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Colony forming unit (CFU) enumeration of ST in tumors of mice treated with and without PEGPH20. Mice with 14-day orthotopic KPC tumors (n=3) were given indicated treatment regimens. Tumors were then extracted at indicated time points post-treatment, homogenized in PBS containing a protease inhibitor mix (Roche), plated on LB-0 plates containing appropriate antibiotics, and then incubated at 37ï‚°C overnight. Data is representative of multiple experiments utilizing identical treatments and post-treatment analyses. (A) CFU comparison of mice treated with ST, with and without PEGPH20, at various time points after treatment. (B) Effects of Gr-1 depletion (Gr1-) on accumulation of shIDO-ST in orthotopic KPC tumors in mice treated with and without PEGPH20. Mice were depleted 24hr prior to treatment. Tumors were extracted 48hr post-treatment and homogenized for CFU enumeration. *p

Evaluation of inflammatory cytokines and ICAM-1 in tumors of mice treated with PEGPH20/shIDO-ST. Mice bearing 14 day orthotopic KPC tumors were treated with indicated regimens. Tumors were excised 96 hours post-treatment and used for RT-qPCR to detect cytokines or immunofluorescence to detect ICAM-1. Data are representative of multiple experiments. (A) Total mRNA was isolated (Qiagen) to generate cDNA libraries using a RevertAid First Strand DNA kit (Thermo Scientific) and analyzed for indicated cytokine mRNAs (Power Syber Green kit, 7300 RT-PCR cycler (Applied Biosystems)). Experimental Ct values were normalized to GAPDH. Relative expression levels were calculated using the equation 2-delta deltaCt and using the CT values of the PEGPH20 only treatment group as the comparator . *p{less than or equal to}0.05, **p{less than or equal to}0.01, ***p{less than or equal to}0.001 using 1-way ANOVA. (B) Tissue sections from indicated treatment groups were stained for ICAM-1 (green), PMN (red) and nuclei (DAPI, blue). Arrows represent leading edges of PMN infiltration associated with ICAM-1. Magnification, 10X.

Key replicate treatment groups (A) Replicate IVIS images of key treatment groups emphasizing reproducibility of treatment in KPC orthotopic model. Numbers above images represent days post-tumor implantation. (B) Quantitation of IVIS luciferase signal for individual mice in key groups represented in Fig. 1A and Supplementary Fig. S3A.

Pathology of peripheral organs in treated mice. Selected organs from mice given indicated treatments were reviewed histopathologically by hematoxylin/eosin stain. The liver shows portal and parenchymal inflammation with occasional foci of coagulative necrosis (N) in mice treated with PEGPH20/shIDO-ST or PEGPH20/shScr-ST, which also show minimal inflammatory changes within kidney (not shown) and pancreas. These findings can be ascribed to the use of Salmonella as a vector, since they are not seen in the PEGPH20 control mice. These findings have been documented previously using VNP20009 (13). Mild splenic hyperplasia is seen in all treated animals. Images shown are representative of multiple experiments.

Western blot analysis of cleaved caspase 3 (CC3). Mice bearing 14 day orthotopic KPC tumors were treated with indicated regimens. Tumors were excised 96 hours post-treatment, homogenized, and total protein was extracted using cell lysis buffer containing a protease inhibitor cocktail mix (Roche). Equal amounts of protein (Bradford) were loaded. Blots were stained separately for pro-caspase 3 and CC3. Equal loading of samples was confirmed by stripping CC3 membranes and reprobing for β-actin. Data are representative of multiple experiments.

Depletion of adaptive immunity. Mice with 14 day tumors were injected intraperitoneal with depleting antibody (CD4, CD8, NK, IgG: 200 ug/mouse; Gr1: 60 ug/mouse) against indicated immune subsets 24hr prior to treatment with PEGPH20/shIDO-ST. Depletion antibodies were re-administered every 3 days. IVIS data are presented as (A) luminescent images, (B) quantitatively for each individual animal (n=6) and (C) collectively from multiple experiments. Groups were euthanized when {greater than or equal to}1 mouse reached endpoint (see Materials and Methods). Combined data only represents time points for groups where the total number of mice was still 6 (n=6). Statistical analyses were also performed for time points under these same criteria. **p{less than or equal to}0.01, Student's t test.

Isolation and characterization of splenic PMN from PEGPH20/ shIDO-ST treated mice. (A) PMN were purified from crude spleen homogenates of treated mice through negative bead selection. Percentage of PMN in crude and purified preparations were determined by flow cytometry. (B) PEGPH20/shIDO-ST treatment was administered to mice that were depleted of PMN using Gr-1 antibody or administered IgG control. KPC-luc tumor growth was monitored by IVIS at indicated time points and quantitated using Living Image® software.

Chromium release assay utilizing a range of PMN (effectors) against various tumor targets. Purified splenic PMN from treated mouse groups (n=3) were used in a 51Cr cytotoxicity assay. Tumor targets consisted of KPC (pancreatic cancer), 4T1 (breast cancer), and B16F10 (melanoma) which were co-incubated directly with effectors at indicated E:T ratios. Each E:T ratio with specific tumor targets were done in triplicate. Percent lysis within the PEGPH20/shIDO-ST group was compared to the PEGPH20/shScr-ST treated group at each E:T ratio using Student's t test. Data shown is representative of multiple experiments.

PMN tracking in control mice. (A) Normal C57BL/6 non-tumor bearing mice were injected intravenously with 50 uL of a 20 uM working solution of NS-NIR reagent. Mice were imaged 1 hr proceeding injection of NS-NIR. Mice were also injected with 300 ug D-luciferin in 200 µL PBS 5 min prior to imaging. Image depicts representative mouse. Imaged with a Xenogen IVIS 100. (B) Mice treated with shScr-ST or shIDO-ST alone show no intratumoral PMN infiltration. C57BL/6 mice implanted with orthotopic KPC-luc tumors were treated with shIDO-ST or shScr-ST as outlined in Supplementary Fig. S2. Mice were imaged at the indicated time points following first treatment with ST. Imaged with a Xenogen IVIS 100. (C) Key replicate data of PMN infiltration (brown) and tumor (rainbow) for indicated treatment groups. Imaged with a Spectral Ami-X.

Preliminary testing of PEGPH20/shIDO-ST in autochthonous KPC-Brca1 mouse model. PMN and DAPI immunofluorescence staining of an autochthonous tumor section from a KPC-Brca1 mouse treated with PEGPH20/shIDO-ST. Tumor was excised, flash frozen, sectioned, and stained 5 days following treatment. Central DAPI negative region of tumor with neutrophils (red) is depicted at 10X magnification with DAPI+ tumor cells (blue) at upper right (T).

Detection of PMN in extracted tumors of PEGPH20/shIDO-ST treated mice. Mice treated with PEGPH20/shScr-ST or PEGPH20/shIDO-ST were euthanized and tumors extracted 96 hours post-ST treatment. One hour prior to euthanization, mice were injected with NS-NIR reagent. Tumors were imaged using a Xenogen 100 equipped with an ICG filter set.

Generation of KPC-luc orthotopic model. (A) 28 day orthotopic KPC tumor (top, 2050 mm3) extracted from untreated C57BL/6 mouse versus normal C57BL/6 pancreas (bottom, 84 mm3). (B) KPC-luc cell line incubated with increasing titration of D-luciferin and imaged using a Xenogen 100 IVIS (C) 5x105 KPC-luc cells were implanted into the pancreas of C57BL/6 mice and growth monitored for 21 days. Representative mouse shown. (D) Kaplan-Meier curve for mice (n=10) implanted with 5x105 KPC-luc cells.

Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8+ T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC. Cancer Immunol Res; 3(9); 1096–107. ©2015 AACR.

Purpose: Human neural stem cells (NSC) are inherently tumor tropic, making them attractive drug delivery vehicles. Toward this goal, we retrovirally transduced an immortalized, clonal NSC line to stably express cytosine deaminase (HB1.F3.CD.C21; CD-NSCs), which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).Experimental Design: Recurrent high-grade glioma patients underwent intracranial administration of CD-NSCs during tumor resection or biopsy. Four days later, patients began taking oral 5-FC every 6 hours for 7 days. Study treatment was given only once. A standard 3 + 3 dose escalation schema was used to increase doses of CD-NSCs from 1 × 107 to 5 × 107 and 5-FC from 75 to 150 mg/kg/day. Intracerebral microdialysis was performed to measure brain levels of 5-FC and 5-FU. Serial blood samples were obtained to assess systemic drug concentrations as well as to perform immunologic correlative studies.Results: Fifteen patients underwent study treatment. We saw no dose-limiting toxicity (DLT) due to the CD-NSCs. There was 1 DLT (grade 3 transaminitis) possibly related to 5-FC. We did not see development of anti-CD-NSC antibodies and did not detect CD-NSCs or replication-competent retrovirus in the systemic circulation. Intracerebral microdialysis revealed that CD-NSCs produced 5-FU locally in the brain in a 5-FC dose-dependent manner. Autopsy data indicate that CD-NSCs migrated to distant tumor sites and were nontumorigenic.Conclusions: Collectively, our results from this first-in-human study demonstrate initial safety and proof of concept regarding the ability of NSCs to target brain tumors and locally produce chemotherapy. Clin Cancer Res; 23(12); 2951–60. ©2016 AACR.

Supplementary Figure S1. Catheter position with respect to resection site.

Purpose: Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors, such as prostate cancers. However, targeting MDSCs proved challenging due to their phenotypic heterogeneity.Experimental Design: Myeloid cell populations were evaluated using flow cytometry on blood samples, functional assays, and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects, localized and metastatic castration-resistant prostate cancer patients.Results: Here, we identify a population of Lin−CD15HICD33LO granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer–associated MDSCs potently inhibit autologous CD8+ T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3, which is a central immune checkpoint regulator. The granulocytic pSTAT3+ cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA, thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8+ T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1, a downstream STAT3 target gene and a potent T-cell inhibitor.Conclusions: Overall, we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. Clin Cancer Res; 21(16); 3771–82. ©2015 AACR.

Supplementary Figure S5. T cell detection in tumor tissue from UPN031.

Supplementary methods. These are more detailed methods than can be included in the body of the article.

Supplementary Figure S2. Consort flow diagram.

Supplementary Methods from Neural Stem Cell–Based Anticancer Gene Therapy: A First-in-Human Study in Recurrent High-Grade Glioma Patients

Supplementary Tables S1-3. Table S1. T cell Product Release Criteria. Table S2. Manufacturing Feasibility. Table S3. Characteristics of Patients Who Received Study Treatment.

Supplementary Figure S4. 18F-Fluorodeoxyglucose ( F-FDG)-CT/PET of subjects UPN031 and UPN033

Supplementary Figure S3. Treatment regimens for each patient, with relapse designated day 0.

Purpose: To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem–like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8+ CTLs (IL13-zetakine+ CTL) to target this therapeutically resistant glioma subpopulation.Experimental Design: A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine+ CTL-mediated killing in vitro and in vivo.Results: We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2pos cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine+ CTL were capable of efficient recognition and killing of both IL13Rα2pos GSCs and IL13Rα2pos differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine+ CTL displayed robust antitumor activity against established IL13Rα2pos GSC TS-initiated orthotopic tumors in mice.Conclusions: Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine+ CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. Clin Cancer Res; 18(8); 2199–209. ©2012 AACR.

Supplementary Table 1: A standard 3 + 3 dose escalation design (19) was used to investigate 3 dose levels of CD-NSCs and 5-FC. Supplementary Table 2: Characteristics of the 15 patients who received study treatment.

Purpose: A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)–engineered, autologous primary human CD8+ cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM).Experimental Design: Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8+ CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 108 CAR-engineered T cells via a catheter/reservoir system.Results: We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine+ CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine+ CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine+ T-cell administration.Conclusions: These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. Clin Cancer Res; 21(18); 4062–72. ©2015 AACR.

Supplementary Tables 1-3 from Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Supplementary Figures 1-5 from Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8+ CTLs. Compared with serum-differentiated CD133low tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population. [Cancer Res 2009;69(23):8886–93]

Neural stem cells (NSCs) are tumor tropic and can be genetically modified to produce anti-cancer therapies locally in the brain. In a prior first-in-human study we demonstrated that a single dose of intracerebrally administered allogeneic NSCs, which were retrovirally transduced to express cytosine deaminase (CD), tracked to glioma sites and converted oral 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). The next step in the clinical development of this NSC-based anti-cancer strategy was to assess the feasibility of administering multiple intracerebral doses of CD-expressing NSCs (CD-NSCs) in patients with recurrent high-grade gliomas. CD-NSCs were given every 2 weeks using an indwelling brain catheter, followed each time by a 7-d course of oral 5-FC (and leucovorin in the final patient cohort). Fifteen evaluable patients received a median of 4 (range 2–10) intracerebral CD-NSC doses; doses were escalated from 50 × 106 to 150 × 106 CD-NSCs. Neuropharmacokinetic data confirmed that CD-NSCs continuously produced 5-FU in the brain during the course of 5-FC. There were no clinical signs of immunogenicity, and only three patients developed anti-NSC antibodies. Our results suggest intracerebral administration of serial doses of CD-NSCs is safe and feasible and identified a recommended dose for phase II testing of 150 × 106 CD-NSCs.

Despite the use of first-line therapies like fluoropyrimidine and platinum-based cytotoxic chemotherapy, gastric cancer (GC) continues to carry a poor prognosis. Recent subgroup analyses of first-line phase III trials have demonstrated that patients with microsatellite instability–high (MSI-H) metastatic GC derive significant improvement in survival rates when immune checkpoint inhibitors (ICIs) are combined with chemotherapy compared with chemotherapy alone. However, it remains to be seen whether the success of ICIs in the metastatic setting can be translated into earlier stages of GC with resectable disease. We report 6 cases of locally advanced, nonmetastatic MSI-H GC that all demonstrated favorable response following treatment with pembrolizumab in addition to neoadjuvant chemotherapy. With the exception of immune-related colitis in one patient, pembrolizumab was well-tolerated. To our knowledge, this is the first reported US case series of patients treated with an ICI in combination with neoadjuvant chemotherapy for advanced, nonmetastatic, resectable or unresectable MSI-H GC.

High-grade (WHO grades III-IV) glioma remains one of the most lethal human cancers. Adoptive transfer of tumor-targeting chimeric antigen receptor (CAR)-redirected T cells for high-grade glioma has revealed promising indications of anti-tumor activity, but objective clinical responses remain elusive for most patients. A significant challenge to effective immunotherapy is the highly heterogeneous structure of these tumors, including large variations in the magnitudes and distributions of target antigen expression, observed both within individual tumors and between patients. To obtain a more detailed understanding of immunotherapy target antigens within patient tumors, we immunochemically mapped at single cell resolution three clinically-relevant targets, IL13Rα2, HER2 and EGFR, on tumor samples drawn from a 43-patient cohort. We observed that within individual tumor samples, expression of these antigens was neither random nor uniform, but rather that they mapped into local neighborhoods - phenotypically similar cells within regions of cellular tumor - reflecting not well understood properties of tumor cells and their milieu. Notably, tumor cell neighborhoods of high antigen expression were not arranged independently within regions. For example, in cellular tumor regions, neighborhoods of high IL13Rα2 and HER2 expression appeared to be reciprocal to those of EGFR, while in areas of pseudopalisading necrosis, expression of IL13Rα2 and HER2, but not EGFR, appeared to reflect the radial organization of tumor cells around hypoxic cores. Other structural features affecting expression of immunotherapy target antigens remain to be elucidated. This structured but heterogeneous organization of antigen expression in high grade glioma is highly permissive for antigen escape, and combinatorial antigen targeting is a commonly suggested potential mitigating strategy. Deeper understanding of antigen expression within and between patient tumors will enhance optimization of combination immunotherapies, the most immediate clinical application of the observations presented here being the importance of including (wild-type) EGFR as a target antigen.

Chlorotoxin (CLTX), a peptide component of scorpion venom, exhibits selective and broad binding to glioblastoma (GBM) and other tumors with minimal activity against non-malignant cells. We have developed a novel CAR that utilizes CLTX as the tumor targeting domain. Preclinical studies established that CLTX-CAR T cells target GBM through recognition of a receptor complex incorporating membrane-bound matrix metalloprotease-2 (MMP-2). Here, we report initial clinical findings for our phase I trial evaluating safety and bioactivity of CLTX-CAR T cells in patients with MMP2+ recurrent GBM (NCT04214392). Weekly infusions of CLTX-CAR T cells are delivered locoregionally, either directly into the tumor cavity (ICT; Arm 1), or in combination with intracerebroventricular (ICV) delivery (dual ICT/ICV; Arm 2). At this interim analysis, four participants have received at least three cycles of CLTX-CAR T cells ICT (Arm 1; 3-8 cycles) at dose level 1 (DL1; 4M, 20M, 20M CAR T cells per cycle). None of the participants experienced dose limiting toxicity (DLT) during the DLT evaluation period of 28-days, although one participant experienced a serious adverse event of grade 3 cerebral edema, possibly attributed to CAR T cells. Overall, Arm 1-DL1 was well-tolerated, and the next patient cohort will be treated on Arm 2-DL1 (dual ICT/ICV; 8M, 40M, 40M CAR T cells per cycle), as per protocol design. Disease response was assessed by RANO, overall survival, and time to progression; three of four participants achieved a best response of stable disease. Liquid biopsy detected persistent CAR T cells in the tumor cavity throughout treatment, suggesting that the therapeutic cells are not immunogenic. Ongoing studies are evaluating biomarkers of response and resistance, including CAR T cell activation and inflammatory cytokines. This clinical study provides first-in-human evidence for the safety and feasibility of CLTX-CAR T cells as a new class of toxin-based CARs for treatment of GBM.

Acute appendicitis is one of the most common emergency surgical procedure, yet atypical presentation sometimes can be challenging for clinician. I present a case of 19-year-old gentleman that initially presented with 1day history of bilateral testicular pain and lower abdominal pain. His past history includes a positive sexual history. Initial ultrasound of the testis showed bilateral orchitis and an equivocal appendix. With a significantly raised inflammatory marker and highly suspicious for appendicitis, a CT scan was obtained which showed perforated appendicitis and the patient underwent laparoscopic appendicectomy with resolution of symptoms after that. We encourage clinician to be aware of this clinical pitfall as patient can sometimes be managed in other department to minimise any delayed diagnosis or any unnecessary procedure

Background Glioblastoma (GBM) remains an almost universally fatal brain tumor. While CAR T cell immunotherapy has shown promising clinical efficacy, therapeutic failure may reflect our incomplete understanding of target antigen expression. We previously examined variations in antigen expression at the level of individual patients (inter-patient or inter-tumor heterogeneity), focusing on immunotherapy targets IL13Rα2, HER2 and EGFR. We concluded that antigen expression diverged from expectations from random expression. Because antigen escape may arise from GBM cell heterogeneity, we have mapped target antigen expression within individual tumors (intra-tumor heterogeneity). Methods Serial sections from a 43 patient cohort were immunostained (DAB with hematoxylin counterstain) for target antigens IL13Rα2, HER2 and EGFR. Each section was annotated directly from the slide by a neuropathologist. Sections were scanned (0.46 µm/pixel; Hamamatsu), and then working within Fiji/ImageJ, images were segmented by color deconvolution into hematoxylin (nuclei) and DAB layers. Images of nuclear layers were aligned, and used to align the DAB layers. Two schemes were used to examine the spatial distributions of the three target antigens. When tumor domains could be identified, we determined expression of each antigen as optical density (OD). In the second scheme, a 10 µm grid was superimposed on each section, and OD was determined for each position and assembled into spreadsheets (Origin v2019b). Maps for expression were generated from the OD in each position. Results Approaching these maps from the perspective of antigen escape, we examined the extent to which expression of target antigens was spatially mixed, how rapidly antigen dominance could shift (spatial frequency), and whether spatial distributions were arrayed in a coordinated manner. When tumor domains could be identified, we calculated the Shannon diversity index (H) for each domain within a section. While values of H clustered within some tumors, usually values of H varied widely. The superimposed grid was used to examine heterogeneity within entire tumor sections. Expression was intermixed, and EGFR and IL13Rα2/HER2 displayeds complementary expression patterns. In tumors with large EGFR+ areas, IL13Rα2+/HER2+ areas could overlap, while when EGFR+ areas were smaller, IL13Rα2+ and HER2+ areas were more distinct. Borders could be quite diffuse, or quite sharp (a few cell diameters). Conclusions Our results indicate that expression of IL13Rα2, HER2 and EGFR is highly heterogeneous and not always spatially distinct. Because GBM tumors adapt to the selection pressures of immunotherapies, we suggest that combination therapies should be designed accordingly, and immunotherapies targeting IL13Rα2/HER2 could benefit from inclusion of EGFR.

91 Background: Chimeric antigen receptor (CAR)-engineered T cell therapies are being pursued for the treatment of mCRPC. Prostate Stem Cell Antigen (PSCA) is highly expressed on the surface membrane in mCRPC and with limited expression on normal tissues. We undertook a phase 1, first-in-human study of a PSCA-targeted 4-1BB-co-stimulated CAR T cell therapy in mCRPC. Methods: CAR T cells were manufactured at City of Hope’s cGMP facility. The trial followed the Target equivalence range design with an equivalence range of.20-.35 and too toxic level of 0.51 following participants in cohorts of 3. The plan was to begin at a dose of 100 million (M) without lymphodepletion (LD) chemotherapy consisting of fludarabine and cyclophosphamide, then add LD to 100M prior to dose escalation to maximum 600M. Patients (pts) were required to have disease progression after at least 1 androgen receptor targeted therapy but there was no limit on prior chemotherapy or other treatments. Primary objective is to define the dose limiting toxicity (DLT) and recommended phase 2 dose as well as to describe preliminary bioactivity and efficacy. Correlative studies include MRI for target bone lesion response, CAR T cell persistence, circulating tumor cells, and serum cytokines. Results: 12 pts have been treated to date, median age 68 (42-72). Three pts were treated at the 100M dose with no DLTs. In the 100M plus LD dose level there 2 pts experienced DLT of grade 3 cystitis non-infective and fatigue. The protocol was amended to reduce the LD dose to 300 mg/m2 cyclophosphamide D1-3 and intensify monitoring with early intervention for cystitis. No DLT occurred in 3 pts treated in the modified LD 100M cohort. Cytokine release syndrome (CRS), DLTs and best response by RECIST are presented by dose level in the table. PSA declines (one >90%) were seen as well as radiographic improvement, though RECIST response was limited to stable disease (SD) by concurrent bone metastases. Correlative studies indicated bioactivity of PSCA-CAR T cells. Conclusions: PSCA-CAR T cell therapy is feasible in pts with mCRPC with DLT of cystitis, and shows preliminary anti-tumor effect at a dose of 100M plus LD. Dose escalation to 300M may proceed. Clinical trial information: NCT03873805. [Table: see text]

Purpose Single agent PD-1 inhibitors have yielded durable responses in a minority of gastroesophageal cancers. Radiation therapy has been recognized to promote antitumor immune responses and may synergize with anti-PD-1 agents. We sought to evaluate if combining palliative radiation therapy with pembrolizumab can augment antitumor immune responses in gastroesophageal cancer. Methods and Materials Patients had metastatic gastroesophageal cancer with indication for palliative radiation therapy with ≥2 disease sites outside of the radiation field assessable for abscopal response and biopsies for laboratory correlative analyses. Palliative radiation was delivered to a dose of 30 Gy over 10 fractions. Pembrolizumab, 200 mg, was administered concurrently intravenously every 3 weeks until disease progression, unacceptable toxicity, or study withdrawal, for up to 2 years. Endpoints included PD-L1 expression in pre- and posttreatment biopsies and abscopal objective response rate per Response Evaluation Criteria in Solid Tumors. Results Of 14 enrolled patients, the objective response rate was 28.6% (95% confidence interval, 8.4%-58.1%), and the median duration of response was not reached (95% confidence interval, 6.9-NR months). Overall, 2 patients had treatment-related grade 3 to 4 adverse events with no grade 5 events. One patient discontinued therapy due to grade 4 colitis. We did not observe an association between radiation and abscopal changes in PD-L1 expression via assessment of an analogous PD-L1 Combined Positive Score, Tumor Proportion Score, Mononuclear Immune Cell Density Score, or proportion of PD-L1-expressing immune cells between pre- and posttreatment tumor biopsies. Conclusions Combining palliative radiation therapy and pembrolizumab provided promising durable responses in this patient population but we were unable to definitively distinguish abscopal biologic changes. Biomarker analyses beyond PD-L1 expression are needed to better understand putative mechanisms and identify patients who will benefit from this approach.

Tumor metastasis to the pituitary gland is a rare, not well-documented and life-threatening condition associated with a shortened life span. A better understanding of its clinical manifestations could lead to earlier diagnosis, appropriate therapy and potentially improving quality of life. Therefore, we retrospectively studied the charts of patients with pituitary metastases who were treated at the City of Hope National Medical Center (Duarte, CA) from 1984 to 2018. We reviewed and analyzed tumor origin, primary pituitary clinical manifestation, duration between primary tumor diagnosis and pituitary metastasis, type of treatment and patient survival. A total of 11 patients were identified with a mean age of 59.2 years and median survival following the diagnosis of metastasis of 10 months. Breast cancer and lymphoma were the most common primary origins in these cases, and diabetes insipidus and panhypopituitarism were the most common clinical manifestations of their metastasis. We also compared our results with reports in the literature published between 1957 and 2018. A total 289 patients with pituitary metastasis have been reported in the literature. Breast cancer was the most frequent primary origin of the metastasis, and visual symptoms were the most common primary manifestation. The posterior part of the pituitary is more susceptible than the anterior to metastasis. Pituitary metastasis may occur as a consequence of successful primary tumor treatment prolonging the chance of seeding. Future studies are needed to determine the molecular mechanism of metastasis to the pituitary.

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) T cell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory T cells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13ζ-CAR CD8+ T cells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR T cell anti-tumor activity in vivo. Furthermore, we found that local intracranial delivery of CAR T cells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR T cell therapy for the treatment of brain tumors.

Measuring signals in ultra-fast electronic systems requires high-end digital oscilloscopes characterized by extremely large analog bandwidths, up to 100 GHz, and digitization capabilities at least up to the corresponding Nyquist rate, i.e. 200 GS/s or above. This performance can be achieved by means of architectures that implement suitable methodologies, based on synchronous time interleaving, digital bandwidth interleaving, or hybrid approaches merging and exploiting both of the previous concepts. Each of these architectures typically includes a number of innovations, concerning both methods and hardware solutions, which are either covered by patents or classified. The paper presents the architecture of an original high-speed digitizer that exploits frequency conversion and time interleaving techniques. Thanks to the use of 4 time-interleaved channels, each one consisting of a state-of-the-art sampler and an ADC, the proposed solution can increase fourfold the analog bandwidth with respect to the individual ADCs, achieving noise reduction with respect to traditional time interleaved architectures.

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell–intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9ON) or the control LacZ (LacZON). When engrafted into mice and treated with tetracycline, Tlr9ON, but not LacZON, tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL-6-type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9-expressing prostate cancer cells and PMN-MDSCs. Antibody-mediated LIF neutralization reduced the percentage of tumor-infiltrating PMN-MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN-MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF-mediated expansion and activation of PMN-MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide-based inhibitors, such as CpG-STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.

The five-year survival rate of patients diagnosed with advanced pancreatic ductal adenocarcinoma (PDAC) has remained static at

TPS2662 Background: Glioblastoma (GBM) is the most common and most aggressive primary brain tumor. Around 294,900 new cases are diagnosed globally with 241,000 deaths each year. The 5-year survival is only 5%. Median overall survival from first recurrence is only 5-8 months. There is no established standard of care for recurrent GBM. City of Hope (COH) has developed and optimized a CAR T cell therapy utilizing the chlorotoxin peptide (CLTX) as the CAR’s tumor recognition domain against GBM. CLTX-CAR T cells specifically and broadly target GBM through recognition of a receptor complex including membrane-bound matrix metalloprotease 2 (MMP-2). CLTX-CAR T cells do not exhibit off-tumor recognition of normal human or murine cells and tissues in preclinical models. In in vitro studies, COH evaluated patient-derived brain tumor (PBT) cell lines for CLTX binding and expression of IL13Rα2, HER2 and EGFR, three targets of CAR T cell trials for GBMs. Strong CLTX binding to tumor cells was observed in of the majority of primary GBM lines, independent of these other antigens. In preclinical studies using in vivo mouse models, a single intratumoral (ICT) injection of CLTX-CAR T cells (1×106 CAR+ T cells) exhibited robust anti-tumor activity against ffLuc+ PBT106 tumors orthotopically-engrafted in NSG mice. Overall, when compared to mice treated with mock-transduced Tn/mem (no CAR) T cells, the CLTX(EQ)28ζ/CD19t+ T cells reduced tumor burden and significantly increased survival. Taken together, these preclinical findings support the potential safety and efficacy of CLTX-CAR T cells, and provide the rationale for clinical testing of this therapy. As cellular heterogeneity intrinsic to GBM likely contributes to resistance to therapy and limited response rates, CLTX-CAR T cells may provide greater tumor eradication in a higher proportion of patients with GBM. Methods: This study is a phase 1, single center, safety and maximum tolerated dose (MTD) finding study of CLTX-CAR T cells for subjects with MMP2+ recurrent or progressive GBM. A safety lead-in of 3−6 participants receiving CLTX-CAR T cells by ICT delivery will be completed first. Subsequently, subjects would receive cells administered through both ICT and intraventricular (ICV catheters) (i.e. dual delivery) in two dose schedules. Subjects will be evaluated for safety and tolerability, and may continue to receive treatment until disease progression. Time to progression, overall survival, and disease response by Response Assessment in Neuro-Oncology (RANO) criteria, will be evaluated and descriptively compared to historical data. The study is actively enrolling patients. Clinical trial information: NCT04214392.

106 Background: PathR is an efficacy endpoint in Phase II and III neoadjuvant trials and is proposed as a surrogate for disease-free survival (DFS) and overall survival. Machine learning (ML)–based, automated approaches standardize quantification of areas of tumor bed and residual viable tumor. Here we show that automation may provide a scalable alternative to or complementary tool for manual assessment. Methods: We determined inter-reader variability for PathR among pathologists in the LCMC3 (NCT02927301) study and developed an AI-powered digital PathR assessment tool in line with manual consensus recommendations. Study cases were reviewed for PathR by a local site pathologist and 3 central expert pathologists (n = 127). When determined manually, major PathR (MPR) was defined as ≤10% viable tumor averaged per case. ML models were trained and validated by the PathAI research platform using digitized H&E-stained tumor sections. The digital PathR model predicted percent viable tumor for each case as the sum of the cancer epithelium area from each slide divided by the sum of tumor bed area for each slide. DFS (clinical cutoff: Oct 23, 2020) was reported for patients with manual and digital PathR assessment (n = 135). For digital MPR, we used a prevalence-matched cutoff that maintained the same proportion of patients as manual MPR. Results: Inter-reader agreement among 1 local and 3 central pathologists for manual PathR was good (n = 127; ICC = 0.87; 95% CI: 0.84-0.90). Agreement was 91% (κ = 0.82) on manual MPR and 98% (κ = 0.88) on pathologic complete response (pCR). 6 patients had unanimous pCR. Digital and manual PathR were strongly correlated (n = 135, Pearson r = 0.78) and digital PathR demonstrated an outstanding predictability for manual MPR (AUROC = 0.975). The range was 0%-60% for digital PathR and 0%-100% for manual PathR with a regression line slope < 1.0 (m = 0.303) indicating systematic differences between the methods, consistent with digital PathR using a high-resolution segmentation of cancer epithelium from stroma across each slide. Longer DFS was observed for MPR yes vs no with both digital and manual assessment (Table). Conclusions: This analysis showed good inter-reader agreement for manual and strong correlation of AI-powered digital and manual PathR. Comparable DFS rates for manual MPR and digital MPR are encouraging in the preliminary data. These data support further studies of digital PathR as a standardized and scalable tool to determine PathR. Clinical trial information: NCT02927301. [Table: see text]

Objectives: We have maintained a local registry of skin and nail infections in patients receiving pertuzumab and trastuzumab as treatment for HER2 positive breast cancer. Over the past 16 months, we have continued to observe an increase in infectious complications in patients receiving the combination of pertuzumab and trastuzumab with or without chemotherapy. We expand a series of prospectively identified patients who developed infections while on these regimens. Methods: We became concerned about an increased incidence of infections shortly after the FDA approval of pertuzumab, and created an IRB approved registry of these patients. Results: Twenty-eight women were identified to have 32 separate infections (often at more than one site); 9 after cycle 1; 6 after cycle 2, 9 after cycle 3 and 8 after 4 or more cycles. The median age was 51 (Range 25-67); 14 received pertuzumab, trastuzumab, carboplatin, and docetaxel (PTCH), 5 pertuzumab, trastuzumab, and docetaxel, 7 pertuzumab, trastuzumab, and nab-paclitaxel, and 2 pertuzumab and trastuzumab. Folliculitis of the scalp, abdomen, and/or buttocks was observed in 19 patients, abscesses in 8 patients (4 of whom required incision and drainage) and cellulitis in 2. Severe paronychial infections involving one to 16 digits were observed in 4; 2 pt required surgical removal of 2 nails. Quantitative immunoglobulins were found to be low in 8 of 17 women tested; 2 patient had low total protein but did not have an assessment of quantitative immunoglobulins. All patients were initially treated with oral antibiotics, and 6 required hospitalization. Cultures were obtained in 10 patients; Staphylococcus aureus was identified in 4, methicillin resistant Staphylococcus aureus (MRSA) in 5, Enterococcus faecalis in 1. A 57 year old pt receiving neoadjuvant PTCH died on cycle 2 day 7. Autopsy was consistent with sepsis and gram positive cocci were identified. A 62 year old became septic and developed renal failure. Skin biopsies were performed in 3 patients and are consistent with changes associated with EGFR inhibition. Conclusions: We believe these infections are a result of combining pertuzumab with trastuzumab as 2 pts received no concurrent chemotherapy. An awareness of this complication is critical as some infections may be life-threatening. We have initiated patient education to ensure awareness of this potential complication. Citation Format: Mortimer JE, Yuan Y, Jung J, Kruper L, Stewart D, Chung S, Yu WK, Mendelsohn M, D'Apuzzo M, Tegtmeier B, Dadwal S. Skin, and nail, infections associated with the addition of pertuzumab to trastuzumab-based chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-10-19.

The paper deals with the distributed monitoring of harmonic and interharmonic pollution in electrical power delivery systems. In order to measure the disturbances and to identify the sources in a wide grid, a distributed measurement system with hundreds of measurement nodes has to be adopted. With the aim of obtaining the reduction of realization costs, the authors propose an innovative distributed architecture, based on cost-effective nodes, that takes advantage from the compressive sampling strategy. Differently from traditional approach, the network nodes will be only mandated to acquire and to digitize the line voltage directly in a compressed form, whereas a central measurement unit will receive data from all nodes deployed in the grid and will perform the successive signal reconstruction, making possible the use of low-performance hardware to realize them. The assessment of the compliance of the proposed measurement technique with the current power quality standards turns out to be mandatory, thus verifying the absence of artifacts introduced by the adopted compressed sampling approach. © 2015 Elsevier Ltd. All rights reserved.

Glioblastoma (GBM) remains an almost universally fatal brain tumor despite numerous on-going efforts to devise novel and efficacious therapies. We are exploring one approach, immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting specific tumor antigens. While adoptive CAR T cell immunotherapy has shown promising efficacy in pre-clinical and clinical studies, tumor relapses nevertheless occur, a reflection of our incomplete understanding of target antigen expression in GBM. We have previously suggested from analysis of inter-tumoral heterogeneity in primary tissues from patients with GBM that, in principle, a multiple antigen approach targeting IL13Rα2, HER2 and EGFR could effectively target approximately 93% of patient tumors. Our most recent observations, working with this same cohort of patient tumor samples, indicate that individual GBM lesions are composed of numerous subpopulations of tumor cells with differing target antigen expression patterns. This high degree of intra-tumoral heterogeneity, evident in the spatial extent (“granularity”) of target antigen expression patterns and their relations to tumor architecture, may complicate single- and multiple-target immunotherapies by contributing to antigen escape and tumor recurrence.

Brainstem tumors are rare, even rarer is a brainstem tumor containing tissues of an embryologic gynecologic origin. We report a very rare case of presence of a calcified heterogeneously contrast enhancing brainstem mass of Mullerian origin in a patient in a 38 year old female with no female genital tract cancer and past surgical history of ventriculoperitoneal (VP) shunt placement for congenital hydrocephalus. To our knowledge this is the very first and unusual case of a mass of gynecologic origin in the brainstem region especially in the setting of no history of gynecological tumor. The authors also reviewed the literature for all tumors reported for anterograde and retrograde dissemination of tumor cells through VP shunt. This case is a reaffirmation of the importance of brain tumor location and tissue diagnosis for the purpose of adjuvant treatment of neurosurgical lesions in the neurocritical care setting. It also highlights the role of catheters as potential routes of iatrogenic transmission not just in anterograde but also in a retrograde manner to the CNS, which is very unusual. This is the only second case to report retrograde flow of tumor cells from an extraneural source up the VP catheters. The authors suggest that intraperitoneal chemotherapy should be considered in the cases of known extraneural abdominal malignancies of high malignant potential with or without the presence of peritoneal infiltration in order to avoid dissemination through VP shunts.

Background. The aim of this study was to correlate T1-weighted dynamic contrast-enhanced MRI- (DCE-MRI-) derived perfusion parameters with overall survival of recurrent high-grade glioma patients who received neural stem cell- (NSC-) mediated enzyme/prodrug gene therapy. Methods. A total of 12 patients were included in this retrospective study. All patients were enrolled in a first-in-human study (NCT01172964) of NSC-mediated therapy for recurrent high-grade glioma. DCE-MRI data from all patients were collected and analyzed at three time points: MRI#1—day 1 postsurgery/treatment, MRI#2— day 7 ± 3 posttreatment, and MRI#3—one-month follow-up. Plasma volume (Vp), permeability (Ktr), and leakage (λtr) perfusion parameters were calculated by fitting a pharmacokinetic model to the DCE-MRI data. The contrast-enhancing (CE) volume was measured from the last dynamic phase acquired in the DCE sequence. Perfusion parameters and CE at each MRI time point were recorded along with their relative change between MRI#2 and MRI#3 (Δ32). Cox regression was used to analyze patient survival. Results. At MRI#1 and at MRI#3, none of the parameters showed a significant correlation with overall survival (OS). However, at MRI#2, CE and λtr were significantly associated with OS (p<0.05). The relative λtr and Vp from timepoint 2 to timepoint 3 (Δ32λtr and Δ32Vp) were each associated with a higher hazard ratio (p<0.05). All parameters were highly correlated, resulting in a multivariate model for OS including only CE at MRI#2 and Δ32Vp, with an R2 of 0.89. Conclusion. The change in perfusion parameter values from 1 week to 1 month following NSC-mediated therapy combined with contrast-enhancing volume may be a useful biomarker to predict overall survival in patients with recurrent high-grade glioma.

Purpose: A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)–engineered, autologous primary human CD8+ cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Experimental Design: Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8+ CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 108 CAR-engineered T cells via a catheter/reservoir system. Results: We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine+ CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine+ CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine+ T-cell administration. Conclusions: These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. Clin Cancer Res; 21(18); 4062–72. ©2015 AACR.