Dynamic changes in peripheral blood monocytes early after anti-PD-1 therapy predict clinical outcomes in hepatocellular carcinoma.

Immune checkpoint inhibitors are effective for advanced hepatocellular carcinoma (HCC), but there remains a need for peripheral blood biomarkers to predict the clinical response. Here, we analyzed the peripheral blood of 45 patients with advanced HCC who underwent nivolumab. During treatment, frequency of classical monocytes (CD14+CD16-) was increased on day 7, and the fold increase in the frequency on day 7 over day 0 (cMonocyteD7/D0) was significantly higher in patients with durable clinical benefit (DCB) than in patients with non-DCB (NDB). When we analyzed transcriptomes of classical monocytes, CD274, gene encoding PD-L1, was upregulated in NDB patients compared to DCB patients at day 7. Notably, gene signature of suppressive tumor-associated macrophages, or IL4l1+PD-L1+IDO1+ macrophages, was enriched after treatment in NDB patients, but not in DCB patients. Accordingly, the fold increase in the frequency of PD-L1+ classical monocytes at day 7 over day 0 (cMonocyte-PDL1D7/D0) was higher in NDB patients than DCB patients. The combined biomarker cMonocyteD7/D0/cMonocyte-PDL1D7/D0 was termed the "monocyte index", which was significantly higher in DCB patients than NDB patients. Moreover, the monocyte index was an independent prognostic factor for survival. Overall, our results suggest that early changes of circulating classical monocytes, represented as a monocyte index, could predict clinical outcomes of advanced HCC patients undergoing anti-PD-1 therapy.

Modulation of CD8+ T Cell Responses by Radiotherapy-Current Evidence and Rationale for Combination with Immune Checkpoint Inhibitors.

Radiotherapy for cancer has been known to affect the responses of immune cells, especially those of CD8+ T cells that play a pivotal role in anti-tumor immunity. Clinical success of immune checkpoint inhibitors led to an increasing interest in the ability of radiation to modulate CD8+ T cell responses. Recent studies that carefully analyzed CD8+ T cell responses following radiotherapy suggest the beneficial roles of radiotherapy on anti-tumor immunity. In addition, numerous clinical trials to evaluate the efficacy of combining radiotherapy with immune checkpoint inhibitors are currently undergoing. In this review, we summarize the current status of knowledge regarding the changes in CD8+ T cells following radiotherapy from various preclinical and clinical studies. Furthermore, key biological mechanisms that underlie such modulation, including both direct and indirect effects, are described. Lastly, we discuss the current evidence and essential considerations for harnessing radiotherapy as a combination partner for immune checkpoint inhibitors.

BRD4 Inhibition Enhances the Antitumor Effects of Radiation Therapy in a Murine Breast Cancer Model.

Bromodomain-containing protein 4 (BRD4) is an intracellular protein that regulates expression of various cellular functions. This study investigated whether BRD4 inhibition can alter the immunomodulatory and antitumor effects of radiation therapy (RT). A murine breast cancer cell line was implanted into BALB/c mice. The dual-tumor model was used to evaluate the abscopal effects of RT. A total of 24 Gy was delivered and BRD4 inhibitor was injected intravenously. Tumor size was measured, and in vivo imaging was performed to evaluate tumor growth. Flow cytometry and immunohistochemistry were performed to examine immunologic changes upon treatment. The combination of BRD4 inhibitor and RT significantly suppressed tumor growth compared to RT alone. BRD4 inhibitor reduced the size of the unirradiated tumor, indicating that it may induce systemic immune responses. The expression of HIF-1α and PD-L1 in the tumor was significantly downregulated by the BRD4 inhibitor. The proportion of M1 tumor-associated macrophages (TAMs) increased, and the proportion of M2 TAMs decreased upon BRD4 inhibition. BRD4 inhibitor expanded CD4+ and CD8+ T cell populations in the tumor microenvironment. Additionally, splenic monocytic myeloid derived suppressor cells, which were increased by RT, were reduced upon the addition of BRD4 inhibitor. Therefore, the addition of BRD4 inhibitor significantly enhanced the systemic antitumor responses of local RT.

Uncoupling immune trajectories of response and adverse events from anti-PD-1 immunotherapy in hepatocellular carcinoma.

BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.

Risk Stratification Using Neoadjuvant Rectal Score in the Era of Neoadjuvant Chemoradiotherapy: Validation With Long-term Outcome Data.

BACKGROUND: Despite the widespread use of neoadjuvant chemoradiotherapy, there is no prognostic surrogate marker established in locally advanced rectal cancer. OBJECTIVE: This study evaluated the role of neoadjuvant rectal score as a prognostic factor to stratify individual-level risks of survival and tumor recurrence. DESIGN: This is a retrospective study. SETTINGS: This study was conducted at the Seoul National University Hospital. PATIENTS: A total of 397 patients who underwent chemoradiotherapy plus total mesorectal excision were analyzed. INTERVENTIONS: There was no intervention. MAIN OUTCOME MEASURES: Harrell C statistic and receiver operating characteristic analysis, as well as Cox regression analysis, were used to assess the prognostic strength. RESULTS: The low (<8), intermediate (8-16), and high (>16) neoadjuvant rectal score groups included 91 (23%), 208 (52%), and 98 patients (25%). A high neoadjuvant rectal score was independently associated with inferior overall survival and disease-free survival (p = 0.011 and 0.008). Regarding the prognostic models adjusted for neoadjuvant rectal score (I) and ypT/N stage (II), the c-index was higher in model I (0.799 and 0.787, p = 0.009 for overall survival; 0.752 and 0.743, p = 0.093 for disease-free survival). The predictive ability of the neoadjuvant rectal score was superior to tumor regression grade, ypT, and ypN in the receiver operating characteristic analyses (p < 0.05 for all). Adjuvant chemotherapy was associated with better overall and disease-free survival (p = 0.003 and 0.052) in the high neoadjuvant rectal score group. LIMITATIONS: Potential selection bias attributed to the retrospective study design was a limitation. CONCLUSIONS: We verified the applicability of the neoadjuvant rectal score to stratify the relapse risk at the individual level for patients with stage II/III rectal cancer undergoing neoadjuvant chemoradiotherapy. Additional studies are needed to validate the usability of neoadjuvant rectal score levels as a determinant of adjuvant strategy. See Video Abstract at http://links.lww.com/DCR/B354. ESTRATIFICACIÓN DE RIESGO UTILIZANDO LA PUNTUACIÓN RECTAL NEOADYUVANTE EN LA ERA DE LA QUIMIORRADIOTERAPIA NEOADYUVANTE: VALIDACIÓN CON DATOS DE RESULTADOS A LARGO PLAZO: A pesar del uso generalizado de la quimiorradioterapia neoadyuvante, no existe un marcador subrogado pronóstico establecido en el cáncer de recto localmente avanzado.Este estudio evaluó el papel de la puntuación rectal neoadyuvante como factor pronóstico para estratificar los riesgos a nivel individual de supervivencia y recurrencia tumoral.Este es un estudio retrospectivo.Este estudio se realizó en el Hospital de la Universidad Nacional de Seúl.Se analizaron un total de 397 pacientes que se sometieron a quimiorradioterapia más escisión mesorrectal total.No hubo intervención.El análisis estadístico C de Harrell y las características operativas del receptor, así como el análisis de regresión de Cox, se utilizaron para evaluar la fuerza pronóstica.Los grupos de puntaje rectal neoadyuvante bajo (<8), intermedio (8-16) y alto (> 16) incluyeron 91 (23%), 208 (52%) y 98 (25%) pacientes, respectivamente. Una puntuación rectal neoadyuvante alta se asoció independientemente con una supervivencia general y una supervivencia libre de enfermedad inferiores (p = 0.011 y 0.008, respectivamente). Con respecto a los modelos pronósticos ajustados por la puntuación rectal neoadyuvante (I) y el estadio ypT/N (II), el índice c fue mayor en el modelo I (0.799 y 0.787, p = 0.009 para la supervivencia general; 0.752 y 0.743, p = 0.093 para supervivencia libre de enfermedad). La capacidad predictiva de la puntuación rectal neoadyuvante fue superior al grado de regresión tumoral, ypT y ypN en los análisis de características operativas del receptor (p <0.05 para todos). La quimioterapia adyuvante se asoció con una mejor supervivencia global y libre de enfermedad (p = 0.003 y 0.052, respectivamente) en el grupo de puntaje rectal neoadyuvante alto.El sesgo de selección potencial debido al diseño retrospectivo del estudio fue la limitación.Verificamos la aplicabilidad de la puntuación rectal neoadyuvante para estratificar el riesgo de recurrencia a nivel individual para pacientes con cáncer rectal en estadio II/III sometidos a quimiorradioterapia neoadyuvante. Se necesitan más estudios para validar la usabilidad de los niveles de puntuación rectal neoadyuvante como determinante de la estrategia adyuvante. Consulte Video Resumen en http://links.lww.com/DCR/B354.

Effects of trastuzumab on locoregional recurrence in human epidermal growth factor receptor 2-overexpressing breast cancer patients treated with chemotherapy and radiotherapy.

PURPOSE: In the present study, the ability of adjuvant trastuzumab to reduce locoregional recurrence in patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer receiving adjuvant chemotherapy and radiotherapy (RT) was investigated. MATERIALS AND METHODS: We retrospectively included 520 patients with HER2-overexpressing breast cancer who received surgery followed by adjuvant RT and cytotoxic chemotherapy from 2003 to 2011. Adjuvant trastuzumab was administered to 286 patients. Propensity score matching was conducted to compare trastuzumab-treated and non-treated cohorts. RESULTS: Median follow-up duration was 7.1 years (range 1.1-14.1 years). Propensity score matching yielded 171 matched pairs of patients with no significantly different clinical factors. An improved 7-year locoregional control (LRC) rate was observed in the trastuzumab-treated cohort compared with the non-treated cohort (95.6% vs. 89.9%, p = 0.014). Based on multivariate analysis, hormone receptor negativity (hazard ratio [HR] = 5.348, p = 0.007), positive lymph node ratio > 0.25 (HR = 2.549, p = 0.040), and lack of adjuvant trastuzumab (HR = 3.401, p = 0.017) were identified as significant risk factors for poor LRC. Adjuvant trastuzumab significantly reduced the locoregional recurrence rate in patients with one or two risk factors (7-year LRC = 95.0% vs. 84.2%, p = 0.007); however, the benefit of adjuvant trastuzumab was non-significant in patients with no risk factors (7-year LRC = 95.8% vs. 97.9%, p = 0.75). CONCLUSIONS: Adjuvant trastuzumab improved LRC in patients with HER2-overexpressing breast cancer receiving adjuvant RT and cytotoxic chemotherapy, especially in hormone receptor-negative, HER2-enriched subtype, and high positive lymph node ratio breast cancer.

Air-electron stream interactions during magnetic resonance IGRT : Skin irradiation outside the treatment field during accelerated partial breast irradiation.

PURPOSE: To investigate and to prevent irradiation outside the treatment field caused by an electron stream in the air generated by the magnetic field during magnetic resonance image-guided accelerated partial breast irradiation (APBI). MATERIALS AND METHODS: In all, 20 patients who received APBI with a magnetic resonance image-guided radiation therapy (MR-IGRT) system were prospectively studied. The prescription dose was 38.5 Gy in 10 fractions of 3.85 Gy and delivered with a tri-cobalt system (the ViewRay system). For each patient, primary plans were delivered for the first five fractions and modified plans with different gantry angles from those of the primary plan (in-treatment plans) were delivered for the remaining five fractions to reduce the skin dose. A 1 cm thick bolus was placed in front of the patient's jaw, ipsilateral shoulder, and arm to shield them from the electron stream. Radiochromic EBT3 films were attached to the front (towards the breast) and back (towards the head) of the bolus during treatment. Correlations between the measured values and the tumor locations, treatment times, and tumor sizes were investigated. RESULTS: For a single fraction delivery, the average areas of the measured isodoses of 14% (0.54 Gy), 12% (0.46 Gy), and 10% (0.39 Gy) at the front of the boluses were as large as 3, 10.4, and 21.4 cm2, respectively, whereas no significant dose could be measured at the back of the boluses. Statistically significant but weak correlations were observed between the measured values and the treatment times. CONCLUSION: During radiotherapy for breast cancer with an MR-IGRT system, the patient must be shielded from electron streams in the air generated by the interaction of the magnetic field with the beams of the three-cobalt treatment unit to avoid unwanted irradiation of the skin outside the treatment field.

Efficacy of adjuvant radiotherapy in the intracranial hemangiopericytoma.

We retrospectively evaluated an efficacy of adjuvant radiotherapy (RT) in the intracranial hemangiopericytoma (HPC) and analyzed prognostic factors influencing treatment outcomes. Among 49 patients diagnosed as localized intracranial HPC between 1995 and 2016, 31 patients received adjuvant RT after surgery; 26 with fractionated RT and 5 with stereotactic radiosurgery using Gamma Knife. After gross total resection (GTR) (n = 32) and subtotal resection (STR) (n = 17), histopathological grade was confirmed to be grade II (n = 9) or grade III (n = 40). The median follow-up period was 50 months (range 3-216 months). The local recurrence was defined as intracranial relapse within 15 mm and regional recurrence as beyond 15 mm from the margin of surgical bed. The 10-year overall survival (OS) and progression-free survival (PFS) were 69.9 and 34.4%, respectively. The 10-year local, regional, and distant failure-free rates were 56.6, 88.2, and 73.3%, respectively. Local tumor control was better with GTR followed by RT than GTR alone (p = 0.056), while there was no difference in OS. Local tumor control and OS after STR plus RT were equivalent to those after GTR alone. There were no differences in distant metastasis-free survival (DMFS) among GTR plus RT, GTR alone, and STR plus RT. Tumor volume > 40 cm3 was associated with poor PFS (p = 0.024). The local tumor recurrence was reduced by adjuvant RT after surgery. But OS or DMFS was not improved with adjuvant RT. PFS was better in the tumor with small volume at diagnosis.

Analysis of survival outcomes based on molecular subtypes in breast cancer brain metastases: A single institutional cohort.

PURPOSE: To evaluate the survival outcomes based on molecular subtypes of breast cancer in patients with brain metastasis. MATERIALS AND METHODS: We retrospectively reviewed 106 breast cancer patients treated for brain metastases, from January 2005 to May 2016. Patients were divided into four groups based on the tumor molecular subtype: luminal A (Estrogen Receptor [ER]/Progesterone Receptor [PR] positive, human epithelial growth factor receptor-2 [HER2] negative), luminal B (ER/PR positive, HER2 Positive), HER2 (HER2 positive and ER/PR negative), and Triple negative (TNBC). RESULTS: The median follow-up time for surviving patients was 22 months (range: 11.2-51.1 months). The median survival of all patients was 14 months, with a 1-year overall survival (OS) rate of 57.5% and a 2-year OS rate of 32.1%. Thirty patients (28.3%) had a solitary brain metastasis while 62 (58.5%) patients had multiple metastases. A significant difference was observed in the survival rates of the two groups. Based on the Karnofsky performance score, the performance status of the patients at the time of brain metastasis was also found to affect survival. Patients with different molecular subtypes had different survival rates; the luminal A group showed the highest median survival (luminal A: 23.1, luminal B: 15.0, HER2: 12.5 and TNBC: 6.4 months, respectively), which was statistically significant. CONCLUSION: In breast cancer patients with brain metastasis, survival rates were different based on the molecular subtype of the tumor, despite various local and systemic treatments. Appropriate and tailored treatment approaches should, therefore, be considered for the different molecular subtypes.

Implication of Tumor Location for Lymph Node Metastasis in Maxillary Sinus Carcinoma: Indications for Elective Neck Treatment.

PURPOSE: For either neck irradiation or dissection, the indications for elective neck treatment (ENT) of maxillary sinus carcinoma are still unclear. The purpose of the present study was to investigate the relationship between the anatomic extent of the disease and lymph node metastasis in maxillary sinus carcinoma and to propose a recommendation regarding ENT. MATERIALS AND METHODS: In the present retrospective cohort study, patients with squamous cell carcinoma (SCC) and undifferentiated carcinoma (UDC) of maxillary sinus treated with radical intent from January 1995 to June 2015 in a single institution were recruited by retrospective medical record review. The demographic and tumor characteristics of the patients and maxillary sinus wall invasion, verified on pretreatment volumetric imaging studies, were analyzed. The Cox proportional hazards model was used to find the risk factors for nodal relapse, distant metastasis, and survival. RESULTS: Among a total of 71 identified patients, 66 had SCC and 5 had UDC. In 55 patients with node-negative disease, the risk of ipsilateral nodal relapse was 25.1% without ENT. In contrast, no ipsilateral nodal relapse was reported after ENT. On multivariate analysis, no chemotherapy (hazard ratio [HR] = 7.25; P = .01), posterior wall invasion (HR = 6.51; P = .03), and local failure (HR = 6.42; P = .02) were identified to be the risk factors of nodal relapse. Nodal relapse influenced the risk of distant metastasis with marginal significance (HR = 3.95; P = .07) but did not have an effect on survival. The most common regions of lymph node metastasis, at both initial presentation and relapse, were ipsilateral levels I and II. CONCLUSIONS: For SCC and UDC of the maxillary sinus with posterior wall invasion, ENT involving ipsilateral levels I and II is recommended. Future studies with larger numbers of patients are needed to validate our conclusion.

Combination of local radiotherapy and anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) therapy augments PD-L1 blockade-mediated anti-tumor effects in murine breast cancer model.

PURPOSE: In this study, we investigated whether local radiotherapy (RT) and an anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) agonist could increase the efficacy of PD-L1 blockade. METHODS AND MATERIALS: We analyzed a breast cancer dataset from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) to determine the role of GITR in breast cancer. We used the 4T1 murine TNBC model (primary and secondary tumors) to investigate the efficacy of PD-L1 blockade, local RT, anti-GITR agonist, and their combinations. We assessed tumor growth by tumor volume measurements, in vivo bioluminescence imaging, and metastatic lung nodule counts to evaluate the effects of these treatments. Flow cytometry and immunohistochemistry determined the proportions and phenotypes of CD8+ T-cells and regulatory T-cells (Tregs) in the tumors and spleen. Plasma cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: In the METABRIC cohort, patients with high expression of TNFRSF18, which encodes GITR, had significantly better survival than those with low expression. Adding local RT or anti-GITR agonist to PD-L1 blockade did not significantly augment efficacy compared to PD-L1 blockade alone; however, adding both to PD-L1 blockade significantly reduced tumor growth and lung metastasis. The benefits of the triple combination were accompanied by increased CD8+ T-cells and decreased Tregs in the tumor microenvironment and spleen. CONCLUSIONS: The combination of local RT and an anti-GITR agonist significantly enhanced the anti-tumor immune responses induced by PD-L1 blockade. These results provide the preclinical rationale for the combination of therapy.

Dynamic Responses of Circulating T Cells After Stereotactic Body Radiation Therapy for Bone Metastasis in Patients With Breast Cancer.

PURPOSE: Preclinical studies have shown that radiation therapy modulates antitumor immune responses. However, circulating T-cell responses after radiation therapy in patients with cancer have been poorly characterized. This study aims to explore the changes in circulating T cells after stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: Peripheral blood samples of 30 patients with breast cancer who underwent SBRT for bone metastasis were analyzed using multicolor flow cytometry. Phenotypes of PD-1+ CD8+ T cells and regulatory T (TREG) cells were examined. Additionally, plasma protein levels were analyzed using a bead-based immunoassay. RESULTS: Circulating PD-1+ CD8+ T cells, which are enriched for tumor-specific clonotypes, were activated at 1 week after SBRT. However, circulating TREG cells were also activated after SBRT; this pattern was also evident among effector Foxp3hiCD45RA- TREG cells. We observed no difference in T-cell responses according to the fraction size and number. Notably, activation of TREG cells was more prominent in patients who experienced greater activation of PD-1+ CD8+ T cells. Plasma level changes in TGF-ß1, soluble CTLA-4, and soluble 4-1BB at 1 week after SBRT were associated with PD-1+ CD8+ T-cell responses. Activation of TREG cells at 1 week after SBRT was associated with worse progression-free survival. Clinical factors including molecular subtype were not associated with the T-cell responses. CONCLUSIONS: SBRT induced activation of both potentially tumor-specific CD8+ T cells and TREG cells, which were tightly associated with each other. These results may support the use of TREG cell-modulating strategies with SBRT to improve the antitumor immune response.

In vitro-irradiated cancer vaccine enhances anti-tumor efficacy of radiotherapy and PD-L1 blockade in a syngeneic murine breast cancer model.

BACKGROUND AND PURPOSE: Local radiotherapy (RT) exerts immunostimulatory effects by inducing immunogenic cell death. However, it remains unknown whether in vitro-irradiated tumor cells can elicit anti-tumor responses and enhance the efficacy of local RT and immune checkpoint inhibitors when injected in vivo. METHODS AND MATERIALS: We tested the "in vitro-irradiated cancer vaccine (ICV)", wherein tumor cells killed by varying doses of irradiation and their supernatants are intravenously injected. We examined the efficacy of combining local RT (24 Gy in three fractions), PD-L1 blockade, and the ICV in a murine breast cancer model. The immune cell profiles were analyzed via flow cytometry and immunohistochemistry. The cytokine levels were measured by multiplex immunoassays. RESULTS: The ICV significantly increased the effector memory phenotype and interferon-γ production capacity in splenic CD8+ T cells. The in vitro-irradiated products contained immune response-related molecules. When combined with local RT and PD-L1 blockade, the ICV significantly delayed the growth of irradiated and non-irradiated tumors. The triple combination therapy increased the proportions of CD8+ T cells and effector memory CD8+ T cells while decreasing the proportion of CTLA-4+ exhausted CD8+ T cells within tumor microenvironment. Additionally, plasma level of interferon-γ and proliferation of effector T cells in the spleen and tumor-draining lymph nodes were significantly increased by the triple combination therapy. CONCLUSIONS: The ICV enhanced the therapeutic efficacy of local RT and PD-L1 blockade by augmenting anti-tumor immune responses. Our findings suggest a therapeutic potential of in vitro-irradiation products of tumor cells.

Anti-4-1BB×PDL1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD1 Blockade.

PURPOSE: To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PDL1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PDL1 and 4-1BB and demonstrated strong antitumor T-cell responses without considerable toxicity. In this study, we investigated the mechanisms by which the combination of ABL503 and anti-PD1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TIL) and antitumor efficacy. EXPERIMENTAL DESIGN: Single-cell suspensions of hepatocellular carcinoma and ovarian cancer tissues from treatment-naïve patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD1/hPDL1/h4-1BB triple-knock-in mice were used to evaluate the effects of ABL503 and anti-PD1 blockade in vivo. RESULTS: We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD1 blockade. Importantly, compared with anti-PD1 blockade alone, the combination of ABL503 and anti-PD1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD1 in vivo significantly alleviated tumor growth and induced enhanced infiltration and activation of CD8+ TILs. CONCLUSIONS: ABL503, a PDL1 and 4-1BB dual-targeting bispecific antibody, elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anticancer effects of anti-PD1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD1 inhibitors will likely further enhance therapeutic benefit in clinical trials. See related commentary by Molero-Glez et al., p. 3971.

Artificial Intelligence-Powered Spatial Analysis of Tumor-Infiltrating Lymphocytes as a Potential Biomarker for Immune Checkpoint Inhibitors in Patients with Biliary Tract Cancer.

PURPOSE: Recently, anti-programmed cell death-1/anti-programmed cell death ligand-1 (anti-PD1/L1) immunotherapy has been demonstrated for its efficacy when combined with cytotoxic chemotherapy in randomized phase 3 trials for advanced biliary tract cancer (BTC). However, no biomarker predictive of benefit has been established for anti-PD1/L1 in BTC. Here, we evaluated tumor-infiltrating lymphocytes (TIL) using artificial intelligence-powered immune phenotype (AI-IP) analysis in advanced BTC treated with anti-PD1. EXPERIMENTAL DESIGN: Pretreatment hematoxylin and eosin (H&E)-stained whole-slide images from 339 patients with advanced BTC who received anti-PD1 as second-line treatment or beyond, were employed for AI-IP analysis and correlative analysis between AI-IP and efficacy outcomes with anti-PD1. Next, data and images of the BTC cohort from The Cancer Genome Atlas (TCGA) were additionally analyzed to evaluate the transcriptomic and mutational characteristics of various AI-IP in BTC. RESULTS: Overall, AI-IP were classified as inflamed [high intratumoral TIL (iTIL)] in 40 patients (11.8%), immune-excluded (low iTIL and high stromal TIL) in 167 patients (49.3%), and immune-desert (low TIL overall) in 132 patients (38.9%). The inflamed IP group showed a substantially higher overall response rate compared with the noninflamed IP groups (27.5% vs. 7.7%, P < 0.001). Median overall survival and progression-free survival were significantly longer in the inflamed IP group than in the noninflamed IP group (OS, 12.6 vs. 5.1 months; P = 0.002; PFS, 4.5 vs. 1.9 months; P < 0.001). In the TCGA cohort analysis, the inflamed IP showed increased cytolytic activity scores and IFNγ signature compared with the noninflamed IP. CONCLUSIONS: AI-IP based on spatial TIL analysis was effective in predicting the efficacy outcomes in patients with BTC treated with anti-PD1 therapy. Further validation is necessary in the context of anti-PD1/L1 plus gemcitabine-cisplatin.

Characterization of the gene signature correlated with favorable response to chemoradiotherapy in rectal cancer: A hypothesis-generating study.

PURPOSE: This study aimed to define the gene signature associated with response to neoadjuvant chemoradiotherapy (nCRT), or chemoradiosensitivity (CRS) signature, in rectal cancer, and investigate the correlation between the CRS signature and characteristics of tumor. MATERIALS AND METHODS: Three public microarray datasets of pre-nCRT rectal cancer were used to discover and validate the CRS signature, and the pathway analysis of the CRS signature was performed. Patients in The Cancer Genome Atlas (TCGA) dataset were stratified according to the CRS signature enrichment score, and mutational profile and proportions of infiltrated immune cells were compared. RESULTS: In the discovery dataset (GSE53781), 95 genes were upregulated in complete responders compared to non-complete responders and defined as the CRS signature. Pathways regarding DNA replication and repair processes as well as inflammatory response were enriched in the CRS signature. In the validation datasets (GSE35452 and GSE45404), patients with favorable response to nCRT exhibited higher enrichment score of the CRS. In TCGA-READ cohort, patients with high CRS signature harbored KRAS mutation in lower frequency than those with low CRS signature. In addition, proportions of proinflammatory immune cells were higher, but proportion of immunosuppressive M2 macrophages was lower in patients with high CRS signature than those with low CRS signature. CONCLUSIONS: The current integrative bioinformatic analysis suggests the CRS signature and showed that the CRS signature is associated with dissimilar mutational profile and increased immune response. The discovered CRS signature and related characteristics may serve as candidate of stratification factor in upcoming studies for rectal cancer.

Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers.

PURPOSE: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen. Materials and Methods: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction. RESULTS: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment. CONCLUSION: The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

CEACAM1 Marks Highly Suppressive Intratumoral Regulatory T Cells for Targeted Depletion Therapy.

PURPOSE: Regulatory T cells (Tregs) exert immunosuppressive functions and hamper antitumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. EXPERIMENTAL DESIGN: From 42 patients with renal cell carcinoma and 5 patients with ovarian cancer, immune cells from tumor and peripheral blood were isolated. We performed multicolor flow cytometry and RNA-sequencing to characterize the phenotypes and heterogeneity of intratumoral Tregs. In vitro functional assays were performed to evaluate suppressive capacity of Tregs and effect of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-mediated depletion. The CT26 tumor model was used to evaluate the association between intratumoral Tregs and tumor growth, and examine the in vivo role of CEACAM1+ intratumoral Tregs on antitumor immunity. RESULTS: We found that CEACAM1 was selectively expressed on intratumoral Tregs, whereas its expression on peripheral Tregs or other immune cells was low. The CEACAM1+ intratumoral Tregs accumulated with tumor progression, whereas the CEACAM1- subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of tumor-infiltrating T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1-mediated reinvigoration of exhausted CD8+ T cells. CONCLUSIONS: CEACAM1 marks highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs. These results may inform future studies on CEACAM1-mediated depletion in patients with cancer.

Radiomic models based on magnetic resonance imaging predict the spatial distribution of CD8+ tumor-infiltrating lymphocytes in breast cancer.

Infiltration of CD8+ T cells and their spatial contexture, represented by immunophenotype, predict the prognosis and therapeutic response in breast cancer. However, a non-surgical method using radiomics to evaluate breast cancer immunophenotype has not been explored. Here, we assessed the CD8+ T cell-based immunophenotype in patients with breast cancer undergoing upfront surgery (n = 182). We extracted radiomic features from the four phases of dynamic contrast-enhanced magnetic resonance imaging, and randomly divided the patients into training (n = 137) and validation (n = 45) cohorts. For predicting the immunophenotypes, radiomic models (RMs) that combined the four phases demonstrated superior performance to those derived from a single phase. For discriminating the inflamed tumor from the non-inflamed tumor, the feature-based combination model from the whole tumor (RM-wholeFC) showed high performance in both training (area under the receiver operating characteristic curve [AUC] = 0.973) and validation cohorts (AUC = 0.985). Similarly, the feature-based combination model from the peripheral tumor (RM-periFC) discriminated between immune-desert and excluded tumors with high performance in both training (AUC = 0.993) and validation cohorts (AUC = 0.984). Both RM-wholeFC and RM-periFC demonstrated good to excellent performance for every molecular subtype. Furthermore, in patients who underwent neoadjuvant chemotherapy (n = 64), pre-treatment images showed that tumors exhibiting complete response to neoadjuvant chemotherapy had significantly higher scores from RM-wholeFC and lower scores from RM-periFC. Our RMs predicted the immunophenotype of breast cancer based on the spatial distribution of CD8+ T cells with high accuracy. This approach can be used to stratify patients non-invasively based on the status of the tumor-immune microenvironment.

Combination of OX40 Co-Stimulation, Radiotherapy, and PD-1 Inhibition in a Syngeneic Murine Triple-Negative Breast Cancer Model.

Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.