemistry, qPCR and Western blot. all the data were statistically analyzed with GraphPad Prism 6. Results: There were 0, 1, 2, 4 cases of VTE in sham, tibia fracture, lung cancer, tibia fracture and lung cancer group respectively. The level of PT, APTT, TT and AT-III were significantly decreased after operation (all P<0.05), whereas the level of FIB and D-D were significantly increased after operation (all P<0.05). The level of PT, APTT, TT and AT-III were gradually decreased in tibia fracture, lung cancer, tibia fracture and lung cancer group respectively (all P<0.05), while the level of FIB and D-D were gradually increased in tibia fracture, lung cancer, tibia fracture and lung cancer group respectively (all P<0.05). The medullary lymphocytes increased significantly in the lung cancer group, tibia fracture and lung cancer group (all P<0.05), and the number of medullary lymphocytes in tibia fracture and lung cancer group is significantly higher than that of lung cancer group (P<0.001). The expression of CD31, VEGF and vWF in Tibia fracture and lung cancer group is significantly higher than that of lung cancer group (all P<0.001). The expression ANG and VEGF in tibia fracture and lung cancer group is significantly higher than that of lung cancer group (all P<0.01). Conclusions: PT, TT, APTT, FIB, D-D and AT-III seem to be sensitive molecular markers for identifying the pre-thrombotic state. CD31, VEGF, ANG and vWF play an important role in the progression of the pre-thrombotic state.

Association of biomarkers in pre-invasive and invasive cervical carcinoma

Introduction: Various reports suggest linkage of protein expression to HPV status in cervical carcinomas. The aim of this study was to determine a possible association between HPV type, protein expression and DNA content in both preinvasive (CIN III) and invasive squamous cell carcinoma (SCC) of the cervix, as well as differences between the studied groups in these parameters. Materials and methods: Sections of formalin-fixed paraffin-embedded tumor tissue from 47 cases of CIN III and 60 cases of invasive SCC of the cervix were subjected to HPV genotyping using LiPA (Line immuno-probe assay) and Flow cytometry for DNA content analysis. Also, immunohistochemical staining was performed. Obtained data were analyzed in SPSS using the Chi square test. Results: The major difference (p=0.007) between CIN III and invasive SCC of the cervix was found in DNA content - more aneuploid and tetraploid cases in invasive SCC of the cervix compared to CIN III. Although we observed a similar distribution of studied parameters in both groups, no statistically significant association was found between these parameters, except for p53 and pRb (p=0.018). Conclusion: The studied groups differ in DNA content. A significant association between analyzed parameters was observed in Invasive SCC of the cervix between p53 and pRb expression.

Identification of molecular markers for pre-thrombotic state: validation in the rabbits with tibia fracture or lung cancer

BackgroundCurrently there is no reports on identifying molecular markers for pre-thrombotic state in animal experimental settings. It’s necessary to explore the potential molecular markers for pre-thrombotic state to provide evidence for the pharmacotherapy of venous thromboembolism (VTE).MethodsA total of 36 male rabbits were randomly divided into Sham, Tibia fracture, Lung cancer, Tibia fracture and lung cancer group. Pro-thrombin time (PT), thrombin time (TT), activated partial pro-thrombin time (APTT), fibrinogen (FIB), D-dimer (D-D) and anti-thrombin III (AT-III) at baseline, 1, 7, 14 and 21 days after operation were detected. Immuno-histochemical staining of tumor tissue sections was performed to observe the expression of CD31, VEGF and vWF. Western blot and qPCR were used to detect the expression levels of ANG and VEGF gene in tumor tissues. Furthermore, we analyzed the tumor tissue with HE staining, immunochemistry, qPCR and Western blot. all the data were statistically analyzed with GraphPad Prism 6.ResultsThere were 0, 1, 2, 4 cases of VTE in sham, tibia fracture, lung cancer, tibia fracture and lung cancer group respectively. The level of PT, APTT, TT and AT-III were significantly decreased after operation (all P<0.05), whereas the level of FIB and D-D were significantly increased after operation (all P<0.05). The level of PT, APTT, TT and AT-III were gradually decreased in tibia fracture, lung cancer, tibia fracture and lung cancer group respectively (all P<0.05), while the level of FIB and D-D were gradually increased in tibia fracture, lung cancer, tibia fracture and lung cancer group respectively (all P<0.05). The medullary lymphocytes increased significantly in the lung cancer group, tibia fracture and lung cancer group (all P<0.05), and the number of medullary lymphocytes in tibia fracture and lung cancer group is significantly higher than that of lung cancer group (P<0.001). The expression of CD31, VEGF and vWF in Tibia fracture and lung cancer group is significantly higher than that of lung cancer group (all P<0.001). The expression ANG and VEGF in tibia fracture and lung cancer group is significantly higher than that of lung cancer group (all P<0.01).ConclusionsPT, TT, APTT, FIB, D-D and AT-III seem to be sensitive molecular markers for identifying the pre-thrombotic state. CD31, VEGF, ANG and vWF play an important role in the progression of the pre-thrombotic state.

PD-L1 expression is a predictive biomarker for CIK cell-based immunotherapy in postoperative patients with breast cancer

BackgroundA sequential combination of radiochemotherapy/endocrinotherapy and cytokine-induced killer cell (CIK) infusion has been shown to be an effective therapy for post-mastectomy breast cancer based on statistical analysis of the patient population. However, whether an individual could obtain an improved prognosis from CIK cell-based treatment remains unknown. In the present study, we focused on immune microenvironment regulation and specifically investigated the relationship between PD-L1 expression and survival benefit from CIK immunotherapy in breast cancer.MethodsA total of 310 postoperative breast cancer patients who received comprehensive treatment were enrolled in this retrospective study, including 160 patients in the control group (received chemotherapy/radiotherapy/endocrinotherapy) and 150 patients in the CIK cell treatment group (received chemotherapy/radiotherapy/ endocrinotherapy and subsequent CIK infusion).ResultsWe found that overall survival (OS) and recurrence-free survival (RFS) were significantly better in the CIK group than that in the control group. PD-L1 expression in tumor tissue sections was showed to be an independent prognostic factor for patients in the CIK treatment group using multivariate survival analysis. Further survival analysis in the CIK group showed that patients with PD-L1 tumor expression exhibited longer OS and RFS. In addition, among all patients who were enrolled in this study, only the patients with PD-L1 expression experienced survival benefits from CIK treatment.ConclusionsOur study showed the relationship between PD-L1 expression and CIK therapy and revealed that PD-L1 expression in the tumor is as an indicator of adjuvant CIK therapy for postoperative breast cancer.

Dual-tracer PET of viable tumor volume and hypoxia for identification of necrosis-containing radio-resistant Sub-volumes

Introduction: Positron emission tomography (PET) using hypoxia-selective tracers like FAZA may guide radiation dose-escalation approaches. However, poor resolution combined with slow tracer retention in relatively inaccessible target cells and slow clearance of unbound tracer results in low-contrast images, and areas where viable hypoxic tracer retaining cells and necrosis (no tracer) are intermixed may pass unnoticed during image thresholding. Here we hypothesized that a clinical feasible one-day dual tracer approach that combines a short-lived (e.g., 11C labeled) metabolic tracer that provides voxel-wise information on viable tissue volume (preferably independently of tumor microenvironment) and a hypoxia marker, may limit threshold-based errors.Material and methods: 11C-acetate and 11C-methionine uptake was quantified in tumor cell lines under tumor microenvironment-mimicking conditions of high/low O2 (21%/0%) and pH (7.4/6.7). Next, tumor-bearing mice were administered FAZA and sacrificed 1 h (mimics a clinical low-contrast image scenario) or 4 h (high contrast) later. In addition, all mice were administered pimonidazole (hypoxia) and 14C-methionine 1 h prior to sacrifice. Tumor tissue sections were analyzed using dual-tracer autoradiography. Finally, FAZA, or FAZA normalized to 14C-methionine retention (to adjust for differences in viable tissue volume) was compared to hypoxic fraction (deduced from immune-histological analysis of pimonidazole; ground truth) in PET-mimicking macroscopic pixels with variable extent of necrosis/hypoxia.Results/conclusions: Low pH stimulated 11C-acetate retention in many cell lines, and uptake was further modified by anoxia, compromising its usefulness as a universal marker of viable tumor volume. In contrast, 11C-methionine was largely unaffected by the in vitro microenvironment and was further tested in mice. Necrosis increased the risk of missing hypoxia-containing pixels during thresholding and hypoxic fraction and FAZA signal correlated poorly in the low contrast-scenario. Voxel-based normalization to 14C-methionine increased the likelihood of detecting voxels harboring hypoxic cells profoundly, but did not consistently improve the correlation between the density of hypoxic cells and tracer signal.

Evaluación semicuantitativa de gliomas cerebrales en adultos: un enfoque de las características neuropatológicas.

Gliomas are neoplasms with high recurrence and mortality. Due to the difficulty to apply the World Health Organization (2016) classification, developing countries continue to use histological evaluation to diagnose and classify these neoplasms. To develop a semi-quantitative scale to numerically grade gliomas morphological characteristics. A cohort of patients with gliomas was assessed and followed for 36 months. Tumor tissue sections were analyzed and graded, including aspects such as cell line, cellularity, nuclear pleomorphism, mitosis, endothelial hyperplasia, hypoxic changes, apoptotic bodies, necrosis, hemorrhage and proliferation index. 58 cases were analyzed. Low-grade gliomas median score was 12 points (9 and 13.5 for percentiles 25 and 75, respectively), whereas for high-grade gliomas it was 17 points (16 and 20.5 for percentiles 25 and 75, respectively) (p < 0.0001). Thirty-six-month survival of patients with low (13/17) and high grade gliomas (6/41) was also significantly different (p < 0.0001). The semi-quantitative morphological scale allows an objective evaluation of gliomas, with an adequate correlation between the score, tumor grade and survival time.

Assessment of Long-term Distant Recurrence-Free Survival Associated With Tamoxifen Therapy in Postmenopausal Patients With Luminal A or Luminal B Breast Cancer

Patients with estrogen receptor (ER)-positive breast cancer have a long-term risk for fatal disease. However, the tumor biological factors that influence the long-term risk and the benefit associated with endocrine therapy are not well understood. To compare the long-term survival from tamoxifen therapy for patients with luminal A or luminal B tumor subtype. Secondary analysis of patients from the Stockholm Tamoxifen (STO-3) trial conducted from 1976 to 1990, which randomized postmenopausal patients with lymph node-negative breast cancer to receive adjuvant tamoxifen or no endocrine therapy. Tumor tissue sections were assessed in 2014 using immunohistochemistry and Agilent microarrays. Only patients with luminal A or B subtype tumors were evaluated. Complete long-term follow-up data up to the end of the STO-3 trial on December 31, 2012, were obtained from the Swedish National registers. Data analysis for the secondary analysis was conducted in 2017 and 2018. Patients were randomized to receive at least 2 years of tamoxifen therapy or no endocrine therapy; patients without recurrence who reconsented were further randomized to 3 additional years of tamoxifen therapy or no endocrine therapy. Distant recurrence-free interval (DRFI) by luminal A and luminal B subtype and trial arm was assessed by Kaplan-Meier analyses and time-dependent flexible parametric models to estimate time-varying hazard ratios (HRs) that were adjusted for patient and tumor characteristics. In the STO-3 treated trial arm, 183 patients had luminal A tumors and 64 patients had luminal B tumors. In the untreated arm, 153 patients had luminal A tumors and 62 had luminal B tumors. Age at diagnosis ranged from 45 to 73 years. A statistically significant difference in DRFI by trial arm was observed (log rank, P < .001 [luminal A subtype, n = 336], P = .04 [luminal B subtype, n = 126]): the 25-year DRFI for luminal A vs luminal B subtypes was 87% (95% CI, 82%-93%) vs 67% (95% CI, 56%-82%) for treated patients, and 70% (95% CI, 62%-79%) vs 54% (95% CI, 42%-70%) for untreated patients, respectively. Patients with luminal A tumors significantly benefited from tamoxifen therapy for 15 years after diagnosis (HR, 0.57; 95% CI, 0.35-0.94), and those with luminal B tumors benefited from tamoxifen therapy for 5 years (HR, 0.38; 95% CI, 0.24-0.59). Patients with luminal A subtype tumors had a long-term risk of distant metastatic disease, which was reduced by tamoxifen treatment, whereas patients with luminal B tumors had an early risk of distant metastatic disease, and tamoxifen benefit attenuated over time.

Increased Soluble CrkL in Serum of Breast Cancer Patients Is Associated with Advanced Disease

Over-expression of Crk-like protein (CrkL), an intracellular adaptor protein, in breast cancer biopsies has been linked to poor prognosis. CrkL can be secreted from cancer cells binding to β1 integrin on the cell membrane. In this study, we evaluated, for the first time, the levels of soluble CrkL in serum of breast cancer patients. Expression of CrkL and secreted fractions from human breast cancer cell lines and clinical patient samples were assessed by immunohistochemistry and Enzyme Linked Immuno-Sorbent Assay (ELISA). CrkL levels in tissues and sera of patients with different disease stages were compared and statistically analyzed by Chi-square test and Student’s t-test. Culture media from human breast cancer cell lines SUM159, MDA-MB231, and MCF7 showed over a 21-, 15-, and 11-fold higher concentration of soluble CrkL as compared to normal breast epithelium cell line MCF10A. Expression of CrkL was elevated in 85% of breast tumor tissue sections. Serum levels of CrkL were significantly higher in breast cancer patients than in healthy donors. All patients with metastatic disease had significantly elevated concentration of soluble CrkL in the serum with on average three-fold increase from the baseline. The data suggest that soluble fraction of CrkL can be further evaluated as a serum biomarker for advanced disease in breast cancer patients.

Abstract 1205: Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S., outranking breast and colorectal cancer by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the cancer genome atlas shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhances expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy. Methods: Level of secreted VIP in 1) serum of healthy volunteers, 2) untreated PDAC patients, 3) cell culture supernatant from murine melanoma, breast and PDAC cells and 4) serum from mice bearing these cell lines were tested via VIP specific enzyme immunoassay. Effect of inhibiting VIP signaling in-vivo was tested using VIPhyb, an antagonist for VIP receptor. Mice were subcutaneously inoculated with MT5 or luciferase transfected KPC (KPC luc), PDAC cell lines and treated with VIPhyb and/or anti-PD1 treatment after the tumors were palpable. The treatment regimen involved administering 10µg VIPhyb subcutaneously, every day and 200µg anti-PD1 or IgG2a, intraperitoneally, every three days, for 10 days. The tumors were measured using calipers or imaged via IVIS imaging system. The mice were sacrificed when tumors ulcerated or when they reached IACUC endpoint. Immunohistochemistry was performed on tumor tissue sections harvested at the time of sacrifice for expression of VIP, CD8 and DAPI. Results: Consistent with the human genome atlas, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from untreated human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing MT5 or KPC luc PDAC cells with the combination of VIPhyb and anti-PD1 produced complete and durable regression of tumors in 20% of the mice and delayed tumor progression in both tumor models. Further, upon analysis of the tumor tissues sections, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1. Conclusion: VIP is a novel immune checkpoint pathway in PDAC. Blocking VIP signaling during anti-PD1 therapy, shows improved survival and delayed tumor progression in mouse models of PDAC. Experiments are underway to understand the mechanism of action, as well as, improving the potency of VIPhyb for enhanced clinical outcome. Citation Format: Sruthi Ravindranathan, Yiwen Li, Shuhua Wang, Mohammad Y. Zaidi, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy [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 1205.

High Expression of Angiopoietin-like Protein 4 in Advanced Colorectal Cancer and its Association with Regulatory T Cells and M2 Macrophages.

Colorectal cancer (CRC) is one of the most aggressive tumours in the human digestive system. Most CRC patients have poor prognosis due to metastasis and recurrence. Angiopoietin-like 4 (ANGPTL4) is involved in tumour development. Regulatory T (Treg) cells and M2 macrophages promote tumour growth and metastasis. Herein, we explored the changes of ANGPTL4 expression in CRC patients at different stages and observed whether in situ tumour-Treg and -M2 macrophages are correlated with ANGPTL4 expression. Serum ANGPTL4 (sANGPTL4) levels of 70 CRC patients and 10 healthy controls were detected by ELISA. ANGPTL4, Foxp3 and CD163 expression levels in CRC tissues were measured by immunohistochemistry. Recombinant ANGPTL4 (rANGPTL4) proteins were further added into cell-culture systems for induction of Treg cells and M2 macrophages. The results showed both sANGPTL4 and in situ tumour-ANGPTL4 expression levels increased in Dukes C-D stage CRC patients. Foxp3+ and CD163+ cells in tumour tissue sections were also more intensive in Dukes C-D stage patients than in Dukes A-B stage patients. Foxp3+ and CD163+ cells in tumour tissues were positively correlated with both tissue and sANGPTL4 expression (P < 0.01). Recombinant ANGPTL4 promoted the induction of murine Treg cells and M2 macrophages ex vivo. Therefore, elevated ANGPTL4 expression could be a marker for advanced CRC. Treg cell and M2 macrophage induction could be one of the mechanisms of tumour promotion mediated by ANGPTL4.

Abstract 126: Multidimensional gene expression models for characterizing response and metastasis in solid tumor samples

Abstract Predicting response to standard of care therapies and new immunotherapies is enabled by tools that quantitatively analyze the complex heterogeneity of the tumor microenvironment. Current methods to do so primarily rely either on single gene or protein markers, and often report solely on one feature of the multifaceted immune cycle. Unlike other molecular approaches, the ImmunoPrismTM Immune Profiling Assay has been built using multidimensional gene expression models validated to accurately identify immune cells in a complex mixture with high correlation to flow cytometry measurements, but accessible for formalin-fixed paraffin-embedded (FFPE) tissues. We will report results showing the assay is highly sensitive, capable of detecting immune cells present to as little as 2% of the specimen, important for critical immune cell types such as M1 and M2 macrophages and Tregs. Further, the assay requires as few as two sections of FFPE solid tumor tissue or as little as 20 ng of total RNA which enables the analysis of rare clinical archives. In this study, we apply the ImmunoPrism assay to cohorts of patients with indications including non-small cell lung carcinoma (NSCLC), triple-negative breast cancer (TNBC) and pancreatic adenocarcinoma (ADC) treated with standard of care therapies. Immune profile changes for pre vs. post treatment and primary vs. metastatic tissue are reported in the context of clinical data, such as therapy response. Resulting biomarkers, including a novel machine-learning based multidimensional marker are also reported with predictive accuracy, PPV, NPV, and selection thresholds. Citation Format: Walter P. Carney, Milan Bhagat, Natalie LaFranzo. Multidimensional gene expression models for characterizing response and metastasis in solid tumor samples [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 126.

Abstract 5081: Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer

Abstract Increased beta-oxidation of fatty acids to meet energy demand is a rather unique characteristic of a subset of human prostate cancers. A safe and effective intervention for inhibition of fatty acid synthesis is still a clinically unmet need. We have shown previously that leelamine (LLM), a phytochemical derived from pine tree bark, suppresses expression and activity of full-length androgen receptor (AR) and its splice variants in vitro and in vivo in preclinical models of prostate cancer. Because AR is implicated in regulation of fatty acid metabolism, the present study was undertaken to determine the effect of LLM on this metabolic pathway. Treatment of a castration-resistant (22Rv1) as well as an androgen-responsive (LNCaP) human prostate cancer cell line with LLM (2.5 and 5 µmol/L) resulted in downregulation of key fatty acid synthesis enzyme proteins including ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBP1). LLM treatment also decreased intracellular levels of total free fatty acids and neutral lipid droplets in LNCaP and 22Rv1 cells. Consistent with the in vitro results, we also observed a significant decrease in ACLY and SREBP1 protein expression and number of neutral lipid droplets in vivo in tumor tissue sections of 22Rv1 xenografts after intraperitoneal administration of LLM (9.1 mg/kg bw/ day, 5 times/week) compared to controls. Studies are in progress to determine if overexpression of AR and/or SREBP1 confers protection against fatty acid synthesis inhibition by LLM. In conclusion, it is reasonable to postulate that suppression of AR-SREBP1 regulated fatty acid metabolism is an important mechanism in prostate cancer inhibition by LLM. This study was supported by the grant RO1 CA101753 awarded by the National Cancer Institute. Citation Format: Krishna B. Singh, Shivendra V. Singh. Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer [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 5081.