Identification and Quantification of Radiotherapy-related Protein Expression in Cancer Tissues Using the Qupath Software and Prediction of Treatment Response.

BACKGROUND/AIM: Automated measurement of immunostained samples can enable more convenient and objective prediction of treatment outcome from radiotherapy. We aimed to validate the performance of the QuPath image analysis software in immune cell markers detection by comparing QuPath cell counting results with those of physician manual cell counting. PATIENTS AND METHODS: CD8- and FoxP3-stained cervical, CD8-stained oropharyngeal, and Ku70-stained prostate cancer tumor sections were analyzed in 104 cervical, 92 oropharyngeal, and 58 prostate cancer patients undergoing radiotherapy at our Institution. RESULTS: QuPath and manual counts were highly correlated. When divided into two groups using ROC curves, the agreement between QuPath and manual counts was 89.4% for CD8 and 88.5% for FoxP3 in cervical cancer, 87.0% for CD8 in oropharyngeal cancer and 80.7% for Ku70 in prostate cancer. In cervical cancer, the high CD8 group based on QuPath counts had a better prognosis and the low CD8 group had a significantly worse prognosis [p=0.0003; 5-year overall survival (OS), 65.9% vs. 34.7%]. QuPath counts were more predictive than manual counts. Similar results were observed for FoxP3 in cervical cancer (p=0.002; 5-year OS, 62.1% vs. 33.6%) and CD8 in oropharyngeal cancer (p=0.013; 5-year OS, 80.2% vs. 47.2%). In prostate cancer, high Ku70 group had worse and low group significantly better outcome [p=0.007; 10-year progression-free survival (PFS), 56.0% vs. 93.8%]. CONCLUSION: QuPath showed a strong correlation with manual counting, confirming its utility and accuracy and potential applicability in clinical practice.

Increased ROS levels in mitochondrial outer membrane protein Mul1-deficient oocytes result in abnormal preimplantation embryogenesis.

Reactive oxygen species (ROS) are associated with oocyte maturation inhibition, and N-acetyl-l-cysteine (NAC) partially reduces their harmful effects. Mitochondrial E3 ubiquitin ligase 1 (Mul1) localizes to the mitochondrial outer membrane. We found that female Mul1-deficient mice are infertile, and their oocytes contain high ROS concentrations. After fertilization, Mul1-deficient embryos showed a DNA damage response (DDR) and abnormal preimplantation embryogenesis, which was rescued by NAC addition and ROS depletion. These observations clearly demonstrate that loss of Mul1 in oocytes increases ROS concentrations and triggers DDR, resulting in abnormal preimplantation embryogenesis. We conclude that manipulating the mitochondrial ROS levels in oocytes may be a potential therapeutic approach to target infertility.

Fatty-marrow transformation following radiotherapy for pancreatic cancer detected using dual-energy computed tomography: A case report.

Bone damage, a late side effect of radiotherapy, occurs concurrently with the replacement of fat cells in the bone marrow, causing changes in bone composition. Changes in composition can affect bone quality and disease states, and reduced bone mass can reduce quality of life by increasing the risk of fractures. A 70-year-old woman presented to the orthopedic outpatient clinic with the chief complaint of lower-back pain. The patient reported no history of trauma but was in great pain and had difficulty walking. Since the patient had a history of pancreatic cancer, tumor-marker testing, bone scintigraphy, and dual-energy computed tomography were performed. Although the tumor-marker levels were normal, dual-energy computed tomography and bone scintigraphy revealed fresh compression fractures of the L1 and L3 vertebrae. In addition, dual-energy computed tomography material-discrimination analysis suggested high fat density in the L2 vertebral body. The patient had received approximately 30 Gy radiation to the L2 vertebral body for her pancreatic cancer, which resulted in fatty myelination in the bone. The diagnosis of fatty myelination is made on T1-weighted magnetic resonance images; however, diagnosis remains challenging because of the difficulty in assessing bone morphology on magnetic resonance images. Moreover, some patients are not candidates for magnetic resonance imaging. Dual-energy computed tomography-based material-discrimination analysis can visually depict changes in the bone marrow, and is a valuable diagnostic tool owing to its simplicity.

Predictive value of an exosomal microRNA-based signature for tumor immunity in cervical cancer patients treated with chemoradiotherapy.

Resistance of cervical cancer to radiotherapy with concurrent chemotherapy (CCRT) results in a poor prognosis. To identify new biomarkers for predicting the treatment response and prognosis, we explored exosomal microRNA (miRNA) expression signatures associated with the outcome of cervical cancer patients treated with CCRT. Exosomes were isolated from the plasma of 45 patients prior to CCRT during 2014-2020, and miRNA analysis was performed by next-generation sequencing. At a median follow-up of 38 months, 26 patients were recurrence free, 15 patients had died of the disease, and 4 patients received salvage chemotherapy due to distant metastasis. Of the 2522 miRNAs detected, 9 (miR-148a-5p, 1915-3p, 3960, 183-5p, 196b-5p, 200c-3p, 182-5p, 374a-5p, and 431-5p) showed differential expression between the recurrence-free and recurrence groups. Patients were divided into high- and low-risk groups according to the cutoff of the miRNAs-based risk score calculated from respective expression levels. The high-risk group had significantly worse disease-specific survival than the low-risk group (p < 0.001). In addition, miR-374a-5p and miR-431-5p expression showed a weak inverse correlation with tumor-infiltrating CD8+ and FOXP3+ T cells, suggesting a potential inhibitory effect on CCRT by suppressing tumor immunity. This miRNA signature could improve non-invasive monitoring and personalized treatment for cervical cancer.

Combined chemoradiotherapy and programmed cell death-ligand 1 blockade leads to changes in the circulating T-cell receptor repertoire of patients with non-small-cell lung cancer.

Combined chemoradiotherapy (CRT) and programmed cell death-ligand 1 (PD-L1) blockade is a new care standard for unresectable stage III non-small-cell lung cancer (NSCLC). Although this consolidation therapy has improved the overall survival of patients with NSCLC, the synergistic action mechanisms of CRT and immunotherapy on T cells remain unclear. In addition, there is a paucity of reliable biomarkers to predict clinical responses to therapy. In this study, we analyzed T-cell receptor (TCR) sequences in the peripheral blood of five patients with NSCLC. T-cell receptor analysis was undertaken before treatment, after CRT, and after PD-L1 blockade. Notably, we observed the expansion and alteration of the dominant T-cell clonotypes in all cases with a complete response. In contrast, neither expansion nor alteration of the TCR repertoire was observed in cases with progressive disease. T cell expansion was initiated after CRT and was further enhanced after PD-L1 blockade. Our findings suggest the systemic effect of CRT on circulating T cells in addition to the curative effect on limited tumor sites. Dynamic changes in circulating T-cell clonotypes could have a prognostic significance for combined CRT and PD-L1 blockade.

Radiotherapy for HPV-related cancers: prediction of therapeutic effects based on the mechanism of tumor immunity and the application of immunoradiotherapy.

Human papillomavirus (HPV)-related cancer is one of the diseases entities for which the applications of radiotherapy have been increasing. Recently, the process of carcinogenesis from HPV infection and the mechanism of tumor immunity that develops during disease progression have been elucidated. In this review, we will describe the mechanism of tumor immunity and how chemoradiotherapy may overcome and improve the efficacy of tumor immunity. We will also discuss the usefulness of proteins involved with tumor immunity as a predictive marker of radiotherapy response, and present an overview of ongoing clinical trials of combinations of immune checkpoint inhibitors and radiotherapy to demonstrate the promising combination therapy that has been currently emerging.

Prediction of treatment response from the microenvironment of tumor immunity in cervical cancer patients treated with chemoradiotherapy.

To supplement clinical decision-making in the management of cervical cancer, various prognostic factors, including tumor immune microenvironments, were examined in patients with cervical cancer treated with definitive chemoradiotherapy. We retrospectively analyzed the expression of CD8, FoxP3, HLA-1, PD-L1, and XRCC4 in 100 cases of cervical cancer. The observed tumor immune microenvironments were also classified into three types: inflamed, excluded, and cold type. Less FoxP3+ T cells and cold-type tumor were found to be poor prognostic factors in addition to non-SCC, large pre-treatment tumor volume, and three or less cycles of concurrent chemotherapy based on multivariate analysis. Cold-type tumors had significantly worse prognoses than the other two types, whereas inflamed- and excluded-type tumors showed similar 5-year disease-specific survival (P < 0.001; 0% vs. 60.3% vs. 72.3%). Radiotherapy could overcome the inhibitory immune microenvironment that occurs in excluded type. Individualized combination therapy adapted to pre-treatment tumor immunity may be necessary to improve radiotherapy outcomes in cervical cancer.

Association between cancer immunity and treatment results in uterine cervical cancer patients treated with radiotherapy.

OBJECTIVE: To evaluate proteins related to tumor immune response and treatment outcome from radiotherapy for uterine cervical cancer patients. METHODS: We performed a retrospective immunohistochemical staining of 81 patients with uterine cervical cancer who underwent definitive radiotherapy. We examined the expression of programmed death ligand 1, human leukocyte antigen class I, tumor-infiltrating CD8+, and forkhead box P3+ (FoxP3+) T cells in tumor tissues. RESULTS: In biopsy specimen, patients with a higher number of CD8+ T cells and FoxP3+ T cells had a better disease-specific survival than patients with a lower number of CD8+ T cells and FoxP3+ cells (P = 0.018 and P = 0.009). Multivariate analysis showed that equivalent dose in 2 Gy fractions (EQD2) of the minimum dose to 90% of the high-risk clinical target volume, FoxP3+ T cells and expression of human leukocyte antigen class I were significant prognostic factors. When the EQD2 is 70 Gy or more, a higher local control rate is obtained regardless of the number of CD8- or FoxP3-positive cells. When EQD2 is <70 Gy, the number of CD8-positive cells has a significant impact on treatment outcome: the recurrence rate (local recurrence rate + distant metastasis rate) was 46.2% in the group with a CD8 value of 230 or higher, whereas the recurrence rate was 75.7% in the group with a CD8 value of less than 230. CONCLUSION: The combination of CD8 or FoxP3 with EQD2 can be potentially useful to predict the treatment results of radiotherapy for cervical cancer, leading to individualized optimal selection of treatment for cervical cancer.

Association between radiotherapy-induced alteration of programmed death ligand 1 and survival in patients with uterine cervical cancer undergoing preoperative radiotherapy.

PURPOSE: To evaluate radiotherapy-induced changes in the expression of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), and human leukocyte antigen class I (HLA-1) in patients with uterine cervical cancer, as well as infiltration of CD8+ and Forkhead box P3+ (FoxP3+) T lymphocytes into tumor tissue and the prognostic value of these parameters. MATERIALS AND METHODS: We performed immunohistochemical analysis of pre-radiotherapy biopsies and corresponding post-radiotherapy resected tissues in 104 uterine cervical cancer patients undergoing preoperative chemoradiotherapy or radiotherapy alone. We scored the expression of various proteins to distinguish positive from negative samples. RESULTS: PD-L1-expressing tumor cells (PD-L1 TC) increased significantly after chemoradiotherapy (p = 0.043). CD8+ T cell infiltration (p = 0.002) and FoxP3+ T cell infiltration (p = 0.003) decreased significantly after chemoradiotherapy. Expression of PD­1, PD-L1-expressing immune cells (PD-L1 IC), and HLA­1 did not change after chemoradiotherapy. In biopsy specimens obtained before chemoradiotherapy or radiotherapy, greater infiltration of CD8+ T cells (p = 0.001) and FoxP3+ T cells (p = 0.003) were significant predictors of better overall survival (OS). In surgical specimens obtained after chemoradiotherapy or radiotherapy, greater infiltration of PD-L1 TC was the only significant predictor of better OS (p < 0.001) and was related to a significantly lower probability of out-of-field recurrence (p = 0.005). CONCLUSION: Chemoradiotherapy induced an immunologic shift that increased PD-L1 TC. Chemoradiotherapy has immunological effects that can influence the results of treatment for uterine cervical cancer.

Influence of PD-L1 expression in immune cells on the response to radiation therapy in patients with oropharyngeal squamous cell carcinoma.

BACKGROUND AND PURPOSE: To investigate influences of proteins involved with tumor immunity on outcomes of radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC). MATERIAL AND METHODS: We performed immunohistochemical staining to examine expressions of p16 and proteins involved with tumor immunity in 92 OPSCC patients treated with radiotherapy. RESULTS: Patients with abundant infiltrating CD8-positive cells had the significantly better overall survival (OS) rate than patients with fewer CD8-positive cells (p = 0.026). Patients with higher PD-L1 expression in tumor cells (TC 1-3) had a better outcome than those with low PD-L1 expression in tumor cells (TC 0) for both OS (p = 0.019) and progression-free survival (PFS) rate (p = 0.032). Patients with high PD-L1 expression in infiltrating immune cells (IC 3) showed significantly better OS (p = 0.009) and PFS (p = 0.011) than those with low PD-L1 expression (IC 0-2). Patients with p16-negative and IC 3 showed similar OS to patients with p16-positive and IC 0-2. P16-positive tumors had a significantly higher CD8-positive cell infiltration and PD-L1 expression in tumor cells than p16-negative tumors. CONCLUSIONS: In addition to tumor p16 expression, PD-L1 expression in TC and IC can be useful for predicting the response of OPSCC to radiotherapy.

Influence of XRCC4 expression in esophageal cancer cells on the response to radiotherapy.

DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation and is mainly repaired by the non-homologous end joining (NHEJ) repair. Immunohistochemical analysis of proteins involved in NHEJ, such as XRCC4 (X-ray repair cross-complementing protein 4), Ku86 and DNA-PKcs (DNA-dependent protein kinase, catalytic subunits), may be useful for predicting tumor radiosensitivity. We examined 92 patients with esophageal squamous cell carcinoma (ECSS) who were treated by radiotherapy between 1999 and 2008. Immunohistochemical examination of tumor tissue for Ki-67 and DSB-related proteins, including XRCC4, Ku86, and DNA-PKcs, was performed using pretreatment biopsy specimens. Low expression of XRCC4 was detected in 31 of 92 examined samples (33.7 %). The 5-year overall survival (OS) rate was 67.7 % in the low expression group and 31.0 % in the high expression group (P = 0.00). Multivariate analysis confirmed that advanced T-stage (HR 3.24, P = 0.01), radiation dose less than 66 Gy (HR 2.23, P = 0.02), absence of systemic chemotherapy (HR 2.59, P = 0.05), and high expression of XRCC4 (HR 12.0, P = 0.02) were independent prognostic factors for predicting poor OS. Other DSB-related proteins and Ki-67 were not predictive factors. XRCC4 expression might have an influence on results of radiotherapy for patients with ESCC.