Platinum-based regimens versus cetuximab in definitive chemoradiation for human papillomavirus-unrelated head and neck cancer.

For patients ineligible for cisplatin with definitive radiotherapy (CP-CRT) for locally advanced head and neck squamous cell carcinoma (LA-HNSCC), concurrent cetuximab (C225-RT) is a popular substitute. Carboplatin-based chemoradiation (CB-CRT) is another option; however, relative efficacies of CP-CRT, CB-CRT and C225-RT are unclear, particularly in the human papillomavirus (HPV)-unrelated population. We identified 316 patients with stage III-IVB cancers of the oropharynx (24.7%), larynx (58.2%) and hypopharynx (17.1%) undergoing definitive C225-RT (N = 61), CB-CRT (N = 74) or CP-CRT (N = 181). Kaplan-Meier and cumulative incidence functions were generated to estimate overall survival (OS), locoregional failure (LRF) and distant metastasis (DM). Cox proportional hazards were used to determine the association of survival endpoints with clinical characteristics. Respectively, 3-year cumulative incidences for CP-CRT, CB-CRT and C225-RT were: LRF (0.19, 0.18 and 0.48, p ≤ 0.001), DM (0.17, 0.12 and 0.25, p = 0.32). Kaplan-Meier estimates for 3 year OS were: CP-CRT: 71%; CB-CRT: 59% and C225-RT: 54%; p = 0.0094. CP-CRT (hazard ratio [HR] 0.336; 95% confidence interval [CI] 0.203-0.557, p < 0.01) and CB-CRT (HR 0.279; 95% CI 0.141-0.551, p < 0.01) were associated with reduced hazard for LRF on multivariable analysis. CP-CRT (HR 0.548; 95% CI 0.355-0.845, p < 0.01) and CB-CRT (HR 0.549; 95% CI 0.334-0.904, p = 0.02) were associated with a reduced hazard for death on multivariable analysis. Propensity matching confirmed reduced hazards with a combined CP/CB-CRT group compared to C225-RT for LRF: HR 0.384 (p = 0.018) and OS: HR 0.557 (p = 0.045) and CB-CRT group compared to C225-RT for LRF: HR 0.427 (p = 0.023). In conclusion, CB-CRT is an effective alternative to CP-CRT in HPV-unrelated LA-HNSCC with superior locoregional control and OS compared to C225-RT.

Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a.

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

RAS-RAF-miR-296-3p signaling axis increases Rad18 expression to augment radioresistance in pancreatic and thyroid cancers.

Oncogenic RAS and RAF signaling has been implicated in contributing to radioresistance in pancreatic and thyroid cancers. In this study, we sought to better clarify molecular mechanisms contributing to this effect. We discovered that miRNA 296-3p (miR-296-3p) is significantly correlated with radiosensitivity in a panel of pancreatic cancer cells, and miR-296-3p is highly expressed in normal cells, but low in cancer cell lines. Elevated expression of miR-296-3p increases radiosensitization while decreasing the expression of the DNA repair enzyme RAD18 in both pancreatic and thyroid cancer cells. RAD18 is overexpressed in both pancreatic and thyroid tumors compared to matched normal controls, and high expression of RAD18 in tumors is associated with poor prognostic features. Modulating the expression of mutant KRAS in pancreatic cancer cells or mutant BRAF in thyroid cancer cells demonstrates a tight regulation of RAD18 expression in both cancer types. Depletion of RAD18 results in DNA damage and radiation-induced cell death. Importantly, RAD18 depletion in combination with radiotherapy results in marked and sustained tumor regression in KRAS mutant pancreatic cancer orthotopic tumors and BRAF mutant thyroid heterotopic tumors. Overall, our findings identify a novel coordinated RAS/RAF-miR-296-3p-RAD18 signaling network in pancreatic and thyroid cancer cells, which leads to enhanced radioresistance.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Discovery of the inhibitor of DNA binding 1 as a novel marker for radioresistance in pancreatic cancer using genome-wide RNA-seq.

Purpose/Objective(s): Discovery of genetic drivers of radioresistance is critical for developing novel therapeutic strategies to combine with radiotherapy of radioresistant PDAC. In this study, we used genome-wide RNA-seq to identify genes upregulated in generated radioresistant PDAC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a potential regulator of radioresistance in PDAC. Materials/Methods: Radioresistant clones of the PDAC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) in vitro over two weeks (total 20 Gy) followed by standard clonogenic assays following one week from the end of IR. The generated RR and parental cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to the control. Genes with an absolute fold change > 1 were considered significant. RNA sequencing expression data from the Cancer Genome Atlas (TCGA) database was analyzed through the online databases GEPIA, cBioPortal, and the Human Protein Atlas. Results: Following exposure to two weeks of 2 Gy daily IR in vitro, the two PDAC cell lines showed significantly greater clonogenic cell survival than their parental cell lines, indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed increased expression of the ID1 protein in the RR cell lines compared to their parental cell lines. We found that ID1 mRNA was significantly higher in PDAC tumors compared to matched normal and high ID1 expression correlated with significantly worse disease-free survival (DFS) in PDAC patients (HR = 2.2, log rank P = 0.009). ID1 mRNA expression was also strongly correlated in tumors with TP53 mutation, a known driver of radioresistance. Conclusion: Our analysis indicates a novel role of ID1 in PDAC radioresistance. ID1 expression is higher in tumor tissue compared to normal, and high expression correlates with both worse DFS and association with the TP53 mutation, suggesting that targeting ID1 prior to IR is an attractive strategy for overcoming radioresistance in PDAC.

Predictors of Response, Progression-Free Survival, and Overall Survival in Patients With Lung Cancer Treated With Immune Checkpoint Inhibitors.

Monoclonal antibodies that target immune checkpoint proteins, so-called immune checkpoint inhibitors, prevent tumor evasion of the immune system and are often effective in the treatment of lung cancer. Studies have revealed improved objective response rates, progression-free survival, and overall survival with immune checkpoint inhibitors when used in both first and subsequent-line settings. Unfortunately, only a subset of unselected patients with lung cancer responds to these therapies. An important area of ongoing research is to identify biomarkers that can predict which patients are most likely to derive clinical benefit. This review will discuss established and emerging biomarkers from some of the clinical trials that have demonstrated the efficacy of immune checkpoint inhibitors for the treatment of both NSCLC and SCLC.

Morphologic changes associated with neoadjuvant-treated pancreatic ductal adenocarcinoma and comparison of two tumor regression grading systems.

Pancreatic ductal adenocarcinoma (PDAC) is aggressive, with an overall five-year survival rate of 9%, and few patients are candidates for pancreatectomy at presentation. The role of neoadjuvant therapy (NAT) is evolving, especially for high-risk potentially resectable tumors. Owing to the increasing number of NAT resection specimens, we aim to characterize the histologic changes associated with NAT and to compare two tumor regression grading schemes. One hundred eighteen resections for PDAC were selected from the cases between 2011 and 2018, 59 not treated and 59 treated with NAT. All H&E stained tumor slides were reviewed for histologic changes and graded using the four-tier modified Ryan score (recommended by College of American Pathologists) and the three-tier MD Anderson (MDA) score. The histologic changes evaluated included blue/grey fibrosis, islet cell hyperplasia, dystrophic calcification, amyloid deposition, cholesterol clefts, nerve hypertrophy, elastotic stromal/vascular change, abscess formation, and eosinophilic tumor cell changes. There were statistically significant differences for dystrophic calcification, eosinophilic tumor cell changes, elastotic stromal/vascular change, islet cell hyperplasia, and nerve hypertrophy between the two groups, with these features seen more frequently in NAT cases. Blue/grey stromal fibrosis was present in all cases regardless of NAT, except few complete regression cases and one treated case with intraneural invasion only. Blue/grey fibrosis is a useful histologic visual clue to suggest the possibility of adjacent tumor in the majority of PDAC cases regardless of NAT. By Kaplan-Meier analysis, neither grading scheme correlated with overall survival in our cohort. However, the MDA score was significantly correlated with both time to primary tumor recurrence (p = 0.002) and time to distant recurrence (p = 0.04), whereas the modified Ryan score was not.

BRAF mutation correlates with worse local-regional control following radiation therapy in patients with stage III melanoma.

BACKGROUND: In patients with stage III melanoma, the use of adjuvant radiation therapy (RT) after lymph node dissection (LND) may be currently considered in selected high-risk patients to improve tumor control. Melanomas harbor BRAF mutations (BRAF+) in 40-50% of cases, the majority of which are on the V600E residue. This study sought to compare the clinical outcomes after RT between patients with BRAF+ and BRAF- melanoma. METHODS: This was a retrospective review of 105 Stage III melanoma patients treated at our institution with LND followed by adjuvant RT from 2006 to 2019. BRAF mutational status was determined on the primary skin or nodal tissue samples from all patients. We compared characteristics of the BRAF+ and BRAF- groups using Fisher's exact test and Wilcoxon rank sum test and performed univariate and multivariate analysis using Kaplan-Meier estimates, log-rank tests, and Cox proportional hazards modeling with the clinical outcomes of local-regional lymph node control, distant metastasis-free survival (DMFS), recurrence-free survival (RFS), and overall survival (OS). RESULTS: Fifty-three (50%) patients harbored a BRAF mutation (92%, pV600E). BRAF+ patients were younger and had primary tumors more commonly found in the trunk vs head and neck compared to BRAF- patients (p < 0.05). The 5 year local-regional control in the BRAF + patients was 60% compared to 81% in the BRAF- patients (HR 4.5, 95% CI 1.3-15.5, p = 0.02). There were no significant differences in 5-year DMFS, RFS, and OS rates between the two BRAF patient groups. The presence of 4 or more positive LNs remained a significant prognostic factor for local-regional lymph node control, RFS, and OS in multivariate analysis. CONCLUSIONS: Stage III melanoma patients with BRAF mutation treated with adjuvant RT had > 4 times increased risk of local recurrence or regional lymph node recurrence. These results could be useful for adjuvant RT consideration in lymph node positive melanoma patients and supports other data that BRAF mutation confers radiation resistance.

Increasing neutrophil-to-lymphocyte ratio following radiation is a poor prognostic factor and directly correlates with splenic radiation dose in pancreatic cancer.

PURPOSE: Neutrophil-to-lymphocyte ratio has been correlated with clinical outcomes in many cancers. We investigated whether the delta-NLR (ΔNLR) following radiation therapy (RT) could predict achieving surgical resection and the overall survival (OS) of patients with borderline resectable (BRPC) and locally advanced pancreatic cancer (LAPC), and whether the splenic radiation dose impacted ΔNLR. METHODS/MATERIALS: 101 patients with biopsy-proven BRPC or LAPC who received induction chemotherapy followed by RT were retrospectively enrolled. Following contouring of spleens, dose-volume histograms (DVHs) for splenic dosimetric parameters were calculated. Pre- and post-RT complete blood counts (CBC) within two weeks were recorded. Delta (Δ) values were calculated by subtracting the post-RT value from the pre-RT value. Cox regression survival analysis for pre and postradiation CBC values and OS was performed. Receiver operating curves (ROC) were generated and optimal cutoff points for highest sensitivity and specificity were identified. Kaplan-Meier curves for OS were generated. RESULTS: On univariate Cox regression analysis, the only significant CBC value associated with OS was ΔNLR (HR 1.06, CI 1.03-1.09, p < 0.001). On multivariate analysis, ΔNLR, age, and completed resection all significantly predicted for worse OS (p < 0.05). ΔNLR significantly predicted achieving surgical resection (p = 0.04) and the optimal cutoff point for ΔNLR was 2.5. Patients with ΔNLR < 2.5 had significantly longer OS (log rank p = 0.046). Spleen radiation dose parameters were all significantly higher in patients with a ΔNLR ≥ 2.5. Optimal radiation cutoff points to predict a ΔNLR ≥ 2.5 were splenic Dmean of 308 cGy and V5 of 10.3%. CONCLUSIONS: Among patients with BRPC or LAPC who have received induction chemotherapy, elevated ΔNLR after RT significantly predicts worse OS and decreased odds of achieving resection. Furthermore, ΔNLR is correlated with higher splenic doses, suggesting the spleen may be an important organ at risk.

Altered Gemcitabine and Nab-paclitaxel Scheduling Improves Therapeutic Efficacy Compared with Standard Concurrent Treatment in Preclinical Models of Pancreatic Cancer.

PURPOSE: Concurrent gemcitabine and nab-paclitaxel treatment is one of the preferred chemotherapy regimens for metastatic and locally advanced pancreatic ductal adenocarcinoma (PDAC). Previous studies demonstrate that caveolin-1 (Cav-1) expression is critical for nab-paclitaxel uptake into tumors and correlates with response. Gemcitabine increases nab-paclitaxel uptake by increasing Cav-1 expression. Thus, we hypothesized that pretreatment with gemcitabine would further enhance the sensitivity of PDAC to nab-paclitaxel by increasing Cav-1 expression and nab-paclitaxel uptake. EXPERIMENTAL DESIGN: We investigated the sensitivity of different gemcitabine and nab-paclitaxel treatment regimens in a panel of PDAC cell lines and orthotopic xenograft models. The sensitivity of different treatment regimens was compared with the standard concurrent treatment. RESULTS: Pretreatment with gemcitabine before nab-paclitaxel increased Cav-1 and albumin uptake and significantly decreased proliferation and clonogenicity compared with concurrent treatment, which correlated with increased levels of apoptosis. Cav-1 silencing reduced the uptake of albumin, and therapeutic advantage was observed when cells were pretreated with gemcitabine prior to nab-paclitaxel. In addition, we observed that pretreatment with gemcitabine resulted in partial synchronization of cells in the G2-M-phase at the time of nab-paclitaxel treatment, providing another mechanism for the benefit of altered scheduling. In heterotopic and orthotopic xenograft models, the altered schedule of gemcitabine prior to nab-paclitaxel significantly delayed tumor growth compared with concurrent delivery without added toxicity. CONCLUSIONS: Pretreatment with gemcitabine significantly increased nab-paclitaxel uptake and correlated with an increased treatment efficacy and survival benefit in preclinical models, compared with standard concurrent treatment. These results justify preclinical and clinical testing of this altered scheduling combination.

Development of a MicroRNA Signature Predictive of Recurrence and Survival in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Optimal patient selection for radiotherapy in pancreatic ductal adenocarcinoma (PDAC) is unestablished. Molecular profiling may select patients at high risk for locoregional recurrence (LRR) who would benefit from radiation. METHODS: We included resectable pancreatic cancer (R-PDAC) patients, divided into training and validation cohorts, treated among three institutions with surgery and adjuvant chemotherapy, and borderline resectable or locally advanced pancreatic cancer (BR/LA-PDAC) patients treated with chemotherapy with or without radiation at the primary study institution. We isolated RNA from R-PDAC surgical specimens. Using NanoString, we identified miRNAs differentially expressed between normal and malignant pancreatic tissue. ElasticNet regression identified two miRNAs most predictive of LRR in the training cohort, miR-181b/d and miR-575, which were used to generate a risk score (RS). We evaluated the association of the median-dichotomized RS with recurrence and overall survival (OS). RESULTS: We identified 183 R-PDAC and 77 BR/LA-PDAC patients with median follow up of 37 months treated between 2001 and 2014. On multivariable analysis of the R-PDAC training cohort (n = 90), RS was associated with worse LRR (HR = 1.34; 95%CI 1.27-11.38; p = 0.017) and OS (HR = 2.89; 95%CI 1.10-4.76; p = 0.027). In the R-PDAC validation cohort, RS was associated with worse LRR (HR = 2.39; 95%CI 1.03-5.54; p = 0.042), but not OS (p = 0.087). For BR/LA-PDAC, RS was associated with worse LRR (HR = 2.71; 95%CI 1.14-6.48; p = 0.025), DR (HR = 1.93; 95%CI 1.10-3.38; p = 0.022), and OS (HR = 1.97; 95%CI 1.17-3.34; p = 0.011). Additionally, after stratifying by RS and receipt of radiation in BR/LA-PDAC patients, high RS patients who did not receive radiation had worse LRR (p = 0.018), DR (p = 0.006), and OS (p < 0.001) compared to patients with either low RS or patients who received radiation, irrespective of RS. CONCLUSIONS: RS predicted worse LRR and OS in R-PDAC and worse LRR, DR, and OS in BR/LA-PDAC. This may select patients who would benefit from radiation and should be validated prospectively.

Erratum: Clinical Outcomes and Multidisciplinary Patterns of Failure for Olfactory Neuroblastoma: The Ohio State Experience.

[This corrects the article DOI: 10.1055/s-0039-1692479.].

Neoadjuvant-modified FOLFIRINOX vs nab-paclitaxel plus gemcitabine for borderline resectable or locally advanced pancreatic cancer patients who achieved surgical resection.

We conducted an institutional study to compare the clinical and pathological efficacy between the neoadjuvant therapy (NAT)-modified FOLFIRINOX (mFOLF) vs nanoparticle albumin-bound paclitaxel plus gemcitabine (nab-P/G) for borderline resectable pancreatic cancer (BRPC) and locally advanced pancreatic cancer (LAPC) patients who completed resection. The study retrospectively enrolled patients with pathologically confirmed BRPC or LAPC from 2010 to 2018 at our institution. The survival rates were determined by the Kaplan-Meier method and log-rank test was used to test differences. Cox's proportional hazard model was used to assess survival with respect to covariates. Seventy-two patients who completed at least two cycles of neoadjuvant chemotherapy and surgical resection were included, with 52 (72.2%) patients receiving mFOLF and 20 (27.8%) receiving nab-P/G. Patients treated with mFOLF had statistically higher rates of RECIST 1.1 partial or complete response (16/52 vs 1/20, P = .028). Additionally, mFOLF patients had greater pathological tumor size reduction, fewer positive lymph nodes, and higher treatment response grade compared to the nab-P/G patients (all P < .05). The median overall survival was 33.3 months vs 27.1 months (P = .105), and distant metastasis-free survival (DMFS) was 21.3 months vs 14.6 months (P = .042) in the mFOLF vs nab-P/G groups, respectively. On multivariate analysis, mFOLF (hazard ratio, 0.428; 95% confidence interval [CI], 0.186-0.987) and abnormal postoperative CA 19-9 (hazard ratio, 2.47; 95% CI, 1.06-5.76) were associated with DMFS. Among patients with BRPC and LAPC who complete surgical resection, neoadjuvant mFOLF was associated with improved pathological and clinical outcomes compared with nab-P/G.

A microRNA-based signature predicts local-regional failure and overall survival after pancreatic cancer resection.

Resectable pancreatic adenocarcinoma (PC) is generally managed with surgery followed by chemotherapy, but the role of postoperative chemoradiation (pCRT) is controversial. We sought to identify a microRNA (miRNA) expression profile associated with higher risk for local-regional recurrence (LRR), which might help identify patients that may benefit from pCRT. Total RNA was isolated from viable tumor from 88 patients who underwent PC resection with or without chemotherapy, but did not receive radiation. Digital miRNA expression profiling was performed and risk scores were calculated based on the expression levels of the four most significantly correlated miRNAs, and dichotomized about the median to detect correlations between risk group, LRR and overall survival (OS). Two cohorts from The Cancer Genome Atlas (TCGA) and Seoul National University (SNU) were used for validation. Patients with high-risk scores had significantly worse LRR (p = 0.001) and worse OS (p = 0.034). Two-year OS rates for the high- and low-risk groups were 27.7% and 52.2%, respectively. On multivariable analysis, the risk score remained significantly associated with LRR (p = 0.018). When validated on TCGA data, a high-risk score was associated with worse OS on univariate (p = 0.03) and multivariable analysis (p = 0.017). When validated on the SNU cohort, a high-risk score was likewise associated with worse OS (p = 0.042). We have developed a 4-miRNA molecular signature that is associated with risk of LRR and OS after PC resection and validated on two separate cohorts. This signature has the potential to select patients most likely to benefit from pCRT, and should be tested further.

Clinical Outcomes and Multidisciplinary Patterns of Failure for Olfactory Neuroblastoma: The Ohio State Experience.

Purpose Olfactory neuroblastoma (ONB) is a rare head and neck cancer believed to be originated from neural crest cells of the olfactory membrane located in the roof of the nasal fossa. This study evaluates clinical outcomes and failure patterns in ONB patients of those patients treated with surgical resection at a high-volume tertiary cancer center. Methods and Materials Thirty-nine ONB patients who underwent surgical resection at our institution from 1996 to 2017 were retrospectively identified. Univariate, multivariate, and survival analysis were calculated using Cox regression analysis and Kaplan-Meier log-rank. Results Median follow-up time was 59 months (range: 5.2-236 months). The median overall survival (OS) and disease-free survival (DFS) for the entire cohort were 15 and 7.6 years, respectively. The 5-year cumulative OS and DFS were 83 and 72%, respectively. The 5-year OS for low Hyams grade (LHG) versus high Hyams grade (HHG) was 95 versus 61% ( p = 0.041). LHG was found in 66% of the early Kadish stage patients compared with 28% in the advanced Kadish stage patients ( p = 0.057). On multivariate analysis, HHG and positive node status predicted for worse OS and only HHG predicted for worse DFS. Of note, five patients (all Kadish stage A) who received surgical resection alone had no observed deaths or recurrences with a median follow-up of 44 months (range: 5-235 months). Conclusion In this retrospective cohort, patients with positive nodes or HHG have significantly worse clinical outcomes. Future studies should explore treatment intensification for HHG or positive nodes.

Inhibiting BRAF Oncogene-Mediated Radioresistance Effectively Radiosensitizes BRAFV600E-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair.

PURPOSE: Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. EXPERIMENTAL DESIGN: Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. RESULTS: BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. CONCLUSIONS: BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.

Altering the response to radiation: radiosensitizers and targeted therapies in pancreatic ductal adenocarcinoma: preclinical and emerging clinical evidence.

Radiation therapy continues to have an evolving role in pancreatic ductal adenocarcinoma. While metastatic failure likely contributes to the majority of patient mortality, achieving local control through surgery and/or radiation appears to be important as certain studies suggest that mortality is contributed by local failure. Many studies support that pancreatic cancer is a relatively radiation resistant tumor type. In addition, the ability to further improve radiation through dose escalation strategies in the non-metastatic setting is hampered by closeness of normal organs, including small bowel and stomach, to the tumor. Thus subverting molecular pathways that promote radiation resistance will be critical to further success of radiation in this disease. There is a wealth of preclinical data supporting the targeting of various molecular pathways in combination with radiation therapy, including DNA repair, cell cycle checkpoint proteins, receptor tyrosine kinases, oncoproteins, stem cells, and immunomodulation. A number of clinical trials have been completed or are on-going with novel molecular inhibitors. In this review, we summarize existing preclinical and clinical molecular strategies for improving the efficacy of radiation in pancreatic cancer, and highlight recent and ongoing clinical trials combining radiation and various targeted therapies.

Blocking Interleukin (IL)4- and IL13-Mediated Phosphorylation of STAT6 (Tyr641) Decreases M2 Polarization of Macrophages and Protects Against Macrophage-Mediated Radioresistance of Inflammatory Breast Cancer.

PURPOSE: To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. METHODS AND MATERIALS: The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. RESULTS: Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. CONCLUSIONS: These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

Lack of Breastfeeding History in Parous Women with Inflammatory Breast Cancer Predicts Poor Disease-Free Survival.

Purpose: Breastfeeding alters the breast microenvironment, and several lines of evidence suggest the breast microenvironment contributes to the clinical phenotype of inflammatory breast cancer. We investigated breastfeeding history as a modifier of locoregional recurrence (LRR), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS) in parous women with inflammatory breast cancer. Methods: Parous women with inflammatory breast cancer were identified from a prospective registry at The University of Texas MD Anderson Cancer Center. We compared patient and tumor characteristics, LRR, DM, DFS, and OS patients with (BF+) and without (BF-) a history of breastfeeding. Results: Eighty-two patients were included. At a median follow-up of 50 months, BF+ patients had significantly lower risk of LRR (9.0% vs. 23.6%; p=0.01), a lower risk of DM (26.8% vs. 53.8%; p=0.008), and better DFS (73.1% vs. 48.1%; p=0.006) than BF- patients. On multivariate analysis, BF+ history was associated with significantly lower risk of DM (hazard ratio 0.38, 95% confidence interval 0.15-0.97; p=0.04) and better DFS (hazard ratio 0.37, 95% confidence interval 0.15-0.93; p=0.04) after adjusting for established predictive and prognostic variables. The prognostic significance of breastfeeding may be most pronounced in women with triple-negative IBC. Conclusion: A lack of breastfeeding history in parous women with inflammatory breast cancer may predict worse prognosis. We speculate that breastfeeding-induced alterations in the breast microenvironment may alter the aggressiveness of inflammatory breast cancer.

Histone deacetylase inhibitor-induced cancer stem cells exhibit high pentose phosphate pathway metabolism.

PURPOSE: We recently demonstrated that histone deacetylase (HDAC) inhibitors can "reprogram" differentiated triple-negative breast cancer cells to become quiescent stem-like cancer cells. We hypothesized that the metabolic state of such cells differs from that of their differentiated progeny. RESULTS: In untreated cells, glucose uptake was higher in ALDH+ cells than in ALDH- cells (p = 0.01) but lactate production was not different; treating ALDH- or ALDH+ cells with VA or SAHA similarly increased glucose uptake without changing lactate production but upregulated G6PD, a rate-limiting enzyme in pentose phosphate pathway metabolism. NADPH production was higher in HDAC inhibitor-treated stem-like cells than in vehicle-treated cells (p < 0.05). Two G6PD inhibitors, 6-aminonicotinamide and dehydroepiandrosterone, decreased mammosphere formation efficiency and ALDH activity and 6-aminonicotinamide reduced the VA-induced increase in ALDH+ cells. Finally, patients expressing high G6PD mRNA had significantly worse overall survival (p < 0.001), and patients with high G6PD protein showed a similar trend towards worse disease-specific survival (p = 0.06). METHODS: Glucose consumption, lactate and NADPH production, and reactive oxygen species generation were compared in aldehyde dehydrogenase (ALDH)-positive and -negative cells in the presence or absence of the HDAC inhibitors valproic acid (VA) or suberoylanilide hydroxamic acid (SAHA). Glucose-6-phosphate dehydrogenase (G6PD) expression was evaluated in a tissue microarray from 94 patients with node-positive invasive breast carcinoma and in two publically available databases and correlated with overall survival. CONCLUSIONS: Energy metabolism in HDAC inhibitor-induced stem-like cancer cells differed sharply from that of differentiated cell types. HDAC inhibitor-induced dedifferentiation promoted metabolic reprogramming into the pentose phosphate pathway, which is targeted effectively by G6PD inhibition. These findings highlight a potential dual-therapy approach to targeting bulk differentiated cells with HDAC inhibitors and CSCs with G6PD inhibitors.