Marilyn M. Bui

Combination of External Beam Radiotherapy (EBRT) With Intratumoral Injection of Dendritic Cells as Neo-Adjuvant Treatment of High-Risk Soft Tissue Sarcoma Patients

PURPOSE The goal of this study was to determine the effect of combination of intratumoral administration of dendritic cells (DC) and fractionated external beam radiation (EBRT) on tumor-specific immune responses in patients with soft-tissue sarcoma (STS). METHODS AND MATERIAL Seventeen patients with large (>5 cm) high-grade STS were enrolled in the study. They were treated in the neoadjuvant setting with 5,040 cGy of EBRT, split into 28 fractions and delivered 5 days per week, combined with intratumoral injection of 10(7) DCs followed by complete resection. DCs were injected on the second, third, and fourth Friday of the treatment cycle. Clinical evaluation and immunological assessments were performed. RESULTS The treatment was well tolerated. No patient had tumor-specific immune responses before combined EBRT/DC therapy; 9 patients (52.9%) developed tumor-specific immune responses, which lasted from 11 to 42 weeks. Twelve of 17 patients (70.6%) were progression free after 1 year. Treatment caused a dramatic accumulation of T cells in the tumor. The presence of CD4(+) T cells in the tumor positively correlated with tumor-specific immune responses that developed following combined therapy. Accumulation of myeloid-derived suppressor cells but not regulatory T cells negatively correlated with the development of tumor-specific immune responses. Experiments with (111)In labeled DCs demonstrated that these antigen presenting cells need at least 48 h to start migrating from tumor site. CONCLUSIONS Combination of intratumoral DC administration with EBRT was safe and resulted in induction of antitumor immune responses. This suggests that this therapy is promising and needs further testing in clinical trials design to assess clinical efficacy.

MK1775, a Selective Wee1 Inhibitor, Shows Single-Agent Antitumor Activity against Sarcoma Cells

Wee1 is a critical component of the G2–M cell-cycle checkpoint control and mediates cell-cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 can abrogate G2–M checkpoint, resulting in premature mitotic entry and cell death. MK1775 has recently been tested in preclinical and clinical studies of human carcinoma to enhance the cytotoxic effect of DNA-damaging agents. However, its role in mesenchymal tumors, especially as a single agent, has not been explored. Here, we studied the cytotoxic effect of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo. Our data show that MK1775 treatment at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. In MK1775-treated cells, CDC2 activity was enhanced, as determined by decreased inhibitory phosphorylation of tyrosine-15 residue and increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells seems to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to MK1775 treatment. Finally, in patient-derived sarcoma samples, we showed that MK1775 as a single agent causes significant apoptotic cell death, suggesting that Wee1 inhibition may represent a novel approach in the treatment of sarcomas. Mol Cancer Ther; 11(1); 174–82. ©2011 AACR.

Soft Tissue Sarcoma, Version 2.2016, NCCN Clinical Practice Guidelines in Oncology.

Soft tissue sarcomas (STS) are rare solid tumors of mesenchymal cell origin that display a heterogenous mix of clinical and pathologic characteristics. STS can develop from fat, muscle, nerves, blood vessels, and other connective tissues. The evaluation and treatment of patients with STS requires a multidisciplinary team with demonstrated expertise in the management of these tumors. The complete NCCN Guidelines for STS provide recommendations for the diagnosis, evaluation, and treatment of extremity/superficial trunk/head and neck STS, as well as intra-abdominal/retroperitoneal STS, gastrointestinal stromal tumors, desmoid tumors, and rhabdomyosarcoma. This portion of the NCCN Guidelines discusses general principles for the diagnosis, staging, and treatment of STS of the extremities, superficial trunk, or head and neck; outlines treatment recommendations by disease stage; and reviews the evidence to support the guidelines recommendations.

Systemic Buffers Inhibit Carcinogenesis in TRAMP Mice

PURPOSE Hypoxia and acidosis develop in in situ tumors as cellular expansion increases the diffusion distance of substrates and metabolites from blood vessels deep to the basement membrane. Prior studies of breast and cervical cancer revealed that cellular adaptation to microenvironmental hypoxia and acidosis is associated with the transition from in situ to invasive cancer. We hypothesized that decreased acidosis in intraductal tumors would alter environmental selection pressures for acid adapted phenotypes and delay or prevent evolution to invasive cancer. MATERIALS AND METHODS A total of 37 C57BL/6 TRAMP mice were randomized to a control group or to 1 of 4 treatment groups. In the latter groups 200 mM sodium bicarbonate were added to drinking water starting between ages 4 and 10 weeks. RESULTS In all 18 controls prostate cancer developed that was visible on 3-dimensional ultrasound at a mean age of 13 weeks. They died within 52 weeks (median 37). When sodium bicarbonate therapy commenced before age 6 weeks in 10 mice, all reached senescence (age 76 weeks) without radiographic evidence of prostate cancer. Histological sections of the prostates in this cohort showed hyperplasia but no cancer in 70% of mice and minimal well differentiated cancer in the remainder. When therapy commenced after age 6 weeks in 9 mice, prostate cancer development was no different from that in controls. CONCLUSIONS Immunohistochemical staining for carbonic anhydrase 9 in regions of ductal hyperplasia showed increased expression in controls vs the early treatment group. Regional pH perturbation in in situ tumors may be a simple, inexpensive and effective cancer prevention strategy.

The Cyclin-Dependent Kinase Inhibitor SCH 727965 (Dinacliclib) Induces the Apoptosis of Osteosarcoma Cells

Although rare, osteosarcoma is an aggressive cancer that often metastasizes to the lungs. Toward the goal of developing new treatment options for osteosarcoma, we show that the cyclin-dependent kinase (CDK) inhibitor SCH 727965 (SCH) induces the apoptosis of several osteosarcoma cell lines including those resistant to doxorubicin and dasatinib. Cell lines prepared in our laboratory from patients who had received adjuvant chemotherapy and explants derived from a human osteosarcoma xenograft in mice were also responsive to SCH. Apoptosis occurred at low nanomolar concentrations of SCH, as did CDK inhibition, and was p53-independent. SCH activated the mitochondrial pathway of apoptosis as evidenced by caspase-9 cleavage and accumulation of cytoplasmic cytochrome c. Amounts of the apoptotic proteins Bax and Bim increased in mitochondria, whereas amounts of the antiapoptotic proteins Mcl-1 and Bcl-xL declined. Osteosarcoma cells apoptosed when codepleted of CDK1 and CDK2 but not when depleted of other CDK combinations. We suggest that SCH triggers the apoptosis of osteosarcoma cells by inactivating CDK1 and CDK2 and that SCH may be useful for treatment of drug-resistant osteosarcomas. SCH also induced the apoptosis of other sarcoma types but not of normal quiescent osteoblasts or fibroblasts. Mol Cancer Ther; 10(6); 1018–27. ©2011 AACR.

Noninvasive Detection of Breast Cancer Lymph Node Metastasis Using Carbonic Anhydrases IX and XII Targeted Imaging Probes

Purpose: To develop targeted molecular imaging probes for the noninvasive detection of breast cancer lymph node metastasis. Experimental Design: Six cell surface or secreted markers were identified by expression profiling and from the literature as being highly expressed in breast cancer lymph node metastases. Two of these markers were cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) and were validated for protein expression by immunohistochemistry of patient tissue samples on a breast cancer tissue microarray containing 47 normal breast tissue samples, 42 ductal carcinoma in situ, 43 invasive ductal carcinomas without metastasis, 46 invasive ductal carcinomas with metastasis, and 49 lymph node macrometastases of breast carcinoma. Targeted probes were developed by conjugation of CAIX- and CAXII-specific monoclonal antibodies to a near-infrared fluorescent dye. Results: Together, these two markers were expressed in 100% of the lymph node metastases surveyed. Selectivity of the imaging probes were confirmed by intravenous injection into nude mice-bearing mammary fat pad tumors of marker-expressing cells and nonexpressing cells or by preinjection of unlabeled antibody. Imaging of lymph node metastases showed that peritumorally injected probes detected nodes harboring metastatic tumor cells. As few as 1,000 cells were detected, as determined by implanting, under ultrasound guidance, a range in number of CAIX- and CAXII-expressing cells into the axillary lymph nodes. Conclusion: These imaging probes have potential for noninvasive staging of breast cancer in the clinic and elimination of unneeded surgery, which is costly and associated with morbidities. Clin Cancer Res; 18(1); 207–19. ©2011 AACR.

Using image analysis as a tool for assessment of prognostic and predictive biomarkers for breast cancer: How reliable is it?

Background : Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) are important and well-established prognostic and predictive biomarkers for breast cancers and routinely tested on patient′s tumor samples by immunohistochemical (IHC) study. The accuracy of these test results has substantial impact on patient management. A critical factor that contributes to the result is the interpretation (scoring) of IHC. This study investigates how computerized image analysis can play a role in a reliable scoring, and identifies potential pitfalls with common methods. Materials and Methods : Whole slide images of 33 invasive ductal carcinoma (IDC) (10 ER and 23 HER2) were scored by pathologist under the light microscope and confirmed by another pathologist. The HER2 results were additionally confirmed by fluorescence in situ hybridization (FISH). The scoring criteria were adherent to the guidelines recommended by the American Society of Clinical Oncology/College of American Pathologists. Whole slide stains were then scored by commercially available image analysis algorithms from Definiens (Munich, Germany) and Aperio Technologies (Vista, CA, USA). Each algorithm was modified specifically for each marker and tissue. The results were compared with the semi-quantitative manual scoring, which was considered the gold standard in this study. Results : For HER2 positive group, each algorithm scored 23/23 cases within the range established by the pathologist. For ER, both algorithms scored 10/10 cases within range. The performance of each algorithm varies somewhat from the percentage of staining as compared to the pathologist′s reading. Conclusions : Commercially available computerized image analysis can be useful in the evaluation of ER and HER2 IHC results. In order to achieve accurate results either manual pathologist region selection is necessary, or an automated region selection tool must be employed. Specificity can also be gained when strict quality assurance by a pathologist is invested. Quality assurance of image analysis by pathologists is always warranted. Automated image analysis should only be used as adjunct to pathologist′s evaluation.

Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.

Long Non-coding RNAs (LncRNA) Regulated by Transforming Growth Factor (TGF) β: LncRNA-HIT-MEDIATED TGFβ-INDUCED EPITHELIAL TO MESENCHYMAL TRANSITION IN MAMMARY EPITHELIA*

Background: Long noncoding RNAs (LncRNA) are emerging as key regulators in various biological processes. However, their role in epithelial-to-mesenchymal transition (EMT) remains elusive. Results: A subset of lncRNAs are dysregulated upon transforming growth factor (TGF) β-induced EMT, and lncRNA-HIT mediates this process. Conclusion: LncRNAs such as lncRNA-HIT ((HOXA transcript induced by TGFβ) play a pivotal role in EMT and breast cancer progression. Significance: Here we profiled lncRNAs in TGFβ-induced EMT and identified a novel conserved lncRNA-HIT. Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Targeting immune suppression with PDE5 inhibition in end-stage multiple myeloma.

Noonan and colleagues report that addition of the phosphodiesterase-5 inhibitor, tadalafil, reduced the expression of immunosuppressive mediators in myeloid-derived suppressor cells, restored the patients responsiveness to lenalidomide-based therapy, and elicited a durable antimyeloma clinical response. Myeloid-derived suppressor cells (MDSC) play a significant role in tumor-induced immune suppression. Targeting their function could improve antitumor therapies. Previously, we demonstrated that phosphodiesterase 5 (PDE5) inhibition in MDSCs augmented antitumor immunity in murine models. Here, we show how the addition of the PDE5 inhibitor, tadalafil, in a patient with end-stage relapsed/refractory multiple myeloma reduced MDSC function and generated a dramatic and durable antimyeloma immune and clinical response. Strategies targeting MDSC function with PDE5 inhibitors represent a novel approach that can augment the efficacy of tumor-directed therapies. Cancer Immunol Res; 2(8); 725–31. ©2014 AACR.