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Metastatic disease in the brain is difficult to control and predicts poor prognosis. Here, we analyze human brain metastases and demonstrate their robust infiltration by CD8

Spatial transcriptomics is an emerging technology that measures gene expression while preserving spatial information. Here, we present a method to determine localization of specific T cell clones by obtaining T cell receptor (TCR) sequences from spatial transcriptomics assays. Our method uses an existing commercial spatial transcriptomics platform and open-source software for analysis, allowing simple and inexpensive integration with archived samples and existing laboratory workflows. Using human brain metastasis samples, we show that TCR sequences are readily obtained from human tumor tissue and that these sequences are recapitulated by single-cell sequencing methods. This technique will permit detailed studies of the spatial organization of the human T cell repertoire, such as the identification of tumor-infiltrating and tumor-excluded T cell clones.

Despite improved outcomes with checkpoint blockade immunotherapy, patients with brain metastases have the worst prognosis among patients with metastatic cancer. Immune checkpoint blockade agents target inhibitory receptors, such as PD-1, on exhausted CD8+ T cells to restore their anti-cancer function. Many patients, however, either do not respond or progress after an initial response to immune checkpoint blockade, and distant intracranial failure is common despite excellent options for local treatment of brain metastasis. To develop more effective therapeutic strategies for the treatment of brain metastases, an understanding of the phenotype of brain metastasis-infiltrating CD8+ T cells is essential. Here we performed a detailed characterization of the CD8+ T cells contained in brain metastases. Brain metastases were densely infiltrated by CD8+ T cells; blood contamination of tumor samples was rare. Compared to patient-matched circulating cells, brain metastasis-infiltrating CD8+ T cells had a distinct phenotype characterized by more frequent expression of PD-1, with subpopulations defined by expression of additional co-inhibitory molecules and the residence marker CD69. Single cell RNA-sequencing identified four phenotypic subpopulations within brain metastasis-infiltrating PD-1+ CD8+ T cells. Two of these populations — a terminally-differentiated and a dividing population — were characterized by high expression of co-inhibitory molecules and lacked expression of progenitor markers such as TCF-1. There was significant T cell receptor (TCR) overlap between the terminally-differentiated and dividing populations, suggesting that the dividing cells give rise to the terminally-differentiated cells. There was minimal TCR overlap between these two populations and other brain metastasis-infiltrating PD-1+ CD8+ T cells. T cell clones from brain metastasis-infiltrating CD8+ T cells were rare in circulation, particularly clones from the terminally-differentiated and dividing populations. We systematically identified bystander CD8+ T cells specific for microbial antigens; these cells infiltrated brain metastases and expressed genes shared with exhausted progenitor CD8+ T cells, such as TCF7 and IL7R. We performed spatial transcriptomics on brain metastases and used a novel method to obtain TCR sequences from spatial transcriptomics data. These data revealed distinct niches within the TME defined by their gene expression patterns and cytokine profiles. Terminally-differentiated CD8+ T cells preferentially occupied niches within the tumor parenchyma. Together, our results show that antigen-specificity restricts the spatial localization, phenotypic states, and differentiation pathways available to CD8+ T cells within the brain metastasis TME.

Brain metastases from non-small cell lung cancer often cause neurologic symptoms which lead to initial diagnosis or identification of recurrence. In other patients, they are identified on surveillance imaging or when a patient undergoing treatment develops neurological symptoms. Patients with symptomatic lesions should be started on dexamethasone and evaluated by a neurosurgeon as soon as possible. If feasible, surgery should be offered to decrease intracranial pressure, alleviate symptoms, and prevent irreversible neurological damage. Postoperative stereotactic radiosurgery (SRS) to the resection cavity and any additional brain metastases should follow within 4 weeks of surgery, as early as 2 weeks post-op. Tissue from surgery is used to confirm the diagnosis and test for targetable oncogenic driver mutations. Treatment response and surveillance for development of additional lesions is assessed with MRI of the brain 1 month after SRS and every 3 months thereafter. Patients who are not surgical candidates or who have small, asymptomatic brain metastases should proceed with SRS, the preferred treatment, or sometimes whole-brain radiation therapy (WBRT) if multifocal disease requires more extensive treatment, such as for leptomeningeal spread of disease. The number of brain metastases that warrants use of WBRT over SRS is controversial and a topic of ongoing investigation, and is discussed in this review. When possible, SRS is preferred over WBRT due to reduce morbidity and cognitive side effects. When patients are already on systemic therapy at time of brain metastases diagnosis, systemic therapy should continue, with radiation therapy occurring between cycles. Regarding systemic therapy for new diagnosis at time of brain metastases presentation, molecular testing will guide treatment choice, when available. If there is no neurosurgical intervention, biopsy of another site of disease may provide tissue for molecular testing. If there are no targetable oncogenic driver mutations, concurrent immune checkpoint blockade (ICB) and chemotherapy is preferable for patients who can tolerate it. Single-agent ICB is an alternative option for patients who cannot tolerate chemotherapy. Systemic therapy should start as soon as possible. In some patients with poor performance status, best supportive care may be the most appropriate choice. Treatment decisions should always incorporate patients’ goals of care and in many cases should be discussed in a multidisciplinary setting.

Purpose Pediatric high-grade glioma (HGG) is a devastating disease with a poor prognosis. The purpose of this analysis is to evaluate the impact of Radiation Therapy (RT) variables on outcomes of pediatric HGG patients in the National Cancer Database (NCDB). Methods The NCDB was used to select patients age 6 weeks), and RT modality were analyzed along with baseline demographic, tumor and treatment variables to assess the impact on overall survival in univariate and multivariable analyses. Results 498 pediatric HGG patients were included. Histologies included glioblastoma (30%), astrocytoma (55%), oligogendroglioma (5%) and gliomas not otherwise specific (10%). The median RT dose was 59.4 Gy (SD 2.9 Gy) starting a median of 4.4 weeks from diagnosis (SD 2.5 weeks). Median follow-up was 19.6 months with 1- and 3-year OS of 78.4% and 40.4%, respectively. On Multivariable analysis, female gender, older age, and private insurance remained independently associated with lower rate of overall death. Radiation initiation ≤ 4 weeks from diagnosis, and glioblastoma histology were significantly associated with higher rate of overall death. There was no relationship between radiation dose or whether radiation was delivered with proton or photon therapy and overall survival. Conclusions Outcomes for pediatric HGG are poor. Early initiation of RT within 4 weeks from diagnosis was negative associated with overall survival and may be related to unknown prognostic factors.

Limited stage small cell lung cancer (LS-SCLC) remains a challenging disease, with 5-year overall survival ranging from 30–35% with current standard of care treatment consisting of thoracic radiation to 45 Gy in 30 fractions delivered twice daily, with concurrent platinum/etoposide chemotherapy, followed by prophylactic cranial irradiation (PCI). The randomized, phase III CONVERT study confirmed 45 Gy delivered twice daily to be the optimal radiation fractionation regimen, without significantly increased toxicity when compared to daily radiation to 66 Gy. Immunotherapy is now being studied in addition to chemoradiation, in both the concurrent and consolidative setting. These randomized trials are ongoing. Additionally, the role of PCI compared to MRI surveillance is being evaluated in patients with LS-SCLC in both the North America and Europe. Ideally these ongoing studies will continue to improve outcomes for LS-SCLC.

BACKGROUND Use of immune checkpoint blockade (ICB) therapy has prolonged overall survival in patients with metastatic cancer. One potential strategy to improve the effectiveness of ICB is to target additional inhibitory receptors on exhausted CD8+ T cells, which may promote rescue of CD8+ T cells that do not respond to PD-1 pathway blockade alone. This therapeutic strategy requires knowledge of the inhibitory molecules expressed on tumor-specific CD8+ T cells. Toward achieving this goal, we characterized the phenotype of CD8+ T cells infiltrating brain metastases. METHODS We performed flow cytometry on 45 brain metastases samples, single cell RNA sequencing with T cell receptor (TCR) sequencing on 5 samples, and spatially-resolved transcriptomics on 8 samples. RESULTS Analysis of our scRNA-seq data revealed 4 populations of PD-1+ CD8+ T cells infiltrating brain metastases. One of these populations (cluster A) has a terminal effector exhausted phenotype suggesting that this population contains tumor-specific CD8+ T cells. Two of the other populations (clusters B and C) have a transcriptional profile that suggests they may contain stem-like CD8+ T cells. TCR sequencing shows that cells in cluster A do not express the same TCRs as cells in clusters B and C, suggesting that stem-like cells in clusters B and C are not the progenitors of the terminal effector cells in cluster A. Bystander cells expressing TCRs specific for viral antigens are found predominantly in clusters B and C, further supporting the hypothesis that cluster A contains tumor-specific cells. Spatial transcriptomics reveals that cluster A cells are infiltrating the tumor parenchyma while cluster B and C cells are predominantly in peri-tumoral inflammatory tissue. CONCLUSIONS Brain metastases are infiltrated by a population of terminally-differentiated effector CD8+ T cells which express co-inhibitory molecules that may be potential therapeutic targets to improve control of metastatic disease in the brain.

PURPOSE/OBJECTIVE(S) Brain metastases are a significant source of morbidity and mortality among cancer patients. Stereotactic radiosurgery (SRS) offers excellent local control rates. However, some patients experience in-field recurrences and many develop distant brain failure, making long-term control of disease in the brain a challenge. The immune cell infiltrate of extracranial tumors has been studied and shown to correlate with clinical outcomes in numerous tumor histologies. Here, we aim to characterize the immune cell infiltrate of brain metastases. MATERIALS/METHODS From September 2020 to January 2021, fresh brain metastasis samples were weighed and digested immediately after resection. Immune cells were isolated, washed and stained for the pan-leukocyte marker CD45, the cytotoxic T cell marker CD8, and the B cell marker CD19. Cells were analyzed by flow cytometry. RESULTS Twenty-one brain metastases were analyzed (NSCLC, melanoma, breast, esophageal, renal cell carcinoma, and uterine). The median number of CD45+ lymphocytes per gram of tumor was 5.7 × 105 (range: 3.2 × 104 - 9.1 × 106); 19% of the CD45+ lymphocytes were CD8+. The median number of CD8+ T cells per gram of tumor was 1.1 × 105 (range: 5.6 × 103- 2.7 × 106). Most of these tumors were also infiltrated by B cells; the median number of CD19+ B cells per gram of tumor was 1.0 × 104 (range: 0- 4.9 × 105). One study patient had local recurrence after SRS. This patient underwent surgical resection of the dominant brain metastasis on presentation (prior to treatment). There was progression of a separate lesion after SRS, which was subsequently resected. The initial untreated tumor contained 1.7 × 106 CD45+ lymphocytes, 9.5 × 105 CD8+ T cells, and 1.8 × 104 CD19+ B cells per gram of tumor. After SRS, the recurrent tumor contained 2.1 × 105 CD45+ lymphocytes, 5.9 × 104 CD8+ T cells, and 6.4 × 103 CD19+ B cells per gram of tumor. Thus, the number and frequency of CD45+ CD8+ T cells decreased after SRS (5.9 × 104 vs 9.5 × 105 and 28% vs 56%, respectively). CONCLUSION Brain metastases contain variable immune infiltration of cytotoxic T cells and B cells. SRS may decrease the density of lymphocytic immune cell infiltration in brain metastases. Given that CD8+ T cells comprise a smaller proportion of the post-radiation lymphocytic infiltrate, other populations, such as regulatory T cells, might be enriched after treatment.

BACKGROUND The combination of oral pentoxifylline (Ptx) and vitamin E (VitE) has been used to treat radiation-induced fibrosis and soft tissue injury. A 2008 study showed an average decrease in edema with the use of Ptx + VitE in 11 patients with suspected radiation necrosis after stereotactic radiosurgery (SRS). Here we review outcomes in patients prescribed Ptx + VitE at our institution for the treatment of radiation necrosis. METHODS 48 patients were included in this analysis. All patients were treated with SRS, had evidence of radiation necrosis, and had MR imaging before and after starting Ptx + VitE. The radiation oncologist’s impression of the imaging in the electronic medical record was used to score response to treatment. Official radiology reports were also reviewed. RESULTS 43.8% of patients showed evidence of improvement, 18.8% showed no change, and 25% showed worsening radiation necrosis on imaging after starting Ptx + VitE. One patient had a mixed response; 5 patients (10.4%) had disease progression. The median time to response assessment after starting Ptx + Vit E was 3.17 months. 9 patients progressed significantly on Ptx + VitE and required Bevacizumab, hyperbaric oxygen therapy or surgery. Patients who had multiple lesions treated with SRS were less likely to show improvement on imaging after Ptx + VitE (p=0.037). 34 patients were also prescribed dexamethasone, either before (7), with (16), or after starting (11) Ptx + VitE. Use of dexamethasone was not associated with improved response to Ptx + VitE (p=0.471). 3 patients stopped Ptx + VitE due to reported side effects. CONCLUSION Ptx + VitE appears safe for the treatment of radiation necrosis, but randomized data are needed to assess efficacy.

Background Radiotherapy boost to the entire posterior fossa (PF) is standard of care for high-risk (H-R) medulloblastoma patients; the utility of tumor bed (TB)-only boost is unclear. The purpose of this study was to examine the impact of PF versus TB boost volume on tumor control and survival in the H-R medulloblastoma population. Methods Single-institution records for patients with H-R medulloblastoma were reviewed. The median craniospinal irradiation dose was 36 Gy (range, 23.4-45 Gy), and boost doses to either PF or TB were 54 to 55.8 Gy. PF (local) failures were scored as in-field, marginal (between 80% and 95% isodose lines), or distant. Kaplan-Meier methods and Cox proportional hazards were used to assess the impact of radiation boost technique on local control (LC) and survival endpoints. Results Thirty-two patients with H-R medulloblastoma were treated between 1990 and 2015, with a median follow-up length of 5.12 years. Twenty-two patients received PF boost, and 10 received TB boost. Patient and disease characteristic were comparable between groups. A total of 11 PF failures occurred, including 3 isolated LFs (2 in the PF and 1 in the TB group). Most PF failures were in-field: three of four in the TB group and six of seven in the PF group; the remainder were marginal failures. TB boost was not associated with inferior LC (hazard ratio [HR] 0.86, log-rank P = 0.81) or overall survival (HR 1.40, P = 0.56) compared with PF boost. Conclusion Reduced-volume radiotherapy boost to the TB does not appear to compromise LC or survival in patients with H-R medulloblastoma; it may reduce the risk of ototoxicity.

Purpose Studies have shown that an additional mean dose of 1 Gy to the heart can increase the relative risk of cardiac events. The purpose of this study was to quantify the dose delivered to the heart and left anterior descending artery (LAD) in a series of patients with left-sided breast cancer (BC) or ductal carcinoma in situ treated with multicatheter-accelerated partial breast irradiation (MC-APBI) at a single institution. Methods and Materials Patients with left-sided BC or ductal carcinoma in situ treated consecutively from 2005 to 2011 with MC-APBI were retrospectively identified. Cardiac and LAD contours were generated for each patient. Cardiac dosimetry and distance to the planning target volume were recorded. Patient health records were reviewed and cardiac events were recorded based on Common Terminology Criteria for Adverse Events version 4.0. Results Twenty consecutive patients with left-sided BC treated with MC-APBI were retrospectively identified. Median followup was 41.4 months. Mean equivalent dose in 2 Gy fractions delivered to the heart and LAD were 1.3 (standard deviation: 0.7, range: 0.2–2.9) and 3.8 (standard deviation: 3.0, range: 0.4–11.3) Gy, respectively. There was an inverse linear relationship (R2 = 0.52) between heart-to-lumpectomy cavity distance and mean heart equivalent dose in 2 Gy fractions. One patient (5%) experienced symptomatic cardiac toxicity. Conclusions MC-APBI consistently delivers average doses to the heart and LAD that are similar to those achieved in most series with deep inspiration breath-hold and lower than free-breathing radiotherapy techniques. Distance from the heart to the lumpectomy cavity and the availability of other heart-sparing technologies should be considered to minimize the risk of cardiac toxicity.