An atlas of transcribed enhancers across helper T cell diversity for decoding human diseases.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Real-World Patient Characteristics, Treatment Patterns, and Outcomes of HR+, HER2- Early Breast Cancer Patients in Japan: An Analysis with National Database(NDB).

Real-world evidence for clinical outcomes and treatment patterns in patients with hormone receptor-positive(HR+)and human epidermal growth factor receptor 2-negative(HER2-)early breast cancer(EBC)in Japan is limited. We aimed to provide recent evidence in this population using the National Database of Health Insurance Claims and Specific Health Check-ups of Japan(NDB). Adults ≥20 years old who were diagnosed with HR+/HER2- breast cancer and underwent breast resection surgery were followed up. Patient characteristics and treatment patterns were evaluated. Durations of overall post-operative endocrine therapy(ET)and luteinizing hormone-releasing hormone(LH-RH)agonist therapy, and time to metastasis/recurrence after surgery were analyzed using Kaplan-Meier method. Overall, 294,904 patients were included. Cyclophosphamide and tamoxifen were the most common peri-operative chemotherapeutic and ET drugs. Median(95% confidence interval[CI])duration of post-operative ET and LH-RH agonist therapy was 5.01(5.01-5.01)years and 2.13 (2.12-2.14)years, respectively. Five-year cumulative rate(95% CI)of any recurrence was 8.6%(8.5-8.7), visceral metastasis being the most common. Nation-wide treatment patterns were described, which were consistent with guideline recommendations for patients with HR+, HER2- EBC. Further discussion is required to delay metastasis/recurrence and improve clinical outcomes(Fig. 1: Plain language summary of the study).

An adaptive planning strategy in carbon ion therapy of pancreatic cancer involving beam angle selection.

BACKGROUND AND PURPOSE: In carbon-ion radiotherapy for pancreatic cancer, altered dose distributions due to changes in the gastrointestinal gas volume and anatomy during irradiation are an unresolved therapeutic issue. We developed and investigated an adaptive strategy involving beam angle selection to improve dose distributions in pancreatic cancer. MATERIALS AND METHODS: In the adaptive strategy, multiple beams were prepared with angles similar to those of the conventional strategy, and the beam that best reproduces the dose distribution of the treatment plan was used. The dose distributions of the adaptive strategy were compared with those of the conventional strategy for five patients. Patients underwent computed tomography (CT) before every irradiation. The adaptive strategy was evaluated using the same irradiation schedule as that of the conventional method and an adjusted method based on anatomical changes per fraction. Dose distributions on the pre-treatment CT and accumulated dose distributions on the treatment planning CT were evaluated using the volume receiving ≥95% of the prescription dose (V95) from the clinical target volume (CTV) between strategies. RESULTS: There were significant differences in the CTV V95 values for the pre-treatment CT between all strategies. The median (range) CTV V95 for the conventional strategy was 92.7% (87.1-96.1%), for the proposed adaptive strategy without adjusted schedules was 96.9% (95.1-97.8%), and for the proposed strategy with adjusted schedules was 97.8% (96.5-99.2%). CONCLUSIONS: The adaptive strategy can improve target coverage for the pre-treatment CT and accumulated dose distributions for the treatment planning CT without increasing the dose to critical organs.

Deterioration of pancreatic exocrine function in carbon ion radiotherapy for pancreatic cancer.

BACKGROUND AND PURPOSE: In radiotherapy (RT) for pancreatic cancer, the pancreas is considered an important organ at risk. However, there are insufficient reports on pancreatic function deterioration after X-ray RT as organ at risk, and there are no reports on those after carbon ion (C-ion) RT. Here, we evaluated pancreatic exocrine insufficiency (PEI) after C-ion RT using dose-volume histogram (DVH) analysis. MATERIALS AND METHODS: Data were retrospectively collected from patients who had undergone C-ion RT for pancreatic cancer between July 2013 and June 2019. The prescribed C-ion doses were 55.2 Gy (relative biological effectiveness) in 12 fractions. Serum pancreatic amylase and lipase values were measured before and after C-ion RT. In DVH analysis, we assessed V5Gy-50Gy and V<5 Gy-50Gy of pancreatic volume and analyzed whether these DVH parameters involved PEI. RESULTS: Thirty-three patients were included in the analysis. The median follow-up duration after the initiation of C-ion RT in these patients was 15.8 months (range, 4.3-64.8). During and after treatment, 57.6% of patients developed PEI within 13.6 months, defined as pancreatic amylase and lipase deficiencies. In DVH analysis, V<5Gy was the most effective factor for the PEI, and the cutoff value for developing PEI in V<5Gy was 4.57 cm3. CONCLUSION: We showed that pancreatic exocrine function declined after C-ion RT for pancreatic cancer and that PEI was initiated early in the course of C-ion RT. Additionally, a low dose of DVH parameters, such as V<5Gy, was a prognostic factor of PEI.

Prospective Evaluation of Quality of Life and Functional Outcomes after Carbon Ion Radiotherapy for Inoperable Bone and Soft Tissue Sarcomas.

Carbon-ion radiotherapy (CIRT) represents a definitive treatment for inoperable bone and soft tissue sarcoma (BSTS). This prospective study analyzed 61 patients with inoperable BSTS who were treated with CIRT to evaluate QOL, functional outcomes, and predictive factors in patients with inoperable BSTS treated with definitive CIRT. The Musculoskeletal Tumor Society (MSTS) scoring system and the Short Form (SF)-8 questionnaire were completed before and at 1, 3, 6, 12, and 24 months after CIRT. The median follow-up period was 38 months. The main site of primary disease was the pelvis (70.5%), and the most common pathologic diagnosis was chordoma (45.9%). The 3-year overall survival and local control rates were 87.8% and 83.8%, respectively. The MSTS score and physical component score (PCS) of SF-8 did not change significantly between the baseline and subsequent values. The mental component score of SF-8 significantly improved after CIRT. Multivariate analysis showed that the normalized MSTS and normalized PCS of SF-8 at the final follow-up were significantly affected by performance status at diagnosis and sex. CIRT showed clinical efficacy, preserving the physical component of QOL and functional outcomes and improving the mental component of QOL, suggesting its potential value for the treatment of patients with inoperable BSTS.

Comparison of Oncologic Outcomes between Carbon Ion Radiotherapy and Stereotactic Body Radiotherapy for Early-Stage Non-Small Cell Lung Cancer.

Lung cancer is a leading cause of cancer-related deaths worldwide. Radiotherapy is an essential treatment modality for inoperable non-small cell lung cancer (NSCLC). Stereotactic body radiotherapy (SBRT) is the standard treatment for early-stage NSCLC because of its favorable local control (LC) compared to conventional radiotherapy. Carbon ion radiotherapy (CIRT) is a kind of external beam radiotherapy characterized by a steeper dose distribution and higher biological effectiveness. Several prospective studies have shown favorable outcomes. However, there is no direct comparison study between CIRT and SBRT to determine their benefits in the management of early-stage NSCLC. Thus, we conducted a retrospective, single-institutional, and contemporaneous comparison study, including propensity score-adjusted analyses, to clarify the differences in oncologic outcomes. The 3-year overall survival (OS) was 80.1% in CIRT and 71.6% in SBRT (p = 0.0077). The 3-year LC was 87.7% in the CIRT group and 79.1% in the SBRT group (p = 0.037). Multivariable analyses showed favorable OS and LC in the CIRT group (hazard risk [HR] = 0.41, p = 0.047; HR = 0.30, p = 0.040, respectively). Log-rank tests after propensity score matching and Cox regression analyses using propensity score confirmed these results. These data provided a positive efficacy profile of CIRT for early-stage NSCLC.

Skin Dose Reduction by Layer-Stacking Irradiation in Carbon Ion Radiotherapy for Parotid Tumors.

Background: Layer-stacking irradiation (LSI) results in the accumulation of multiple small spread-out Bragg peaks along the beam direction. Although the superiority of LSI to conventional passive irradiation (CPI) regarding normal tissue sparing is theoretically evident, the clinical benefit of LSI has not been demonstrated. Here, we compared LSI with CPI using the same treatment planning-computed tomography images used for carbon ion radiotherapy (CIRT). Methods: Twenty-one parotid tumors were analyzed. The clinical target volume (CTV) 1 and CTV2 encompassed the parotid grand and the tumor, respectively. CTV1 and CTV2 received 36 Gy (RBE: relative biological effectiveness) in nine fractions and 64 Gy (RBE) in 16 fractions, respectively, using either LSI or CPI. CTV coverage was assessed by DX%, which is the dose covering at least X% of the target volume. Skin dose was assessed by SX, which is the skin surface area receiving at least X Gy (RBE). Results: For CTV1 and CTV2, there were no significant differences in D2% between LSI and CPI. D50% and D98% were slightly higher for CPI; however, the absolute difference between the two methods was <3%. S10-S60 (in increments of 10) were significantly lower for LSI than for CPI (P < 0.001 for all parameters). LSI was associated with a significant trend toward dose reduction at the skin area irradiated with a higher dose by CPI (P < 0.001). Conclusions: LSI achieved better skin sparing than CPI without sacrificing target volume coverage in parotid tumor patients.

Reporting of methodologies used for clonogenic assays to determine radiosensitivity.

Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

Clinical impact of Hypofractionated carbon ion radiotherapy on locally advanced hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) involving a major branch of the portal or hepatic vein is in a locally advanced stage and remains difficult to cure. This study aimed to evaluate the clinical effects of carbon ion radiotherapy (C-ion RT) in locally advanced HCC (LAHCC). METHODS: The data of 11 consecutive patients with LAHCC who received C-ion RT were analyzed. The C-ion RT doses of 52.8 Gy (relative biological effectiveness [RBE]) and 60.0 Gy (RBE) were delivered in 4 fractions for standard cases, and the 60.0 Gy dose was delivered in 12 fractions for close-to-gastrointestinal-tract cases. Survival and local control probabilities were calculated using the Kaplan-Meier method. RESULTS: The median follow-up duration after C-ion RT was 36.4 months. The median age at the time of registration for C-ion RT was 76 years. The median tumor size was 53 mm. The numbers of treatment-naive and recurrent HCC patients were 1 and 10, respectively. Direct invasion of the major branch of the portal vein, hepatic vein, or both portal and hepatic veins was observed in three, five, and three patients, respectively. The 3-year overall survival, local control, and progression-free survival rates were 64, 78, and 18%, respectively. No patient developed radiation-induced liver diseases or grade 3 or higher toxicities in the acute and late phases. CONCLUSIONS: C-ion RT showed favorable clinical outcomes with a high rate of local control and minimal toxicities in LAHCC. Our findings suggest that C-ion RT is a promising multidisciplinary treatment option in LAHCC.

Evaluation of Intensity- and Contour-Based Deformable Image Registration Accuracy in Pancreatic Cancer Patients.

We aimed to clarify the accuracy of rigid image registration and deformable image registration (DIR) in carbon-ion radiotherapy (CIRT) for pancreatic cancer. Six patients with pancreatic cancer who were treated with passive irradiation CIRT were enrolled. Three registration patterns were evaluated: treatment planning computed tomography images (TPCT) to CT images acquired in the treatment room (IRCT) in the supine position, TPCT to IRCT in the prone position, and TPCT in the supine position to the prone position. After warping the contours of the original CT images to the destination CT images using deformation matrices from the registration, the warped delineated contours on the destination CT images were compared with the original ones using mean displacement to agreement (MDA). Four contours (clinical target volume (CTV), gross tumor volume (GTV), stomach, duodenum) and four registration algorithms (rigid image registration [RIR], intensity-based DIR [iDIR], contour-based DIR [cDIR], and a hybrid iDIR-cDIR ([hDIR]) were evaluated. The means ± standard deviation of the MDAs of all contours for RIR, iDIR, cDIR, and hDIR were 3.40 ± 3.30, 2.2 1± 2.48, 1.46 ± 1.49, and 1.46 ± 1.37 mm, respectively. There were significant differences between RIR and iDIR, and between RIR/iDIR and cDIR/hDIR. For the pancreatic cancer patient images, cDIR and hDIR had better accuracy than RIR and iDIR.

Deep learning-assisted literature mining for in vitro radiosensitivity data.

BACKGROUND AND PURPOSE: Integrated analysis of existing radiosensitivity data obtained by the gold-standard clonogenic assay has the potential to improve our understanding of cancer cell radioresistance. However, extraction of radiosensitivity data from the literature is highly labor-intensive. To aid in this task, using deep convolutional neural networks (CNNs) and other computer technologies, we developed an analysis pipeline that extracts radiosensitivity data derived from clonogenic assays from the literature. MATERIALS AND METHODS: Three classifiers (C1-3) were developed to identify publications containing radiosensitivity data derived from clonogenic assays. C1 uses Faster Regions CNN with Inception Resnet v2 (fRCNN-IRv2), VGG-16, and Optical Character Recognition (OCR) to identify publications that contain semi-logarithmic graphs showing radiosensitivity data derived from clonogenic assays. C2 uses fRCNN-IRv2 and OCR to identify publications that contain bar graphs showing radiosensitivity data derived from clonogenic assays. C3 is a program that identifies publications containing keywords related to radiosensitivity data derived from clonogenic assays. A program (iSF2) was developed using Mask RCNN and OCR to extract surviving fraction after 2-Gy irradiation (SF2) as assessed by clonogenic assays, presented in semi-logarithmic graphs. The efficacy of C1-3 and iSF2 was tested using seven datasets (1805 and 222 publications in total, respectively). RESULTS: C1-3 yielded sensitivity of 91.2% ±â€¯3.4% and specificity of 90.7% ±â€¯3.6%. iSF2 returned SF2 values that were within 2.9% ±â€¯2.6% of the SF2 values determined by radiation oncologists. CONCLUSION: Our analysis pipeline is potentially useful to acquire radiosensitivity data derived from clonogenic assays from the literature.

Robustness of Clonogenic Assays as a Biomarker for Cancer Cell Radiosensitivity.

Photon radiation therapy is a major curative treatment for cancer. However, the lack of robust predictive biomarkers for radiosensitivity precludes personalized radiation therapy. Clonogenic assays are the gold standard method for measuring the radiosensitivity of cancer cells. Although a large number of publications describe the use of clonogenic assays to measure cancer cell radiosensitivity, the robustness of results from different studies is unclear. To address this, we conducted a comprehensive detailed literature search of 256 common cancer cell lines and identified the eight cell lines most-frequently examined for photon sensitivity using clonogenic assays. Survival endpoints and experimental parameters from all 620 relevant experiments were compiled and analyzed. We found that the coefficients of variation for SF2 (surviving fraction after 2 Gy irradiation) and for D10 (dose that yields a surviving fraction of 10%) were below 30% for all cell lines, indicating that SF2 and D10 have acceptable inter-assay precision. These data support further analysis of published data on clonogenic assays using SF2 and D10 as survival endpoints, which facilitates robust identification of biological profiles representative of cancer cell sensitivity to photons.

Dose-volume parameters and local tumor control in cervical cancer treated with central-shielding external-beam radiotherapy and CT-based image-guided brachytherapy.

Definitive radiotherapy for cervical cancer consists of external-beam radiotherapy (EBRT) and brachytherapy. In EBRT, a central shield (CS) reduces the dose to the rectum and bladder. The combination of whole-pelvic (WP)- and CS-EBRT and brachytherapy is the standard radiotherapy protocol in Japan. Despite clinical studies, including multi-institutional clinical trials, showing that the Japanese treatment protocol yields favorable treatment outcomes with low rates of late radiation toxicities, dose-volume parameters for the Japanese treatment protocol remain to be established. We conducted a retrospective dose-volume analysis of 103 patients with uterine cervical cancer treated with the Japanese protocol using computed tomography-based adaptive brachytherapy. The 2-year overall survival and 2-year local control rates according to FIGO stage were 100% and 100% for Stage I, 92% and 94% for Stage II, and 85% and 87% for Stage III-IV, respectively. Late adverse effects in the rectum and bladder were acceptable. Receiver operating characteristic analysis discriminated recurrence within the high-risk clinical target volume (HR-CTV) (n = 5) from no local recurrence (n = 96), with the optimal response obtained at a dose of 36.0 GyEQD2 for HR-CTV D90 and 28.0 GyEQD2 for HR-CTV D98. These values were used as cut-offs in Fisher exact tests to show that high HR-CTV D90 and HR-CTV D98 doses for brachytherapy sessions were significantly associated with tumor control within the HR-CTV. These data suggest a contribution of brachytherapy to local tumor control in WP- and CS-EBRT and brachytherapy combination treatment, warranting validation in multi-institutional prospective studies.

Mutational analysis of uterine cervical cancer that survived multiple rounds of radiotherapy.

Radiotherapy is an essential component of cancer therapy. Despite advances in cancer genomics, the mutation signatures of radioresistant tumors have not yet been fully elucidated. To address this issue, we analyzed a unique set of clinical specimens from a uterine cervical cancer that repeatedly locally recurred after multiple rounds of radiotherapy. Exon sequencing of 409 cancer-related genes in the treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy identified (i) activating mutations in PIK3CA and KRAS, and putative inactivating mutations in SMAD4, as trunk mutation signatures that persisted over the clinical course; and (ii) mutations in KMT2A, TET1, and NLRP1 as acquired mutation signatures observed only in recurrent tumors after radiotherapy. Comprehensive mining of published in vitro genomics data pertaining to radiosensitivity revealed that simultaneous mutations in KRAS and SMAD4, which have not been described previously in uterine cervical cancer, are associated with cancer cell radioresistance. The association between this mutation signature and radioresistance was validated by isogenic cell-based experiments. These results provide proof-of-principle for the analytical pipeline employed in this study, which explores clinically relevant mutation signatures for radioresistance, and demonstrate that this approach is worth pursuing with larger cohorts in the future.

Inter-assay precision of clonogenic assays for radiosensitivity in cancer cell line A549.

Clonogenic assays are the gold standard for determining radiosensitivity, which governs tumor response to radiation therapy. Although multiple studies of clonogenic assays on cancer cell lines have been published, the robustness of this technique has not been examined by comparative analysis of data from different studies. To address this issue, we investigated the inter-assay precision of clonogenic assays by analyzing in-house and published data on A549, a cell line frequently studied in this context. The coefficients of variation for SF2, the surviving fraction after 2 Gy irradiation, and D10, the radiation dose that reduces survival to 10%, were below 30% for both in-house data obtained from 20 independent experiments performed under consistent experimental settings (i.e., radiation type, dose rate, and timing of cell seeding) and data collected from 192 publications using diverse experimental settings. Multivariate analyses of the published data revealed that timing of cell seeding significantly affected SF2. These data indicate that SF2 and D10 of clonogenic assay have acceptable inter-assay precision, and that timing of cell seeding influences the inter-assay precision of SF2. These results provide a rationale for combined analysis of published clonogenic assay data, which may help to discover robust biological properties associated with tumor radiosensitivity.