Predictors of Acute Hematologic Toxicity in Women Receiving Extended-Field Chemoradiation for Cervical Cancer: Do Known Pelvic Radiation Bone Marrow Constraints Apply?

Purpose: Patients with cervical cancer who are at high risk for para-aortic lymphatic involvement may receive extended-field chemoradiation (EF-CRT), with inclusion of the para-aortic region. Increased radiation to bone marrow (BM) may heighten hematologic toxicity (HT) and affect timely delivery of chemoradiation. Factors associated with HT in this setting have not been well studied. Methods and Materials: This study was a retrospective analysis of women treated with EF-CRT from 2012 to 2018 with platinum-based chemotherapy. Factors including age, body mass index (BMI), race, Charlson Comorbidity Index (CCI), and nadirs for white blood cell count, absolute neutrophil count, hemoglobin, and platelet count were collected. The BM metrics included V5Gy, V10Gy, V15Gy, V20Gy, V25Gy, V30Gy, V35Gy, V40Gy and V45Gy (VxGy was defined as the percentage of BM volume receiving x Gy). Hematologic toxicity was defined as grade ≥2 (Cooperative Group Common Toxicity Criteria) leukopenia, anemia, neutropenia, or thrombocytopenia. Univariate analysis (UVA) and multivariate analysis (MVA) were performed using the χ2 test, the Fisher exact test, and logistic regression. Previously published dosimetric BM constraints were examined as detailed in each respective study. Results: Fifty-two women underwent EF-CRT with cisplatin. UVA showed no association between HT and age, BMI, or CCI. When accounting for race, V5Gy ≥98% was associated with grade ≥2 leukopenia (P = .02) and grade ≥2 HT (P = .05). Most previously described radiation metrics were not reproduced in our cohort, but a similar constraint, V20Gy <70%, was associated with reduced leukopenia of grade ≥2 on UVA (P = .02) and MVA (P < .05). Conclusions: Acute HT in patients receiving EF-CRT was associated with large volumes of low-dose radiation to the BM and was also associated with race. Restricting the BM V20Gy to less than 70% to 75% may be beneficial in reducing HT, but other pelvic radiation BM constraints may not be applicable to this population.

Opioid Use in Patients With Cervical Cancer at Two Urban Medical Centers.

Purpose: Patients with cervical cancer are at high risk for opioid use. This study aimed to characterize opioid prescribing patterns at 2 urban hospitals. Methods and Materials: Data from patients with cervical cancer treated with curative intent from 2011 to 2018 were retrospectively collected. Women with unrelated chronic opioid use before diagnosis, persistent/recurrent disease at 3 months after initiation of treatment, or initiation of opioids >6 months after treatment were excluded. Demographics, disease characteristics, treatment, and outpatient prescription practices were collected. Endpoints included duration of opioid use ≥6 and ≥12 months. Results: There were 106 women included, of whom 83% received definitive radiation. Most patients (n = 91, 85.8%) received outpatient opioids. Most common timing of prescriptions were before cancer therapy (35.9%), postprocedure (26.4%), and during radiation therapy (17.0%). Median duration was 3 (interquartile range, 1-11) months; 35.2% of these patients received opioids ≥6 months and 22% received opioids ≥12 months. Greater International Federation of Gynaecology and Obstetrics (FIGO) stage, recurrent/residual disease, initiation of opioids before treatment, history of depression or anxiety, and use of gabapentin or steroids were associated with long-term opioid use. Conclusions: Most patients were prescribed outpatient opioids, many of whom used opioids for 12 months. Improvement in provider communication and education, increased posttreatment monitoring, and further evaluation of nonopioid therapies are needed in this patient population to reduce long-term opioid use.

Local Control and Use of External Beam Parametrial Boost in the Era of Image-Guided Brachytherapy for Locally Advanced Cervical Cancer.

OBJECTIVE: Historically, external beam parametrial boost (EBPB) has been used in locally advanced cervical cancers to supplement radiation dose. However, it has become controversial in the era of image-guided brachytherapy. Modern 3D imaging and brachytherapy techniques have improved delineation and coverage of tumor. Outcomes with and without parametrial boost were analyzed. METHODS: Women with cervical cancer involving the parametria (clinically or radiographically) diagnosed between 2001 and 2017 were identified. Clinicopathologic and treatment features, survival and patterns of failure data were collected. Univariate and multivariable data analysis was performed to evaluate association of these variables, including parametrial boost, with local failure-free survival and overall survival. Competing risks analysis was performed for cumulative incidence of local failure, with death and other failures treated as competing events. RESULTS: A total of 100 women were identified (median follow-up 26.8 mo). Forty-one (41%) received EBPB; these patients were less likely to have received magnetic resonance imaging, positron emission tomography, interstitial, or high-dose rate brachytherapy. Magnetic resonance imaging, positron emission tomography, dose rate, and treatment era were highly correlated (Cramer's V: 0.43 to 0.68, P<0.01). Two-year overall survival and local failure were 78% and 12% for the entire cohort. While the use of EBPB was not associated with any outcome on multivariable analysis, treatment year after 2009 was highly associated with improved outcomes in all models. CONCLUSIONS: In this study, omission of EBPB did not compromise local control or survival in the modern era, supporting a decreased need for standardized use of parametrial boost.

DNA Methylation Controls Metastasis-Suppressive 14q32-Encoded miRNAs.

Expression of 14q32-encoded miRNAs is a favorable prognostic factor in patients with metastatic cancer. In this study, we used genomic inhibition of DNA methylation through disruption of DNA methyltransferases DNMT1 and DNMT3B and pharmacologic inhibition with 5-Aza-2'-deoxycytidine (5-Aza-dC, decitabine) to demonstrate that DNA methylation predominantly regulates expression of metastasis-suppressive miRNAs in the 14q32 cluster. DNA demethylation facilitated CCCTC-binding factor (CTCF) recruitment to the maternally expressed gene 3 differentially methylated region (MEG3-DMR), which acts as a cis-regulatory element for 14q32 miRNA expression. 5-Aza-dC activated demethylation of the MEG3-DMR and expression of 14q32 miRNAs, which suppressed adhesion, invasion, and migration (AIM) properties of metastatic tumor cells. Cancer cells with MEG3-DMR hypomethylation exhibited constitutive expression of 14q32 miRNAs and resistance to 5-Aza-dC-induced suppression of AIM. Expression of methylation-dependent 14q32 miRNAs suppressed metastatic colonization in preclinical models of lung and liver metastasis and correlated with improved clinical outcomes in patients with metastatic cancer. These findings implicate epigenetic modification via DNA methylation in the regulation of metastatic propensity through miRNA networks and identify a previously unrecognized action of decitabine on the activation of metastasis-suppressive miRNAs. SIGNIFICANCE: This study investigates epigenetic regulation of metastasis-suppressive miRNAs and the effect on metastasis.

The Evolution of Radiation Therapy in Metastatic Breast Cancer: From Local Therapy to Systemic Agent.

Radiation therapy is a mainstay of treatment in early and locally advanced breast cancer but is typically reserved for palliation of symptomatic lesions in patients with metastatic breast cancer. With new advances in the field of tumor biology and immunology, the role of radiation in the metastatic setting is evolving to harness its immune-enhancing properties. Through the release of tumor antigens, tumor DNA, and cytokines into the tumor microenvironment, radiation augments the antitumoral immune response to affect both the targeted lesion and distant sites of metastatic disease. The use of immunotherapeutics to promote antitumoral immunity has resulted in improved treatment responses in patients with metastatic disease and the combination of radiation therapy and immunotherapy has become an area of intense investigation. In this article, we will review the emerging role of radiation in the treatment of metastatic disease and discuss the current state of the science and clinical trials investigating the combination of radiation and immunotherapy.

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase.

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma.

Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant.

BACKGROUND: Current therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP) protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A) has been shown to block Survivin, inducing caspase activation and apoptosis. METHODS: In this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2) cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work. RESULTS: Here we show that exosomes collected in the absence of tetracycline (tet-off) from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines. CONCLUSION: This exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas.

Yin Yang 1 regulates the transcriptional repression of Survivin.

The mechanisms for regulation of the Inhibitor of Apoptosis (IAP) Survivin in cells undergoing stress associated with tumor development and the tumor microenvironment are not well understood. The stress response transcription factors HIF-1α and Yin Yang 1 (YY1) were hypothesized to contribute to the upregulation of Survivin in tumor cells. As expected, U2OS cells overexpressing HIF-1α showed a 2- to 3-fold transactivation when transfected. Surprisingly, when YY1 was overexpressed in this survivin promoter reporter system, luciferase expression was repressed 30- to 40-fold. YY1 involvement in survivin promoter repression was confirmed using siRNA directed against YY1. These studies showed that knockdown of YY1 releases the survivin promoter from the observed repression and leads to a 3- to 5-fold increase in promoter activity above basal levels. A U2OS cell line containing a stable YY1 Tet-off system was used to determine whether a temporal increase in YY1 expression affects Survivin protein levels. A low to moderate decrease in Survivin protein was observed 24h and 48h after Tet removal. Studies also confirmed that YY1 is capable of directly binding to the survivin promoter. Collectively, these findings identify novel basal transcriptional requirements of survivin gene expression which are likely to play important roles in the development of cancer and resistance to its treatment.

Tumor-released survivin induces a type-2 t cell response and decreases cytotoxic T cell function, in vitro.

Clinical studies of T cell profiles from cancer patients have shown a skewing toward a type-2 T cell response with decreased cytotoxic T cell function. However, the primary cause of this shift remains unknown. Here we show that tumor-released Survivin, an inhibitor of apoptosis (IAP) protein and tumor-specific antigen, is taken up by T cells and alters their response. The addition of Survivin to T cell cultures resulted in decreased T cell proliferation and reduced cytotoxic CD8(+) T cell function. Additionally, type 1 cell numbers and IFN-γ and IL-2 production were significantly reduced, while IL-4 release and type 2 T cell numbers increased. In contrast, the function and numbers of Th17 and T regulatory cells were not affected. These studies show that tumor-released Survivin modulates T cells resulting in a phenotype similar to that observed in cancer patients with a polarity shift from a type 1 to a type 2 response.

Plasma-derived exosomal survivin, a plausible biomarker for early detection of prostate cancer.

BACKGROUND: Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment. METHODS: Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively. RESULTS: Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls. CONCLUSIONS: These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.

Survivin is released from cancer cells via exosomes.

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.