Compassionate use of recombinant human IL-7-hyFc as a salvage treatment for restoring lymphopenia in patients with recurrent glioblastoma

Purpose Lymphopenia is frequently observed and is associated with poor prognosis in glioblastoma (GBM) patients. Restoring lymphopenia in cancer patients has been suggested as a novel immunotherapeutic strategy. As interleukin-7 (IL-7) is necessary for proliferation of lymphocytes and to amplify the total lymphocyte count (TLC), IL-7 therapy has been tried for various cancers, although the results are inconclusive. Here, we describe the clinical results of recurrent GBM treated with long-acting engineered version of recombinant human IL-7 (rhIL-7-hyFc). Methods This prospective case series based on compassionate use was approved by the Ministry of Food and Drug Safety in South Korea. Patients with recurrent GBM were enrolled to Seoul St. Mary's Hospital. Primary outcomes were the safety profile and elevated total lymphocyte count (TLC). Secondary outcomes were overall survival (OS) and progressionbfree survival (PFS). The duration of median follow up was 372.6 days (range 98-864 days). Results Among 18 patients enrolled, 10 received rhIL-7-hyFc with temozolomide, 5 received rhIL-7-hyFc with bevacizumab, 1 received rhIL-7-hyFc with PCV chemotherapy, and 2 received rhIL-7-hyFc alone. The mean TLC of enrolled patients after the first treatment with rhIL-7-hyFc was significantly increased from 1,131 cells/mm^3 (range 330-2,989) at baseline to 4,356 cells/mm^3 (range 661-22,661). Similar increase was observed in 16 of 18 patients (88.8%), only after the first treatment of rhIL-7-hyFc. TLCs of these patients were maintained higher while rhIL-7-hyFc was repeatedly administered. Most common adverse events were injection sites reactions (64.7%) including urticaria and itching sensation, however, there were no serious adverse events more than grade III. Median OS and PFS were 378 days (range 107-864 days) and 231 days (55-726 days), respectively. Conclusion Our study first reports that IL-7 immunotherapy can restore lymphopenia and maintain TLC with various salvageable chemotherapies in recurrent GBM patients without serious adverse toxicities. This outcome warrants further larger and randomized clinical trials to validate the clinical benefits of rhIL-7-hyFc for GBM patients.

Comparison of Tumor Immune Environment Between Newly Diagnosed and Recurrent Glioblastoma in Matched Patients

Purpose: Glioblastoma (GBM) is the most lethal primary brain tumor in adult patients. The disease progression, response to chemotherapy and radiotherapy at initial diagnosis, and prognosis are profoundly associated with the tumor microenvironment (TME), especially the features of tumor-infiltrating immune cells (TII). Recurrent GBM is even more challenging to manage. Differences in the immune environment between newly diagnosed and recurrent GBM and an association with tumor prognosis are not well defined. Methods: To address this knowledge gap, we analyzed the clinical data and tissue specimens from 24 GBM patients (13 at initial diagnosis and 11 at recurrence). The expression levels of multiple immunobiological markers in patients’ GBM at initial diagnosis versus at recurrence were compared, including five patients with both specimens available (paired). The distribution patterns of TII were evaluated in both the intratumoral and perivascular regions. Results: We found that tumors from recurrent GBM have significantly more tumor-infiltrating lymphocytes (TILs) and macrophages and higher PD-L1 expression than tumors at primary diagnosis and benign brain specimens from epilepsy surgery. The pattern changes of the TILs and macrophages of the five paired specimens were consistent with the unpaired patients, while the CD8 to CD4 ratio remained constant from diagnosis to recurrence in the paired tissues. The levels of TILs and macrophages at initial diagnosis did not correlate with OS. TILs and macrophages were increased in recurrent tumors both in intratumoral and perivascular areas, with higher distribution levels in intratumoral than perivascular regions. Higher CD4 or CD8 infiltration at recurrence was associated with worse prognosis, respectively. Conclusion: Our study elucidated that TIL and TAM tend to accumulate in perivascular region and are more abundant in recurrent GBM than newly diagnosed GBM.

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

PurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs.Experimental designIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Hemoglobin, albumin, lymphocytes and platelets (HALP) score is a useful predictor of survival in patients with recurrent glioblastoma multiforme treated with bevacizumab plus irinotecan

Backgrounds: We aimed to investigate whether the HALP score is a predictive marker in patients with recurrent GBM who were given bevacizumab plus irinotecan.Methods: We compared the survival of patients followed up in our clinic with the diagnosis of recurrent GBM and treated with bevacizumab plus irinotecan, according to HALP score.Results: Median PFS and OS were 4.5 (0.9-14.9) and 8 (0.9-21.3) months, respectively. The median PFS of the low HALP score group was 1.85 (1.3-3.37) months, and of the high HALP score group was 4.96 (0.9-14.9) months (p=0.03). The OS of the high HALP score group (9.63 [7.28-11.9]) was statistically higher compared with low HALP score group (2.26 [0.88-3.65]) (p<0.001). In univariate analysis HALP score was a significant prognostic factor; patients with low HALP score had a poorer prognosis than high HALP score (HR: 0.063, p<0.001). The multivariate analysis showed that HALP score (p=0.003), and residual tumor (p=0.029) were significant prognostic factors. In multivariate Cox regression analysis, low HALP score was a significant poor prognostic factor for OS compared with high HALP score (HR: 0.063, p<0.001). Conclusion: We showed that the HALP score at the start of treatment is an independent prognostic factor for PFS and OS in patients with recurrent GBM treated with bevacizumab plus irinotecan. The HALP score, which can be easily calculated by routine tests before chemotherapy, can be used as a predictive marker for bevacizumab treatment decision.

Sustained Accumulation of Blood-Derived Macrophages in the Immune Microenvironment of Patients with Recurrent Glioblastoma after Therapy

The cell composition of the glioblastoma (GBM) microenvironment depends on the recruitment of myeloid cells from the blood, promoting tumor progression by inducing immunosuppression. This phenomenon hampers immunotherapies and investigating its complexity may help to tailor new treatments. Peripheral blood and tissue specimens from the central and marginal tumor areas were collected from 44 primary and 19 recurrent GBM patients. Myeloid and lymphoid cell subsets and the levels of immunosuppressive markers were defined by multiparametric flow cytometry. Multiplexed immunohistochemistry was used to confirm the differences in the immune infiltrate and to analyze the cell spatial distribution. Relapsing GBM showed an increased presence of blood-derived macrophages in both tumor areas and a higher frequency of infiltrating lymphocytes, with a high level of exhaustion markers. The expansion of some myeloid-derived suppressor cell (MDSC) subsets in the blood was found in both primary and recurrent GBM patients. A significant inverse correlation between infiltrating T cells and an MDSC subset was also found. In patients with recurrent GBM after standard first-line therapy, the immune-hostile tumor microenvironment and the levels of some MDSC subsets in the blood persisted. Analysis of the immune landscape in GBM relapses aids in the definition of more appropriate stratification and treatment.

A Comparison of Single- and Multiparametric MRI Models for Differentiation of Recurrent Glioblastoma from Treatment-Related Change

To evaluate single- and multiparametric MRI models to differentiate recurrent glioblastoma (GBM) and treatment-related changes (TRC) in clinical routine imaging. Selective and unselective apparent diffusion coefficient (ADC) and minimum, mean, and maximum cerebral blood volume (CBV) measurements in the lesion were performed. Minimum, mean, and maximum ratiosCBV (CBVlesion to CBVhealthy white matter) were computed. All data were tested for lesion discrimination. A multiparametric model was compiled via multiple logistic regression using data demonstrating significant difference between GBM and TRC and tested for its diagnostic strength in an independent patient cohort. A total of 34 patients (17 patients with recurrent GBM and 17 patients with TRC) were included. ADC measurements showed no significant difference between both entities. All CBV and ratiosCBV measurements were significantly higher in patients with recurrent GBM than TRC. A minimum CBV of 8.5, mean CBV of 116.5, maximum CBV of 327 and ratioCBV minimum of 0.17, ratioCBV mean of 2.26 and ratioCBV maximum of 3.82 were computed as optimal cut-off values. By integrating these parameters in a multiparametric model and testing it in an independent patient cohort, 9 of 10 patients, i.e., 90%, were classified correctly. The multiparametric model further improves radiological discrimination of GBM from TRC in comparison to single-parameter approaches and enables reliable identification of recurrent tumors.

IM-4 Impact of oHSV activated NOTCH signaling in tumor microenvironment and its impact on anti-tumor immunity

Oncolytic herpes simplex virus-1 (oHSV) is novel FDA-approved immunotherapy for advanced melanoma patients in US. Also, oHSV is recently approved for the treatment of recurrent GBM in Japan. We have shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells via HSV-1 microRNA-H16 (Otani Y and Yoo JY, Clin Cancer Res, 2020), however, the consequences of NOTCH on immunotherapy in GBM is unknow. Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment (TME). Analysis of TCGA GBM data and experimental murine models revealed NOTCH induced immunosuppressive myeloid cell recruitment and limited anti-tumor immunity. In oHSV treated tissue, viral infection educated tumor associated macrophages to secrete CCL2 which recruited monocytic myeloid derived suppressor cell (MDSC) that attenuated anti-tumor immunity. Consistent with this, CCL2 induction was also observed in serum of recurrent GBM patients treated with oHSV (NCT03152318). Importantly, blockade of NOTCH signaling reduced the oHSV induced immunosuppressive environment and activated a CD8 dependent anti-tumor memory response. These findings present the opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity in GBM.

Ribonucleotide Reductase Regulatory Subunit M2 as a Driver of Glioblastoma TMZ-Resistance through Modulation of dNTP Production

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p<0.0001). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p<0.01), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.

DUSP6 regulates radio-sensitivity in glioblastoma by modulating the recruitment of p-DNAPKcs at DNA double-strand breaks

Glioblastoma (GBM) has poor median survival due to its resistance to chemo-radiotherapy regimen, resulting in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is up-regulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies (n=11) compared to primary biopsies (n=11). Importantly, although reported as cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse population of GBM cell lines generated from in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radio-sensitized primary and importantly, relapse GBM cells by inhibiting the recruitment of p-DNAPKcs, subsequently down-regulating the recruitment of γH2AX and 53BP1. This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

TAMI-49. JAK STAT INHIBITION REVERSES MYELOID CELL INDUCED ANTI TUMOR IMMUNITY IN T CELLS

BACKGROUND Many central questions about the immunosuppressive microenvironment in glioblastoma (GBM) remain unanswered, particularly the interaction with lymphoid and myeloid populations. Here, we combined single-cell (scRNA) and spatial transcriptomics (stRNA) to comprehensively characterize the immune interaction with GBM. MATERIAL AND METHODS We performed scRNA-Seq of 50k CD45+ cells (8 patients) and inferred transcriptional programs and fate decisions in T cells. A novel algorithm (Nearest functionally connected neighbor) was used to predict interacting cells, further validated using spatial transcriptomics and immunofluorescence. Our findings were validated in our human neocortical glioblastoma model with autografted T cells. RESULTS Integration of st/scRNA-seq revealed a transcriptional shift of T cells towards exhaustion/hypoxia induced dysfunction. Pseudo-time analysis revealed increased Interleukin 10 (IL10) response during the Tcell transformation from the effector to the exhausted state. Using NFCN we identified a subset of HMOX1+ myeloid cells (STAT/HMOX axis), responsible for this IL10 release. Computational findings were validated using our human neocortical glioblastoma model with autografted T cells, where IL10R-inhibition/myeloid cell depletion prevented T cell exhaustion/dysfunction (p &lt; 0.01) . In order to target the STAT3/HMOX1 axis we used a JAK/STAT inhibitor in our model which showed a drastic reduction of IL10 release (p&lt; 0.02) and concordant activation of T cells. Clinically, one patient treated with a JAK/STAT-inhibitor in a neoadjuvant setting, 4 weeks prior to the recurrent GBM surgery, led to a significant increase (p&lt; 0.001) in effector T cell population. CONCLUSION Our findings suggest that targeting the myeloid compartment of GBM provides an opportunity to convert a “cold” into “hot” immune environment which might be helpful to improve all T cell based therapies in the future.