Purpose/objectivesDespite mounting evidence for the use of re-irradiation (re-RT) in recurrent high grade glioma, optimal patient selection criteria for re-RT remain unknown. We present a novel scoring system based on radiobiology principles including target independent factors, the likelihood of target control, and the anticipated organ at risk (OAR) toxicity to allow for proper patient selection in the setting of recurrent glioma.Materials/methodsThirty one patients with recurrent glioma who received re-RT (2008–2016) at NCI – NIH were included in the analysis. A novel scoring system for overall survival (OS) and progression free survival (PFS) was designed to include:1) target independent factors (age, KPS (Karnofsky Performance Status), histology, presence of symptoms), 2) target control, and 3) OAR toxicity risk. Normal tissue complication probability (NTCP) calculations were performed using the Lyman model. Kaplan-Meier analysis was performed for overall survival (OS) and progression free survival (PFS) for comparison amongst variables.ResultsNo patient, including those who received dose to OAR above the published tolerance dose, experienced any treatment related grade 3–5 toxicity with a median PFS and OS from re-RT of 4 months (0.5–103) and 6 months (0.7–103) respectively. Based on cumulative maximum doses the average NTCP was 25% (0–99%) for the chiasm, 21% (0–99%) for the right optic nerve, 6% (0–92%) for the left optic nerve, and 59% (0–100%) for the brainstem. The independent factor and target control scores were each statistically significant for OS and the combination of independent factors plus target control was also significant for both OS (p = 0.02) and PFS (p = 0.006). The anticipated toxicity risk score was not statistically significant.ConclusionOur scoring system may represent a novel approach to patient selection for re-RT in recurrent high grade glioma. Further validation in larger patient cohorts including compilation of doses to tumor and OAR may help refine this further for inclusion into clinical trials and general practice.Electronic supplementary materialThe online version of this article (10.1186/s13014-017-0930-9) contains supplementary material, which is available to authorized users.

Introduction: Glioblastoma (GBM) is the most common primary malignant brain tumor in humans and, even with aggressive treatment that includes surgical resection, radiation (IR), and chemotherapy administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem-like cells (GSLCs) exist, which are thought to be therapy resistant and are thus capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg Effect. There is no consensus on the metabolic characteristics of cancer stem cells. GSLCs have been shown to rely more heavily on oxidative phosphorylation, but there is also evidence that cancer stem cells can adapt their metabolism by fluctuating between energy pathways or acquiring intermediate metabolic phenotypes. We hypothesized that the metabolism of GSLCs differs from that of differentiated GBM tumor cell lines, and that the steady state metabolism would be differentially altered following radiation treatment. Materials and Methods: We evaluated the oxygen consumption rate, extracellular acidification rate, and metabolic enzyme levels of GBM cell lines and GSLCs before and after irradiation using extracellular flux assays. We also measured absolute metabolite levels in these cells via mass spectroscopy with and without radiation treatment. Results: GSLCs were found to be significantly more quiescent in comparison to adherent GBM cell lines, highlighted by lower glycolytic and maximal respiratory capacities as well as lower oxygen consumption and extracellular acidification rates. Analysis of individual metabolite concentrations revealed lower total metabolite concentrations overall but also elevated levels of metabolites in different energy pathways for GSLCs compared to GBM cell lines. Additionally, the metabolism of both GSLCs and GBM cell lines were found to be altered by IR. Conclusion: While there is not one metabolic alteration that distinguishes irradiated GSLC metabolism from that of GBM cell lines, therapies targeting more metabolically quiescent tumor cells and thus the resistant GSLC population may increase a cancer's sensitivity to radiotherapy.

Local tumor ablation can be achieved by physical energy-based therapies, such as, irradiation, ultrasound, microwave and radiofrequency. Although tumor antigens released from ablated tumors could induce an anti-tumoral immunity, antigen presentation from ablated tumors are not efficient in inducing a strong systemic immunity. Recently, we demonstrated that immunopriming by LOFU, followed by hypofractionated radiation, reversed immunological tolerance of T cells in draining lymph nodes, induced systemic anti-tumoral immunity, and produced local, regional and metastatic control of murine melanoma (J. Immunol. 196(4):1964-1976). LOFU uses low energy (w500 W/cm 2) and operating frequency of 1 MHz to induce membrane perturbation and mild hyperthermia at the focal zone, without ablating the tissue. Here, we investigate the effect of LOFU on gene expression and cell surface immunomodulation of murine tumor cells. Materials/Methods: LOFU parameters were set at 3-5W of total acoustic output power, 50% duty cycle, 1.5 seconds, and 1 mm spacing (TIPS, Philips Research). After LOFU treatment of a cell pellet, cells were cultured for various time points for RNA sequencing, qPCR, immunofluorescence and flow cytometry studies. Tumor cell lines included human DU145 prostate cancer cells and mouse RM1 and TPSA23 prostate cancer, Lewis lung carcinoma (3LL) and breast cancer (4T1). TPSA23 was derived from TRAMPC1 which was genetically engineered to express human PSA. Statistical evaluation distinguished gene expression changes by 1.5-fold, with false detection rate (FDR) <0.05. Wilcoxon rank sum was used to detect statistical significance in gene expression from RT-PCR results (p<0.05). Results: Gene expression studies (RNAseq and qRT-PCR) demonstrate that LOFU (3W) induced key genes involved in the unfolded protein response (UPR) pathway. Compared to untreated DU145 cells, LOFU induced ERN1 three-folds, HSPA1B and HSPH1 by 8.79 folds and HSP105/110 by 6.18-fold within 6-24 hrs. In separate experiments, there was a significant increase in Hspa1 RNA expression (132-fold) and CHOP expression (2.5 fold) 24 h post-LOFU, in RM-1 and 4T1 cells, respectively. Flow cytometry and immunofluorescence in 3LL, 4T1 and RM1 cells demonstrated the translocation of stress proteins, BiP, calreticulin and HSP70 to the cell surface. There was induction of cell surface death receptors (CD40 and Fas), immune checkpoint inhibitors (PD-L1 and PD-L2) and class I MHC in 3LL, 4T1 and TPSA23 cell lines. Conclusion: These studies demonstrate that LOFU induces expression of genes involved in UPR and endoplasmic stress response. Translocation of stress chaperones to the cell surface along with increased expression of MHC I and death receptors contribute to the immunomodulatory properties of LOFU.

Although most patients (pts) achieve complete remission (CR) following intensive chemotherapy, relapse occurs in about 50% of the cases and is associated with poor prognosis. In this AML subset monitoring of measurable residual disease (MRD) has been shown to identify pts at higher risk of relapse. Aims: To assess the prognostic impact of MRD monitoring in bone marrow (BM) and peripheral blood (PB) in a large cohort of 155 homogeneously treated and clinically well-annotated patients with t(8;21)-AML. Methods: Quantification of RUNX1-RUNX1T1 transcript levels (TL) was performed by RT-qPCR. TL were reported as normalized values of RUNX1-RUNX1T1 per 10 6 transcripts of the housekeeping gene B2 M. Samples were analyzed in triplicate, the sensitivity of our assay was up to 10 -6 . Results: While pretreatment RUNX1-RUNX1T1 TL did not impact prognosis, both reduction of RUNX1-RUNX1T1 TL and achievement of MRD negativity (MRD neg ) at defined time points were of significant prognostic importance. First, achievement of MR 2.5 (>2.5 log reduction) after treatment cycle 1 and achievement of MR 3.0 after cycle 2 were significantly associated with a reduced risk of relapse (P = 0.034 and P = 0.028, respectively). Second, after completion of therapy, achievement of MRD neg in both, BM and PB, was an independent favorable prognostic factor for cumulative incidence of relapse (4-year CIR BM: 17% vs 36%, P = 0.021; PB: 23% vs 55%; P = 0.001) and overall survival (4-year OS rate BM: 93% vs 70%, P = 0.007; PB: 87% vs 47%; P < 0.0001). Finally, during follow-up serial RT-qPCR analyses allowed prediction of relapse in 77% of pts exceeding a cut-off of 150 RUNX1-RUNX1T1 TL in BM, and in 84% of pts with > 50 RUNX1-RUNX1T1 TL in PB, respectively. KIT mutation observed in 28% of pts predicted for lower CR rate and inferior outcome, but its prognostic impact was outweighed by RUNX1-RUNX1T1 TL during treatment. Virtually all relapses occurred within one year after end of treatment (EOT) with a very short latency from molecular to morphologic relapse, necessitating MRD assessment at short intervals during this period. Based on our data we suggest a refined practical guideline for MRD assessment in RUNX1-RUNX1T1-positive AML: Along with the current ELN MRD recommendations, BM and PB should be analyzed after each treatment cycle. According to MRD at EOT and during follow-up we suggest MRD monitoring as follows: (i) MRD neg : PB monthly can be used, (ii) MRD pos (TL < 150 in BM and < 50 in PB): BM 3-monthly and PB monthly, and (iii) MRD pos (TL >150 in BM and/or >50 in PB) or increase of MRD >1-log or conversion from MRD neg to MRD pos : BM and PB, both monthly, should be analyzed. Summary/Conclusion: RUNX1-RUNX1T1 MRD monitoring allows for the discrimination of pts at high and low risk of relapse. MRD neg in both, BM and PB, after completion of therapy was the most valuable independent favorable prognostic factor for relapse risk and OS. During follow-up, serial MRD analyses allowed the definition of cut-offs predicting relapse. Moreover, con...