BackgroundThe dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.MethodsWe compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.FindingsB7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.InterpretationWe demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM.FundAlex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.

Since CNR scales with the square root of imaging dose, changing from TBL + LFB to IBL + LFB and IBL + LFB to IBL + SPA reduces the imaging dose required to obtain a given CNR by factors of 0.38 and 0.37, respectively. MTFs were comparable between imaging system configurations. IBL + SPA patient image quality was always better than that of the TBL + LFB system and as good as or better than that of the IBL + LFB system, for a given dose.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. Deregulation of these systems can lead to conflicts between the transcription and replication machinery leading to DNA damage. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes DNA damage that correlates with RNAPII-dependent transcript elongation and occurs preferentially in S-phase cells.BET bromodomain inhibition also causes accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts, DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET-bromodomain inhibitor-induced DNA damage, and that BET-bromodomain inhibition induces both Rloops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 Cterminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of Rloops in highly replicative cells.

No abstract

Purpose: To quantitatively and qualitatively assess improvement in megavoltage cone beam CT (MVCBCT) image quality afforded by a 4.2 MV imaging beam line (IBL) with a carbon electron target and new pixelated ultrafast ceramic scintillator (UFC) detector, relative to the 6 MV treatment beam line (TBL). Detector blur is reduced with the UFC since light generated in a pixel is less likely to escape laterally. Methods: A prototype IBL+UFC system was installed on a Siemens ONCOR linear accelerator equipped with the MVision™ IGRT system. A UFC strip consisting of four tiles and measuring ∼40 cm by ∼10 cm was installed on the flat panel imager, with the long dimension in the cross‐plane direction. Phantom images were acquired at doses from 2–60 cGy with the TBL, IBL with conventional scintillator, and IBL+UFC. Several head and neck, thoracic, and pelvic patients were imaged with the three systems at doses from 2–15 cGy. Results: Phantom images indicate that the IBL+UFC images have lower noise and higher contrast than the IBL and TBL images. The contrast‐to‐noise ratio (CNR) for the IBL was 1.5–2 times higher than for the TBL, and the IBL+UFC CNR was 1.5–2 times better than for the IBL at all doses. CNR saturated near 30 cGy for the IBL+UFC and at 60 cGy or greater for the IBL and TBL. IBL+UFC patient images showed improved soft tissue contrast at all doses and sites examined. Conclusions: The IBL+UFC combination increases the CNR by up to a factor of four relative to the TBL with the conventional scintillator, and a factor of two relative to the IBL with the conventional scintillator. Image noise and soft tissue contrast in head and neck, thoracic, and pelvic patients improved dramatically in the IBL+UFC images relative to the TBL images. Sponsored partially by Siemens Oncology Care Systems.

Purpose: Bulky (>40 cc) cervical cancer brachytherapy (BT) tumor dose conformity is often poor since symmetric BT dose is limited by the presence of the nearby bladder, rectum, and sigmoid. Rotating shield intensity modulated brachytherapy (RS‐IMBT) can theoretically improve tumor dose conformity, but at the cost of significantly increased treatment times. We developed a time efficient method of improving cervical cancer dose distributions with multi‐rotating‐shield IMBT (MRS‐IMBT) that drastically reduces the delivery times relative to RS‐IMBT. Methods: We developed a shield sequencing method that optimally divides a set of finely sampled IMBT shielding patterns into a combination of coarse and fine shields that delivers the same dose distribution but with a reduced treatment time. The resulting MRS‐IMBT method was applied to eight cervical cancer patients who were treated with MRI‐guided conventional BT. The treatment planning was done with an in‐house treatment planning system. Results: The MRS‐IMBT method significantly reduced treatment times relative to finely‐sampled IMBT without losing tumor coverage. When using shields that provided adequate coverage (22.5°), the sequencing method on average decreased the treatment time by a factor of 4. For the finest shields considered (5.625°), MRS‐IMBT decreased the treatment time by a factor of 12 on average. Conclusions: While RS‐IMBT causes treatment times to rise exponentially with the number of emission directions when a single shield emission angle is used, treatment times for the MRS‐IMBT method increase linearly with the number of emission directions. MRS‐IMBT makes IMBT delivery clinically feasible, which could lead to mean better tumor dose conformity and improved outcomes.

Purpose: To demonstrate that megavoltage cone beam CT (MVCBCT) image quality can be significantly improved without increasing imaging dose or reducing spatial resolution for both head‐ and pelvis‐sized volumes. The improvement is achieved with the combination of an imaging beam line (IBL) with a low atomic number electron target and a novel sintered pixelated array (SPA) detector. Methods: Three Siemens Oncor linear accelerators were equipped with an IBL+SPA system, an IBL system with a conventional Kodak Lanex Fast B scintillator (IBL+LFB), and a 6 MV treatment beam line system with an LFB (TBL+LFB). Head‐ and pelvis‐sized phantom images were acquired with all three systems at imaging doses ranging from 2‐60 cGy. Contrast to noise ratio (CNR) and modulation transfer function (MTF) were calculated from the phantom images. Head and neck, prostate, and lung cancer patients were imaged with the three imaging systems at doses ranging from 2–15 cGy. Results: For head‐ and pelvis‐sized phantom images acquired at 5 cGy or above, the CNR average percentage increases for imaging system upgrades from TBL+LFB to IBL+LFB to IBL+SPA were 52% (p < 1E‐7) and 42% (p < 1E‐6), respectively. The MTFs do not change with imaging system by statistically significant percentages. Soft tissue contrast is generally more easily differentiated on IBL+SPA images than TBL+LFB and IBL+LFB in the patient images. Conclusions: Since CNR scales with the square root of imaging dose, each step in the TBL+LFB to IBL+LFB to IBL+SPA upgrade halves the imaging dose required to obtain a given CNR. No statistically significant change in spatial resolution was observed with any upgrade, suggesting that the pixelation of the SPA prevents a loss in spatial resolution. IBL+SPA patient image quality was always better than that of the TBL+LFB system and as good as or better than that of the IBL+LFB system. Sponsored partially by Siemens Oncology Care Systems.