Difference In Tumor Growth Rate Research Articles

LTBK-06. IMPACT OF VORASIDENIB TREATMENT ON MUTANT IDH1 OR IDH2 DIFFUSE GLIOMA TUMOR GROWTH RATE: RESULTS FROM THE RANDOMIZED, DOUBLE-BLIND, PHASE 3 INDIGO STUDY

Abstract INTRODUCTION The INDIGO study (NCT04164901) showed that vorasidenib, an oral, brain-penetrant, dual inhibitor of mutant isocitrate dehydrogenase (mIDH) 1/2, significantly improved imaging-based progression-free survival and time-to-next-intervention compared with placebo in patients with grade 2 mIDH1/2 glioma previously treated with surgery only. Given the limitations of traditional bi-dimensional measurements, evaluating volumetry and tumor growth rate (TGR) is an additional method of measuring treatment effect in these diffuse growing tumors. METHODS Magnetic resonance imaging (MRI) scans were performed at baseline and every 12 weeks on-treatment; up to three pre-treatment MRI scans were requested when available. Tumor volumes were derived per blinded independent review committee using a semi-automated approach. TGR was defined as percentage change in tumor volume every 6 months. Patients with evaluable baseline and ≥ 1 MRI during the corresponding period were included in the analysis. The difference in TGR in each arm was assessed by slope of tumor growth over time using a linear mixed model. RESULTS 331 patients were randomized to vorasidenib (n=168) or placebo (n=163). Median follow-up was 14.2 months. On-treatment TGR was −2.5% (95% CI, −4.7, −0.2) with vorasidenib (n=167) and 13.9% (95% CI, 11.1, 16.8) with placebo (n=161). In patients with available imaging data, TGR pre- and post-treatment with vorasidenib (n=56) was 13.2% (95% CI, 10.3, 16.3) and −3.3% (95% CI, −5.2, −1.2), respectively, while placebo (n=67) was 18.3% (95% CI, 15.0, 21.7) and 12.2% (95% CI, 9.5, 14.9), respectively. In patients who crossed over from placebo with available imaging data (n=38), TGR pre- and post-crossover was 22.4% (95% CI, 15.7, 29.4) and 5.2% (95% CI, −3.8, 15.0), respectively. CONCLUSIONS Tumor growth was observed in patients with mIDH1/2 gliomas before receiving vorasidenib or placebo. Treatment with vorasidenib reduced the TGR and shrunk tumor volume, whereas continued growth in tumor volume was observed in patients receiving placebo.

Preclinical Evaluation of Tumor Treating Fields Combined with Personalized Ultra-Fractionated Stereotactic Adaptive Radiotherapy (PULSAR)

Purpose/Objective(s)The PULSAR paradigm, treats patients using a few large dose “pulses,” delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination.Materials/MethodsThe Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined.ResultsIn the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported.ConclusionThe addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI. The PULSAR paradigm, treats patients using a few large dose “pulses,” delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination. The Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined. In the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported. The addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI.

Using Free-Range Laboratory Mice to Explore Foraging, Lifestyle, and Diet Issues in Cancer

As cancer progresses, its impact should manifest in the foraging behavior of its host much like the effects of endo-parasites that hinder foraging aptitudes and risk management abilities. Furthermore, the lifestyle of the host can impact tumor growth and quality of life. To approach these questions, we conducted novel experiments by letting C57BL/6 laboratory mice, with or without oral squamous cell carcinoma, free range in a large outdoor vivarium. Our goals were to: (1) determine whether one could conduct experiments with a mouse model under free range conditions, (2) measure effects of cancer burden on foraging metrics, (3) compare tumor growth rates with laboratory housed mice, and (4) begin to sort out confounding factors such as diet. With or without cancer, the C57BL/6 laboratory mice dealt with natural climatic conditions and illumination, found shelter or dug burrows, sought out food from experimental food patches, and responded to risk factors associated with microhabitat by foraging more thoroughly in food patches under bush (safe) than in the open (risky). We quantified foraging using giving-up densities of food left behind in the food patches. The mice’s patch use changed over time, and was affected by disease status, sex, and microhabitat. Males, which were larger, consumed more food and had lower giving-up densities than females. Relative to cancer-free mice, mice with growing tumors lost weight, harvested more food, and increasingly relied on patches in the bush microhabitat. The tumors of free-ranging mice in the vivarium grew slower than those of their cohort that were housed in mouse cages in animal facilities. Numerous interesting factors could explain the difference in tumor growth rates: activity levels, stress, weather, food intake, diet, and more. To tease apart one of these intertwined factors, we found that tumors grew faster when mice in the laboratory were fed on millet rather than laboratory mouse chow. While just a start, these novel experiments and framework show how free-ranging mice provide a model that can test a broader range of hypotheses and use a broader range of metrics regarding cancer progression and its consequences for the host.

Abstract PO-219: Examining racial differences in tumor growth rates for metastatic breast cancer to the brain

Abstract INTRODUCTION: Breast cancer is a common cause of brain metastases, occurring in at least 10–16 % of patients. Unfortunately, patients who develop brain metastases tend to have a poor prognosis with short overall survival. Moreover, for reasons we do not fully understand, African American women tend to have a worse prognosis and survival rate of about only 17% after 2 years of initial diagnosis of brain metastases. Our hypothesis in this project is that brain metastases in African American patients will have a higher growth rate compared to their white counterparts and that this may partially explain the worse prognosis for this population. However, measurement of growth rates of metastatic brain cancer has not been performed previously. A secondary objective is to test whether a combination of repeat brain MRI scans and dedicated software for automatic tumor sizing can be used to determine growth rates of individual brain lesions in this patient population and statistically correlate these with race and primary tumor type. METHODS: This was a pilot retrospective study of women who had been diagnosed with primary breast cancer that metastasized to the brain where multiple follow-up brain MRI scans were available. We used in-house software built upon the NIH ImageJ package to detect and accurately size nodules from the T1-weighted, post-contrast brain MRI scans[RA ref]. Patient factors of age, race, and primary tumor type we gathered as well as the type of treatment: surgery, chemotherapy, whole-brain radiation therapy, and/or stereotactic radiosurgery. RESULTS: To-date we have analyzed the growth rates of 20 brain metastases in 6 women: 3 Blacks, 3 Whites, 3 with ER+, 3 with ER- primary tumors, and age range 31-86 years. We have still not received sufficient results from our computational studies but will be getting soon. CONCLUSION: We have demonstrated the measurement of growth rates of individual brain metastases using repeated post-contrast brain MRIs and dedicated image analysis software. We have successfully applied these in 10 tumors from 20 breast cancer patients with known metastases to the brain. We have shown that growth rates differ between racial and ER+/- cohorts but additional datasets are needed to establish a statistical correlation. We hope these initial results motivate larger future studies that can investigate the role in breast cancer outcomes of patient and societal factors such as race, age, presence of comorbidities, primary tumor type, the aggressiveness of initial treatment, access to medical care, and socioeconomic factors. Citation Format: Daniel Sola, Jada Harvey, Walter O'Dell. Examining racial differences in tumor growth rates for metastatic breast cancer to the brain [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-219.

IKBKE Impacts Radiation Resistance Of Glioblastoma Through AKT/GSK3β Pathway

Explore the effect of IKBKE on the radiation resistance of glioblastoma cell lines and its potential molecular mechanism. We used sh-IKBKE lentivirus and IKBKE overexpression lentivirus to construct U87-MG-shIKBKE cell, U251-shIKBKE cell and U118-oeIKBKE cell. In vivo experiments were performed to verify the difference in tumor growth rate between IKBKE-silent mice and IKBKE-expressing mice after radiotherapy, and the differences in γ-H2AX and Bad expression between the groups were confirmed by immunohistochemical staining. In vitro experiments, we analyzed the potential molecular mechanism. 1. After U87-MG-shIKBKE cell , U251-shIKBKE cell and 118 cell receiving radiotherapy, apoptosis inhibitory protein Bcl2 decreased significantly, apoptotic promoting protein Bad increased significantly, colony forming ability was greatly weakened, the number of γ-H2AX focal points and γ-H2AX protein expression level decreased significantly than U87-MG, U251, and U118-oeIKBKE cell respectively. 2. Comparing with U87-MG cell inoculation group mice, the tumor growth of the U87-MG-shIKBKE cell inoculation group mice was significantly reduced, and the γ-H2AX expression in IHC analysis of U87-MG-shIKBKE cell inoculation group mice was significantly increased. 3. We confirmed that high-expressing IKBKE could promote the phosphorylation of AKT serine 473, AKT threonine 308 and GSK-3β serine 9 after receiving radiotherapy by Immunoblot experiments. 4. The direct interaction between IKBKE and AKT was confirmed by Co-Immunoprecipitation experiments. 5. Rescue experiment demonstrated AZD5363 achieved lowest expression level of apoptosis-inhibiting protein Bcl2 and highest expression of γ-H2AX in U87-MG cells and U118 cells receiving radiotherapy. The IKBKE overexpression plasmid achieved highest expression level of Bcl2 and lowest expression of γ-H2AX in U87-MG cells and U118 cells receiving radiotherapy. After using AZD5363 and IKBKE overexpression plasmid, the expression of Bcl2 and γ-H2AX was moderate. 6. AZD5363 achieved highest apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. IKBKE overexpression plasmid achieved lowest apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. AZD5363 and IKBKE overexpression plasmid achieved moderate apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. 1. In vitro experiments, IKBKE significantly inhibited the apoptosis of U87-MG, U251 and U118 cells receiving radiotherapy.

Optimizing Sequence of PD-L1 Immune-Checkpoint Inhibitors and Radiation Therapy in Bladder Cancer

BACKGROUND: New bladder preserving strategies are needed for muscle invasive bladder cancer (MIBC). Combined therapy of immune-checkpoint inhibitors and radiation was shown to have synergistic antitumoral effects in preclinical studies. OBJECTIVES: We aim to evaluate whether the sequence of administration of this combined therapy impacts antitumoral response. METHODS: We developed an in-vivo syngeneic MIBC mouse model where murine bladder cancer cells (MB49) were injected subcutaneously in the right flank of C57BL/6 mice. Mice were then randomized to the following treatments: control, anti-programmed cell death ligand 1 (PD-L1) alone, radiation alone (XRT) consisting of 6.25 Gy x2 fractions, concurrent anti-PD-L1 with XRT, neoadjuvant anti-PD-L1 followed by XRT, or XRT followed by adjuvant anti-PD-L1 therapy. Tumor growth, survival, and rate of response were analyzed. RESULTS: Total of 60 mice were randomized. One-way analysis of variance showed statistically significant difference in tumor growth rate across the treatment arms (p = 0.029). Importantly, timing of immunotherapy (neoadjuvant, concurrent, or adjuvant) did not alter either tumor growth or survival (p > 0.05). The rate of response was also similar in each combination arm (p > 0.05). CONCLUSION: Combining anti-PD-L1 immunotherapy and radiation therapy offers optimal antitumoral responses. Timing of immunotherapy (neoadjuvant, concurrent, or adjuvant) does not appear to affect outcomes. Whether the toxicity profile differs across various sequential deliveries of combination therapy requires further evaluation.

Prediction of tumor doubling time of lung adenocarcinoma using radiomic margin characteristics.

BackgroundBecause shape or irregularity along the tumor perimeter can result from interactions between the tumor and the surrounding parenchyma, there could be a difference in tumor growth rate according to tumor margin or shape. However, no attempt has been made to evaluate the correlation between margin or shape features and tumor growth.MethodsWe evaluated 52 lung adenocarcinoma (ADC) patients who had at least two computed tomographic (CT) examinations before curative resection. Volume‐based doubling times (DTs) were calculated based on CT scans, and patients were divided into two groups according to the growth pattern (GP) of their ADCs (gradually growing tumors [GP I] vs. growing tumors with a temporary decrease in DT [GP II]). CT radiomic features reflecting margin characteristics were extracted, and radiomic features reflective of tumor DT were selected.ResultsAmong the 52 patients, 41 (78.8%) were assigned to GP I and 11 (21.2%) to GP II. Of the 94 radiomic features extracted, eccentricity, surface‐to‐volume ratio, LoG uniformity (σ = 3.5), and LoG skewness (σ = 0.5) were ultimately selected for tumor DT prediction. Selected radiomic features in GP I were surface‐to‐volume ratio, contrast, LoG uniformity (σ = 3.5), and LoG skewness (σ = 0.5), similar to those for total subjects, whereas the radiomic features in GP II were solidity, energy, and busyness.ConclusionsThis study demonstrated the potential of margin‐related radiomic features to predict tumor DT in lung ADCs.Key pointsSignificant findings of the studyWe found a relationship between margin‐related radiomic features and tumor doubling time.What this study addsMargin‐related radiomic features can potentially be used as noninvasive biomarkers to predict tumor doubling time in lung adenocarcinoma and inform treatment strategies.

Characterization of heterogeneity in three lung cancer patients with multiple nodules by whole exome sequencing.

e21530 Background: Tumor heterogeneity is an important characteristic of malignant tumors that reflects the high complexity and diversity in the process of evolution. It can lead to differences in tumor growth rate, invasion, metastasis, drug sensitivity and prognosis. At present, there exist few genome-wide studies on heterogeneous cases of multiple nodules for patients with non-small-cell lung cancer (NSCLC). Methods: In this study, we performed genetic profiling using a combination of targeted panel and whole exon genome sequencing (WES) on all lesions of three NSCLC patients with multiple nodules. Results: Driver gene alterations were detected in all lesion samples of the three patients. One patient presented with three nodules, which harbored EGFR L858R, ERBB2 exon 20 insertion and a EML4-ALK fusion. Evolutionary analysis showed strong heterogeneity among the three lesions. The three lesions of patient 2 showed EGFR L858R in two lesions (2A and 2C), and a ERBB2 exon 20 insertions in the other (2B). Mutational signatures and cluster analysis of somatic mutations also showed commonality between sample 2A and 2C, suggesting that they might arise from the same clone. Patient 3 had seven lesions. Analysis of somatic mutations and copy number variations revealed possible route of metastasis. Conclusions: Detailed analysis of genetic mutation characteristics and biological evolution trees showed that there were substantial heterogeneity and distinct evolutionary relationships among the different lesions of the same individual. Consequently, such information could aid in the determination of primary and metastasis of tumor lesions and guide the selection of treatment options.

Abstract LB-323: Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma

Abstract Soft tissue sarcomas are tumors of the connective tissue that account for an estimated 12,000 new cancer cases annually and carry a poor prognosis with a five year survival rate of 50% despite treatment. An important research objective in improving therapy for soft tissue sarcoma patients is to understand how genetic mutations affect soft tissue sarcoma development and radiation response. Intriguingly, next-generation sequencing data from The Cancer Genome Atlas and other massive cancer sequencing efforts have identified Alpha Thalassemia and Mental Retardation X-linked, or ATRX, as the second most frequently mutated gene in soft tissue sarcoma. The vast majority of these ATRX mutations are frameshift mutations. To dissect the role of ATRX in soft tissue sarcoma development and therapeutic response, we generated a primary mouse model of soft tissue sarcoma. This model utilizes Cre-LoxP technology to conditionally activate oncogenic KrasG12D, delete p53, and delete both alleles of Atrx (KPA) in mouse hindlimb Pax7+ muscle satellite cells. When compared to mice with activation of oncogenic KrasG12D, deletion of p53 and at least one wild-type Atrx allele (KP), KPA mice showed a significant delay in time to tumor formation (35 vs. 58 days median), but no significant difference in tumor growth rate as measured by time to tumor quintupling. Next, we generated primary soft tissue sarcoma cell lines from our KP mouse model, and performed CRISPR-Cas9 mediated knockout of Atrx to generate isogenic cell lines. Testing of these isogenic cell lines revealed that Atrx deletion in soft tissue sarcoma cells increased sensitivity to double strand break inducing chemotherapeutics and ionizing radiation, but not to single strand break inducing chemotherapeutics in vitro. In clonogenic assays, Atrx deletion increased radiosensitivity of soft tissue sarcoma cell lines more than fourfold across all radiation doses tested. We next used our KP and KPA models to test the effect of Atrx deletion on radiosensitivity in vivo. Results showed that, relative to the KP model, Atrx deletion improved the response of soft tissue sarcomas to radiation therapy in vivo, increasing the time to tumor quintupling after radiation by 20%. Further analysis demonstrated that Atrx deletion significantly increases mitotic segregation defects after ionizing radiation, suggesting a potential mechanism for the radiosensitivity observed in our studies. Our in vivo study provides the first insight into how ATRX alters the development and therapeutic response of soft tissue sarcoma. Citation Format: Robert W. Floyd, Lixia Luo, David Kirsch. Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma [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 LB-323.

Advances on Recognizing and Managing Tumor Heterogeneity

肿瘤异质性是恶性肿瘤的特征之一，可使肿瘤的生长速度、侵袭与转移、药物敏感性、预后等各方面产生差异。肿瘤驱动基因和靶向药物的发现发展开启了战胜肿瘤的希望之门，然而异质性的存在又让肿瘤治疗陷入了难以攻克的困境。在肿瘤复发、进展演化的过程中肿瘤异质性如影随形，纷繁复杂。凭借不断进步的检测技术认识和理解肿瘤异质性，针对肿瘤异质性的原因和表型，定制治疗方案已成为当今精准医疗领域的重点范畴。本综述对肿瘤异质性进行了分析和探讨，从而更好的帮助我们了解肿瘤异质性，有利于我们通过多种手段对抗肿瘤异质性。

Abstract 732: The impact of aerobic exercise on the tumor microenvironment

Abstract Intratumoral hypoxia (pO2 < 10mmHg) is associated with aggressive tumor progression, radiation resistance, suppression of the immune anti-tumor response and poor patient outcome. It is often found in solid tumors, and can constitute a major obstacle to anticancer therapy. The primary cause of abnormal oxygen tensions in tumors is the aberrant tumor vasculature, characterized by tortuous, leaky and immature vessels. A number of intervention strategies designed to overcome tumor hypoxia and improve patient outcome have been investigated including high oxygen content breathing and the use of bioreductive prodrugs, but these have had only moderate success in the clinic. The goal of the present investigations was to determine whether aerobic exercise could be applied to improve tumor perfusion, oxygenation and the immune anti-tumor response in breast cancer and fibrosarcoma models. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of mild and moderate intensity treadmill running were studied in mice bearing syngeneic murine mammary carcinomas (4T1 and EMT6) or fibrosarcomas (KHT). The exercise intensities were determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and exercise commenced when tumors reached a size of ∼500 mm3 (single) or ∼200 mm3 (repeated bouts). Once size was attained, mice were exposed to either mild (12 m/min) or moderate (18 m/min) intensity exercise. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of the exercise period tumors were analyzed by histology to assess for physiological changes. Specifically, blood was collected and tumors were harvested, sectioned and evaluated by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion and while the hypoxia marker (EF5) was used to determine the level of tumor hypoxia. Both markers were quantified using a Chalkley counter and ImageJ NIH software. In addition, the evaluation of tumor infiltrating immune cells after exercise compared to rest is under active investigation by histological analysis. Furthermore, plasma samples from exercised mice are being tested for immune related cytokines, chemokines and growth factors and compared to those from sedentary controls. Results to date indicate no difference in tumor growth rate between sedentary and exercising mice. Mild daily bouts of aerobic exercise do not affect the total number of tumor blood vessels but do increase the number of blood vessels that are actively perfused. These findings suggest that exercise may have potential utility in overcoming the aberrant microenvironmental conditions associated in solid tumors with therapeutic resistance. Citation Format: Jennifer M. Wiggins, Jennifer Lee, Lori Rice, Sharon Lepler, Christine Pampo, Dietmar Siemann. The impact of aerobic exercise on the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 732.

Radiofrequency ablation combined with transcatheter therapy in rabbit VX2 liver tumors: effects and histopathological characteristics

Transarterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) treatment (TACE-RFA) has been confirmed superior to TACE or RFA alone in animal liver tumors. TACE before RFA was shown to increase hepatocellular damage. Further optimization of the combination strategy for transcatheter arterial embolization (TAE) or TACE combined with RFA is warranted. To determine the optimal strategy for radiofrequency ablation combined with transcatheter therapies in VX2 liver tumors in a rabbit model. Twenty-four Japanese White rabbits with VX2 liver tumors were randomly divided into four groups: TACE-RFA (TACE-RFA group), transcatheter arterial embolization (TAE) combined with RFA treatment (TAE-RFA group), RFA only group, and TACE only group. Blood samples were collected 1 day before the operation and at 3 and 7 days postoperatively. Seven days after the operation, maximal diameters of coagulation or infarcted zones in the gross specimens, CT images, histopathological characteristics, tumor necrotic rate, and growth rate were compared. Significantly larger mean long-axis (P < 0.05) and short-axis (P < 0.05) diameters of coagulation and infarction were observed in the TACE-RFA group compared with the TAE-RFA, RFA, and TACE groups on day 7; and the TAE-RFA group showed a significant (P < 0.05) increase versus the RFA and TACE groups on day 7. There were no significant differences in tumor growth rate (109.3 ± 37.5 vs. 119.0 ± 43.1%, P = 0.45) and necrotic rate (89.5 ± 12.0 vs. 83.5 ± 9.3%, P = 0.73) between the TACE-RFA and TAE-RFA groups. TACE-RFA was more effective for achieving tumor destruction than the other treatment strategies, but led to increased rabbits discomfort and more severe liver dysfunction compared with TAE-RFA. TAE-RFA appears to be a beneficial therapeutic modality for treating VX2 liver tumors in a rabbit model.

Depletion of tumor-associated macrophages slows the growth of chemically induced mouse lung adenocarcinomas.

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4–6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

NI-32 * ENHANCING TUMOR RESPONSE USING QUANTITATIVE VOLUMETRY FROM T1 SUBTRACTION MAPS IN MALIGNANT GLIOMA TREATED WITH DENDRITIC CELL VACCINATION

PURPOSE: To ascertain the radiologic response and serial volumetric changes in patients with glioblastoma multiforme (GBM) treated with dendritic cell (DC) vaccine using contrast enhanced (CE) T1-weighted subtraction maps. METHODS: MR images were acquired for fourteen patients with newly diagnosed or recurrent GBM. Genetic analyses and intratumoral histopathology were performed on all but one patient. Changes in CE tumor volumes were assessed with CE T1-weighted subtraction maps generated via voxel-wise subtraction of Gaussian-normalized signal intensities T1-weighted and CE T1-weighted images. Response criterion was determined as 25% reduction in the T1 subtraction CE tumor volume with respect to pre-vaccination baseline. Response stratification was tested as a predictor of PFS using log-rank univariate analysis. RESULTS: Four patients responded to the treatment within the first three months of treatment. Ten patients did not respond within the same period of time. One patient exhibited a growth pattern consistent with pseudo-progression between 100 and 300 days after vaccination. A significant difference in tumor growth rate (P = 0.0064) months between responders and non-responders was found within the first three months via linear regression. A preference for unfavorable response in MGMT unmethylated, EGFR amplified, and EGFRVIII mutated tumors, and favorable response in IDH1 mutant tumors was observed. A significantly longer PFS was found both from the opening date of the trial for each patient (Log-rank, P = 0.0149) and from the date of first vaccination (Log-rank, P = 0.0440) for responders versus non-responders. CONCLUSION: The clinical utility of T1 subtraction maps for tumor visualization and burden evaluation was shown. Response outcomes to DC vaccine therapy become apparent within the first three months of following treatment. Mischaracterization of pseudo-progression in the context of DC vaccine therapy for malignant glioma may not be a major concern. Genetic analyses and histopathological considerations may be a component in assessing treatment response to immunotherapy in malignant gliomas.

Abstract 3930: Recapitulating the microenvironment in vitro for comparative study of factors affecting tumor growth and vascularization

Abstract Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting cells such as the stroma and vasculature, mechanical factors, such as interstitial flow and extracellular matrix and aspects of the tumor mass itself, including shape. However, current tumor growth models, including xenograft models, 2D and/or simplistic 3D cultures, are unable to address these interactions in a high throughput human-derived system. We have developed a novel microfluidic platform that combines human derived perfused microvessels, stroma, and interstitial flow with 3D culture. This platform was used to quantitatively compare the role of these microenvironmental factors on tumor growth. Methods: A microfluidic device was fabricated consisting of two supply channels on either side of a central tissue compartment. The inner stromal compartment consists of normal human fibroblasts (NHLFs) and GFP-labeled human colorectal adenocarcinoma tumor cells, SW620, seeded in a fibrin matrix. To simulate a vasculogenic-like process, human cord blood endothelial colony forming cells endothelial cells (ECFC-ECs) were distributed throughout the stromal channel with the fibroblasts and tumor cells. Tumor growth rate and area was compared across day, interstitial flow rates and tumor shape (fractal dimension, perimeter to area) with ANOVA. Results: Cell viability within the device was maintained under interstitial flow conditions for a period of 21 days. Within one week of culture, microvessel formation and significant tumor growth into spheroids (n=636) were observed. On average, tumor growth rate was 26% ±62% per day with the highest growth rates observed on the first days. By day 7, many tumor masses had died off, with 2-3 large, fast growing tumors remaining per chamber. Highest tumor growth rates and areas were observed in tumor masses with a characteristic morphology of high perimeter to area and lower cohesion. Interstitial flow rates ranging from essentially static to supraphysiologic were generated. Differences in tumor growth rates were not statistically significant across chambers with different mean flow rates. To demonstrate intraluminal flow within the vascular network, fluorescently labeled dextran (40, 70, and 150 kDa) was introduced into the microfluidic lines. Dextran was retained in the vessel network, and showed tumor cells residing in the intraluminal space of the formed vasculature. By day 14, the network eroded, as the tumor masses overgrew and encompassed more than 60% of the chamber volume. Discussion: We have developed a novel 3D microfluidic system of the tumor microenvironment that features perfused capillaries and controlled interstitial flow. Tumor growth was affected by tumor characteristic shape in this model though interstitial flow appeared to play a lesser role. Vascular development was observed and its interaction with tumor growth will be analyzed in future work. Citation Format: Luis F. Alonzo, Claire J. Robertson, Monica L. Moya, Marian L. Waterman, Christopher C. Hughes, Steven C. George. Recapitulating the microenvironment in vitro for comparative study of factors affecting tumor growth and vascularization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3930. doi:10.1158/1538-7445.AM2014-3930

Abstract A076: Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu overexpressing transgenic mouse model

Abstract Fes (a.k.a. Fps) is a cytoplasmic protein tyrosine kinase that is highly expressed in breast epithelial cells during lactation, as well as in vascular endothelial and myeloid cells. Retrovirally encoded oncogenic alleles of Fes are associated with tumors in chickens and cats and cause tumors in transgenic mice; however, a role for Fes in human cancer has not been established. Using an orthotopic mouse mammary engraftment model, we previously found that Fes deficiency correlated with reduction in engrafted tumor growth rates, lung metastasis, and circulating tumor cells. Reduced vascularity and fewer infiltrating macrophages suggested the tumor microenvironment of Fes-deficient mice was less supportive of tumor growth and metastasis. In co-culture with tumor cells, Fes-deficiency in macrophages correlated with a reduced ability to promote tumor cell invasive behavior. These results suggested a tumor promoting role of Fes kinase in breast cancer through roles in cells of the tumor microenvironment. In a transgenic mouse model of breast cancer driven by an activated HER2/Neu allele expressed in the mammary epithelium, we observed delayed tumor onset in Fes-deficient mice compared to wild-type mice. However, there was no difference in tumor growth rates. Taken together, these observations argue that Fes inhibition might provide therapeutic benefits in breast cancer, by attenuating tumor-associated angiogenesis and the metastasis-promoting functions of tumor-associated macrophages, or by delaying breast tumor onset in women with HER2 overexpression. Note:This abstract was not presented at the conference. Citation Format: Connie Shengnan Zhang, Peter A. Greer. Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu overexpressing transgenic mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A076.

Abstract 1405: Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu over-expressing transgenic mouse model.

Abstract Fes (a.k.a. Fps) is a cytoplasmic protein tyrosine kinase that is highly expressed in breast epithelial cells during lactation, as well as in vascular endothelial and myeloid cells. Retrovially encoded oncogenic alleles of Fes are associated with tumors in chickens and cats and cause tumors in transgenic mice; however, a role for Fes in human cancer has not been established. Using an orthotopic mouse mammary engraftment model, we previously found that Fes deficiency correlated with reduction in engrafted tumor growth rates, lung metastasis, and circulating tumor cells. Reduced vascularity and fewer infiltrating macrophages suggested the tumor microenvironment of Fes-deficient mice was less supportive of tumor growth and metastasis. In co-culture with tumor cells, Fes-deficiency in macrophages correlated with a reduced ability to promote tumor cell invasive behavior. These results suggested a tumor promoting role of Fes kinase in breast cancer through roles in cells of the tumor microenvironment. In a transgenic mouse model of breast cancer driven by an activated HER2/Neu allele expressed in the mammary epithelium, we observed delayed tumor onset in Fes-deficient mice compared to wild-type mice. However, there was no difference in tumor growth rates. Taken together, these observations argue that Fes inhibition might provide therapeutic benefits in breast cancer, by attenuating tumor-associated angiogenesis and the metastasis-promoting functions of tumor-associated macrophages, or by delaying breast tumor onset in women with HER2 overexpression. Citation Format: Connie S. Zhang, Peter A. Greer. Deficiency of Fes tyrosine kinase expression correlates with delayed mammary tumor onset in a HER2/Neu over-expressing transgenic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1405. doi:10.1158/1538-7445.AM2013-1405 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract LB-108: Racotumomab antitumor activity in the murine model of non small cell lung carcinoma 3LL is related with the level of expression on NGcGM3

Abstract N-Glycolylneuraminic acid (NGc) is a sialic acid molecule found in most mammalian cells. It is usually found as terminal constituents of different membrane glycoconjugates such as the GM3 ganglioside (NGcGM3) and has been described as tumor antigen in non small cell lung cancer (NSCLC) in humans. However, most mouse cancer cell lines, such as 3LL NSCLC, are negative for the expression of this antigen although mice somatic cells frequently express it. It was reported that minor component of the ganglioside fraction derived from primary tumors of 3LL is NGcGM3. Interestingly in metastatic lesions the presence of NGcGM3 reach the 73.4% of total lipid bound sialic acid. Anti idiotypic antibodies (Ab2) are proposed as therapeutic vaccines when they bear the internal image of tumor antigens. Racotumomab is a gamma type anti idiotypic antibody that is able to trigger a strong anti anti idiotypic antibody (Ab3) response. These Ab3 antibodies have the same antigenic specificity than the Ab1 (P3) but they are substantially different from the internal image of it. Additionally, since it was demonstrated that immunization with Racotumomab elicited an anti NGcGM3 antibody response in humans, the question about to what extent the presence of the vaccine target antigen in the tumor decides the effect of the active treatment remains open. In this work, we evaluated the association of NGcGM3 expression in tumor cells in relation to the antitumor activity of the vaccine. Evaluation of Racotumomab antitumor activity over 3LL primary tumor showed no difference in tumor growth rate between treated and control mice. Moreover, the vaccine showed no antitumor activity in the metastatic dissemination in C57BL/6 mice treated with iv 3LL cells. However, when Racotumomab activity was evaluated in a spontaneous metastasis model it was observed a reduction in lung metastatic nodules in treated group compared to the control. The amount of NGcGM3 in 3LL cells can be increased by preincubation in vitro with purified NGc. Using this NGcGM3 positive metastasis model, we observed that Racotumomab was able to develop an active antitumor activity. The number of experimental metastases was reduced significantly between control and treated group (3.5 (1-18) vs 0.5(1-10), control vs Racotumomab. p&amp;lt;0.05, Kruskal-Wallis test). In a preliminary study of the therapeutic combination or Racotumomab with chemotherapy, mice were injected with 3LL cells and treated with Racotumomab, Pemetrexed or both. The combination of the vaccine and Pemetrexed show a similar antitumor effect than Racotumomab alone. Our results suggest a close association between the expression of NGcGM3 and the antitumor activity of Racotumomab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-108. doi:1538-7445.AM2012-LB-108

Abstract 1304: Specific MUC1 isoforms function to regulate gene expression and tumor growth in pancreatic cancer

Abstract Cancer cells, especially adenocarcinomas, express aberrant forms of mucins, as compared to their normal cell counterparts. These aberrant forms arise as a consequence of transformation-related alterations in expression of mucin mRNAs including alternative splice forms, and deregulation of enzymes that post-translationally modify mucin core proteins. Previous work has established that MUC1 is overexpressed and differentially glycosylated in adenocarcinomas and that this overexpression is associated with aggressive forms of pancreatic cancer. Most studies have focused on the full-length MUC1 isoform that contains the VNTR and all seven exons, but alternative splicing of MUC1 yields a minimum of 30 isoforms, of which several have been linked to malignancy in ovarian, cervical, and breast cancer. We evaluated expression of MUC1 alternate splice forms in a series of isogenic clonal pancreatic cancer cell lines that exhibit different properties of morphological differentiation, tumor growth and metastasis. The results revealed significant variation in splice variant expression between these isogenic cell lines and has implicated an association between isoform expression and metastatic potential. The knockdown of specific splice variants and MUC1 isoform families in S2-007 pancreatic cancer cells suggested that individual isoforms show differential capacity for regulating expression of target genes and miRNAs, which in turn led to differences in tumor growth rate and metastatic potential. Specifically, the stable knockdown of all members of the MUC1 X-family significantly altered expression of miRNAs 193b and 320 and reduced tumor growth in a pancreatic cancer animal model. MUC1 X-family also affected expression levels of several genes associated with Gemcitabine resistance and both pro and anti-inflammatory pathways that are predicted to influence disease progression and tumor associated immunosuppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1304. doi:1538-7445.AM2012-1304

Abstract 790: Are laboratory mice too cold to mount effective anti-tumor immune responses

Abstract During the course of experiments to determine the effects of elevated body temperature on anti-tumor immune responses, we unexpectedly observed that in control animals bearing tumors and maintained under standard room temperatures (IACUC-compliant, 21-23°C), the core temperature actually began to fall significantly as the tumors grew larger. Moreover, using thermal preference analyses, we found that animals bearing tumors exhibit considerable heat-seeking behavior. In an attempt to keep body temperature within the normal range (∼37°C), we began to house tumor bearing mice in a warmer environment (i.e., 28-30°C) considered to be the thermoneutral temperature for mice, as apposed to IACUC compliant temperatures which are considered mildly hypothermic for mice. Decisions that have resulted in the mildly cooler environmental temperature required in animal colonies have been generally based upon optimizing air-handling capacity of fumes and the thermal comfort of personnel rather than the optimal thermal comfort of animals (hence the requirements for multiple mice per cage to allow for huddling). When we analyzed tumor growth under thermoneutral conditions, syngeneic tumor growth was significantly delayed in animals maintained at 28-30°C compared to tumor growth in animals maintained at 21-23 oC. If the same comparison is made using SCID mice, no difference in tumor growth rate is observed. More specifically, BALB/c, C57BL/6 and SCID mice were housed in either standard room temperature (RT = 21-23°C) or in a warmer ambient temperature (WR = 28-30°C). Mice were then implanted with a syngeneic tumor (CT26 for BALB/c and SCID; B16.F10 for C57BL/6) subcutaneously and tumor growth was monitored. Tumor growth in C57BL/6 and BALB/c mice was inhibited when mice were housed in the warmer ambient temperature compared to animals maintained in standard room temperature. There was no difference in CT26 tumor growth in SCID mice. This finding suggested that the T cell-dependent anti-tumor immune response may be at least partly responsible for enhanced tumor growth control under conditions of warmer ambient temperature. Results of depletion studies in C57BL/6 mice bearing B16.F10 tumors, showed that both CD4+ and CD8+ T cells were required for inhibition of tumor growth in the warmer ambient temperature. In BALB/c mice bearing CT26 tumors, only CD8+ T cells were required to inhibit tumor growth. These findings suggest that the natural anti-tumor immune response exhibited by mice housed in standard colony conditions is sub-optimal and can be improved simply by housing mice under thermoneutral conditions. T cell dependent anti-tumor immunity appears to be at least one immune mechanism which is improved when animals are not cold stressed. Supported by NIH R01 CA 135368. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2011-790