Uncertainty estimation using a 3D probabilistic U-Net for segmentation with small radiotherapy clinical trial datasets.

BACKGROUND AND OBJECTIVES: Bio-medical image segmentation models typically attempt to predict one segmentation that resembles a ground-truth structure as closely as possible. However, as medical images are not perfect representations of anatomy, obtaining this ground truth is not possible. A surrogate commonly used is to have multiple expert observers define the same structure for a dataset. When multiple observers define the same structure on the same image there can be significant differences depending on the structure, image quality/modality and the region being defined. It is often desirable to estimate this type of aleatoric uncertainty in a segmentation model to help understand the region in which the true structure is likely to be positioned. Furthermore, obtaining these datasets is resource intensive so training such models using limited data may be required. With a small dataset size, differing patient anatomy is likely not well represented causing epistemic uncertainty which should also be estimated so it can be determined for which cases the model is effective or not. METHODS: We use a 3D probabilistic U-Net to train a model from which several segmentations can be sampled to estimate the range of uncertainty seen between multiple observers. To ensure that regions where observers disagree most are emphasised in model training, we expand the Generalised Evidence Lower Bound (ELBO) with a Constrained Optimisation (GECO) loss function with an additional contour loss term to give attention to this region. Ensemble and Monte-Carlo dropout (MCDO) uncertainty quantification methods are used during inference to estimate model confidence on an unseen case. We apply our methodology to two radiotherapy clinical trial datasets, a gastric cancer trial (TOPGEAR, TROG 08.08) and a post-prostatectomy prostate cancer trial (RAVES, TROG 08.03). Each dataset contains only 10 cases each for model development to segment the clinical target volume (CTV) which was defined by multiple observers on each case. An additional 50 cases are available as a hold-out dataset for each trial which had only one observer define the CTV structure on each case. Up to 50 samples were generated using the probabilistic model for each case in the hold-out dataset. To assess performance, each manually defined structure was matched to the closest matching sampled segmentation based on commonly used metrics. RESULTS: The TOPGEAR CTV model achieved a Dice Similarity Coefficient (DSC) and Surface DSC (sDSC) of 0.7 and 0.43 respectively with the RAVES model achieving 0.75 and 0.71 respectively. Segmentation quality across cases in the hold-out datasets was variable however both the ensemble and MCDO uncertainty estimation approaches were able to accurately estimate model confidence with a p-value < 0.001 for both TOPGEAR and RAVES when comparing the DSC using the Pearson correlation coefficient. CONCLUSIONS: We demonstrated that training auto-segmentation models which can estimate aleatoric and epistemic uncertainty using limited datasets is possible. Having the model estimate prediction confidence is important to understand for which unseen cases a model is likely to be useful.

Partially Ablative Body Radiotherapy (PABR): A novel approach for palliative radiotherapy of locally advanced bulky unresectable sarcomas.

BACKGROUND: Locally advanced, bulky, unresectable sarcomas cause significant tumour mass effects, leading to burdensome symptoms. We have developed a novel Partially Ablative Body Radiotherapy (PABR) technique that delivers a high, ablative dose to the tumour core and a low, palliative dose to its periphery aiming to increase overall tumour response without significantly increasing treatment toxicity. AIM: This study aims to report the safety and oncologic outcomes of PABR in patients with bulky, unresectable sarcomas. METHODS AND MATERIALS: A total of 18 patients with histologically proven sarcoma treated with PABR from January 2020 to October 2023 were retrospectively reviewed. The primary endpoints were symptomatic and structural response rates. Secondary endpoints were overall survival, freedom from local progression, freedom from distant progression, and acute and late toxicity rates. RESULTS: All patients had tumours ≥5 cm with a median tumour volume of 985 cc, and the most common symptom was pain. The median age is 72.5 years and 44.5 % were ECOG 2-3. The most common regimen used was 20 Gy in 5 fractions with an intratumoral boost dose of 50 Gy (83.3 %). After a median follow-up of 11 months, 88.9 % of patients exhibited a partial response with a mean absolute tumour volume reduction of 49.5 %. All symptomatic patients experienced symptom improvement. One-year OS, FFLP and FFDP were 61 %, 83.3 % and 34.8 %, respectively. There were no grade 3 or higher toxicities. CONCLUSION: PABR for bulky, unresectable sarcomas appears to be safe and may provide good symptomatic response, tumour debulking, and local control. Further study is underway.

Selection of motion management in liver stereotactic body radiotherapy and its impact on treatment time.

Background and purpose: Reduction of respiratory tumour motion is important in liver stereotactic body radiation therapy (SBRT) to reduce side effects and improve tumour control probability. We have assessed the distribution of use of voluntary exhale breath hold (EBH), abdominal compression (AC), free breathing gating (gating) and free breathing (FB), and the impact of these on treatment time. Materials and Methods: We assessed all patients treated in a single institution with liver SBRT between September 2017 and September 2021. Data from pre-simulation motion management assessment using fluoroscopic assessment of liver dome position in repeat breath holds, and motion with and without AC, was reviewed to determine liver dome position consistency in EBH and the impact of AC on motion. Treatment time was assessed for all fractions as time from first image acquisition to last treatment beam off. Results: Of 136 patients treated with 145 courses of liver SBRT, 68 % were treated in EBH, 20 % with AC, 7 % in gating and 5 % in FB. AC resulted in motion reduction < 1 mm in 9/26 patients assessed. Median treatment time was higher using EBH (39 min) or gating (42 min) compared with AC (30 min) or FB (24 min) treatments. Conclusions: Motion management in liver SBRT needs to be assessed per-patient to ensure appropriate techniques are applied. Motion management significantly impacts treatment time therefore patient comfort must also be taken into account when selecting the technique for each patient.

Heterosubtypic immune pressure accelerates emergence of influenza A virus escape phenotypes in mice.

Rapid antigenic evolution of the influenza A virus surface antigen hemagglutinin undermines protection conferred by seasonal vaccines. Protective correlates targeted by universal vaccines such as cytotoxic T cells or HA stem directed broadly neutralizing antibodies have been shown to select for immune escape mutants during infection. We developed an in vivo serial passage mouse model for viral adaptation and used next generation sequencing to evaluate full genome viral evolution in the context of broadly protective immunity. Heterosubtypic immune pressure increased the incidence of genome-wide single nucleotide variants, though mutations found in early adapted populations were predominantly stochastic in nature. Prolonged adaptation under heterosubtypic immune selection resulted in the manifestation of highly virulent phenotypes that ablated vaccine mediated protection from mortality. High frequency mutations unique to escape phenotypes were identified within the polymerase encoding segments. These findings suggest that a suboptimial usage of population-wide universal influenza vaccine may drive formation of escape variants attributed to polygenic changes.

Atlantoaxial instability secondary to Bartonella henselae osteomyelitis managed surgically by atlantoaxial instrumentation: A case report and systematic review.

Cat scratch disease (CSD), caused by Bartonella henselae, may atypically present with vertebral osteomyelitis. Antibiotic regimens are tailored to presentation, which is markedly variable and not well defined for any atypical disease. In cases of spinal instability, the use of antibiotics alone may not be sufficient. Atlantoaxial instability caused by osteomyelitis is a rare complication of CSD. In this report, we describe the rare case of vertebral osteomyelitis complicated by atlantoaxial instability, requiring both antibiotics and atlantoaxial fusion. We discuss our case, surgical technique, rationale, and outcome. In addition, we conducted a systematic review of the literature of vertebral osteomyelitis in pediatric secondary to B. henselae. A 2-year-old child presented with a 2-month history of irritability, fever, and rigid neck pain along with a recent history of feline exposure. Physical examination revealed cervical tenderness and decreased range of motion. Computed tomography (CT) showed osteolysis of the right C1 lateral mass and pars articularis; T1-weighted magnetic resonance imaging with contrast showed enhancement around the right C1 lateral mass. The titer for B. henselae was high. A diagnosis of cat scratch osteomyelitis with cervical instability was made, for which the patient underwent surgery with atlantoaxial fusion. Postoperative imaging demonstrated resolution of the contrast-enhanced lesion. At 6-year follow-up, the patient showed no signs of residual complications from surgical intervention with a solid fusion. Our review revealed 44 cases of pediatric CSD vertebral osteomyelitis. Conservative management with antibiotic employed in 86% while antibiotics with surgical intervention in 14% of the cases. Surgical intervention was most often in the form of incision for drainage and decompression without fusion. Average follow-up 10 months with 86% achieved complete resolution. Cervical instability caused by osteolysis is a rare complication of CSD. This can subsequently lead to vertebral instability, requiring definitive surgical intervention.

Impact of Medical Operability and Total Metastatic Ablation on Outcomes After SABR for Oligometastases.

PURPOSE: Medical operability is prognostic for survival after SABR in primary malignancies. This study investigated the prognostic significance of medical operability and total versus subtotal ablation of all oligometastatic disease sites. METHODS AND MATERIALS: Consecutive patients with 1 to 5 sites of active extracranial oligometastases had medical operability status and presence of subtotal versus total metastatic ablation recorded prospectively in an institutional database. We retrospectively compared overall survival (OS) and progression-free survival (PFS) for medically operable or inoperable patients and patients undergoing total or subtotal metastatic ablation. Secondary endpoints were patterns of failure, high-grade treatment toxic effects (Common Terminology Criteria for Adverse Events version 4.0), and freedom from systemic therapy. The threshold dose per fraction considered ablative was 8 Gy. RESULTS: A total of 401 patients with 530 treated oligometastases were included, with a median follow-up of 3 years. Three hundred and two and 99 patients had metachronous and synchronous presentations of oligometastatic disease, respectively. Common histologies included prostate (24%), lung (18%), gastrointestinal (19%), and breast (11%). More than 90% of doses delivered were Biologically Effective Dose [BED10]≥60 Gy. Cumulative incidence at 5 years of local-only failure was 6%, local and distant 2%, and distant-only 58%. The 3- and 5-year OS [95% confidence intervals {CIs}] were 68% [62-73] and 54% [47-61], and PFS was 20% [15-25] and 14% [10-20]. The 3- and 5-year freedom from systemic therapy [95% CIs] were 40% [34-46] and 31% [24-37], respectively. Seventy-six patients were inoperable and 325 were operable. Operability status was not prognostic for OS (adjusted hazard ratio [HR], 1.0; 95% CI, 0.6-1.7; P = .9) or for PFS (adjusted HR, 1.1; 95% CI, 0.8-1.6; P = .5). Total metastatic ablation was prognostic for OS (adjusted HR, 0.8; 95% CI, 0.4-0.9; P = .032) and for PFS (adjusted HR, 0.6; 95% CI, 0.4-0.8; P = .003). CONCLUSIONS: Medical operability was not prognostic in patients with oligometastatic disease treated with SABR. Total metastatic ablation was associated with superior OS and PFS compared with subtotal metastatic ablation. Our data support ablation of all sites of oligometastases wherever feasible.

Long-term Patterns of Failure and the Value of Blood Prognostic Markers in Anal Cancers Treated With Intensity-Modulated Radiation Therapy.

BACKGROUND: To analyze the long-term outcomes and prognostic value of hematological parameters in anal cancer patients receiving intensity-modulated radiation therapy (IMRT). MATERIALS: Hospital records of consecutive patients with anal squamous cell carcinoma who received curative-intent IMRT according to a standardized contouring protocol between 2010 and 2020 were reviewed. Locoregional failure-free survival (LRFS), distant metastasis-free survival (DMFS), progression-free survival (PFS), and overall survival (OS) were estimated using the Kaplan-Meier method. Coverage of locoregional recurrences by the initial IMRT volumes were assessed. The prognostic value of pretreatment blood counts for PFS and OS were determined using Cox regression analysis. RESULTS: A total of 166 patients were analyzed with a median follow-up of 3.3 years. Forty-six percent and 54% of patients had Stage I-II and IIIA-B cancers, respectively. The 5-year LRFS, DMFS, PFS and OS were 81%, 89%, 65% and 76% respectively. Grade ≥ 3 toxicity occurred in 5% of patients. Of all patients who relapsed, 70% had only locoregional recurrence as first site of failure. Ninety percent of locoregional recurrences were in-field. Hemoglobin, neutrophil and platelet counts were associated with PFS on univariable analysis, but only cancer stage and p16 status remained prognostic on multivariable analysis. Patients with more advanced cancer stages also had higher baseline neutrophil counts. Performance status and neutrophil counts were prognostic for OS on multivariable analysis. CONCLUSION: This study affirms the long-term efficacy and safety of IMRT. Treatment resistance, rather than radiation geographic miss, is a major issue underpinning locoregional recurrences. Pretreatment blood counts were not validated to be independently prognostic for disease recurrence.

Complete ophthalmoplegia secondary to idiopathic intracranial hypertension managed successfully with dural sinus stenting: A case and systematic review.

Idiopathic Intracranial Hypertension (IIH) typically occurs in obese (BMI >30 kg/m2) females of childbearing age in the absence of any apparent intracranial space-occupying lesion. Patients typically present with headache, nausea, vomiting, tinnitus, and blurry vision secondary to increased intracranial pressure, with more severe cases involving cranial neuropathies and ophthalmological manifestations. Complete ophthalmoplegia is a rare event in IIH. In such cases, aggressive management with pharmacological, endovascular, and surgical intervention is essential to hasten recovery and limit long-term neurological and visual deficits. Herein, we present a rare case of a patient with IIH associated with third, fourth, and sixth cranial nerve palsies, resulting in complete unilateral ophthalmoplegia, who underwent dural sinus stenting and 2.5-year follow-up revealed complete resolution with full extraocular movements. We also perform a systematic literature review of complete and partial ophthalmoplegia secondary to IIH, highlighting the associated presentations, pathophysiology, management, and outcomes.

AGITG MASTERPLAN: a randomised phase II study of modified FOLFIRINOX alone or in combination with stereotactic body radiotherapy for patients with high-risk and locally advanced pancreatic cancer.

BACKGROUND: Among patients with non-metastatic pancreatic cancer, 80% have high-risk, borderline resectable or locally advanced cancer, with a 5-year overall survival of 12%. MASTERPLAN evaluates the safety and activity of stereotactic body radiotherapy (SBRT) in addition to chemotherapy in these patients. METHODS AND DESIGN: MASTERPLAN is a multi-centre randomised phase II trial of 120 patients with histologically confirmed potentially operable pancreatic cancer (POPC) or inoperable pancreatic cancer (IPC). POPC includes patients with borderline resectable or high-risk tumours; IPC is defined as locally advanced or medically inoperable pancreatic cancer. Randomisation is 2:1 to chemotherapy + SBRT (investigational arm) or chemotherapy alone (control arm) by minimisation and stratified by patient cohort (POPC v IPC), planned induction chemotherapy and institution. Chemotherapy can have been commenced ≤28 days prior to randomisation. Both arms receive 6 × 2 weekly cycles of modified FOLFIRINOX (oxaliplatin (85 mg/m2 IV), irinotecan (150 mg/m2), 5-fluorouracil (2400 mg/m2 CIV), leucovorin (50 mg IV bolus)) plus SBRT in the investigational arm. Gemcitabine+nab-paclitaxel is permitted for patients unsuitable for mFOLFIRINOX. SBRT is 40Gy in five fractions with planning quality assurance to occur in real time. Following initial chemotherapy ± SBRT, resectability will be evaluated. For resected patients, adjuvant chemotherapy is six cycles of mFOLFIRINOX. Where gemcitabine+nab-paclitaxel was used initially, the adjuvant treatment is 12 weeks of gemcitabine and capecitabine or mFOLFIRINOX. Unresectable or medically inoperable patients with stable/responding disease will continue with a further six cycles of mFOLFIRINOX or three cycles of gemcitabine+nab-paclitaxel, whatever was used initially. The primary endpoint is 12-month locoregional control. Secondary endpoints are safety, surgical morbidity and mortality, radiological response rates, progression-free survival, pathological response rates, surgical resection rates, R0 resection rate, quality of life, deterioration-free survival and overall survival. Tertiary/correlative objectives are radiological measures of nutrition and sarcopenia, and serial tissue, blood and microbiome samples to be assessed for associations between clinical endpoints and potential predictive/prognostic biomarkers. Interim analysis will review rates of locoregional recurrence, distant failure and death after 40 patients complete 12 months follow-up. Fifteen Australian and New Zealand sites will recruit over a 4-year period, with minimum follow-up period of 12 months. DISCUSSION: MASTERPLAN evaluates SBRT in both resectable and unresectable patients with pancreatic ductal adenocarcinoma. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry ACTRN12619000409178 , 13/03/2019. Protocol version: 2.0, 19 May 2019.

A novel mechanism of enhanced transcription activity and fidelity for influenza A viral RNA-dependent RNA polymerase.

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.

Safety, Tolerability, and Population Pharmacokinetics of Intravenous and Oral Isavuconazonium Sulfate in Pediatric Patients.

Isavuconazole, administered as the water-soluble prodrug isavuconazonium sulfate, is a new triazole agent used to treat invasive fungal infections. This phase 1 study evaluated the pharmacokinetics (PK), safety, and tolerability of isavuconazole in 46 immunocompromised pediatric patients, stratified by age (1 to <6 [intravenous (i.v.) only], 6 to <12, and 12 to <18 years), receiving 10 mg/kg body weight (maximum, 372 mg) isavuconazonium sulfate either i.v. or orally. A population PK model using weight-based allometric scaling was constructed with the pediatric i.v. and oral data plus i.v. data from a phase 1 study in adults. The best model was a 3-compartment model with combined zero-order and first-order input, with linear elimination. Stepwise covariate modeling was performed in Perl-speaks-NONMEM version 4.7.0. None of the covariates examined, including age, sex, race, and body mass index, were statistically significant for any of the PK parameters. The area under the concentration-time curve at steady state (AUCSS) was predicted for pediatric patients using 1,000 Monte Carlo simulations per age cohort for each administration route. The probability of target attainment (AUCSS range, 60 to 233 µg · h/ml) was estimated; this target range was derived from plasma drug exposures in adults receiving the recommended clinical dose. Predicted plasma drug exposures were within the target range for >80% and >76% of simulated pediatric patients following i.v. or oral administration, respectively. Intravenous and oral administration of isavuconazonium sulfate at the studied dosage of 10 mg/kg was well tolerated and resulted in exposure in pediatric patients similar to that in adults. (This study has been registered at ClinicalTrials.gov under identifier NCT03241550).

Study protocol of the LARK (TROG 17.03) clinical trial: a phase II trial investigating the dosimetric impact of Liver Ablative Radiotherapy using Kilovoltage intrafraction monitoring.

BACKGROUND: Stereotactic Ablative Body Radiotherapy (SABR) is a non-invasive treatment which allows delivery of an ablative radiation dose with high accuracy and precision. SABR is an established treatment for both primary and secondary liver malignancies, and technological advances have improved its efficacy and safety. Respiratory motion management to reduce tumour motion and image guidance to achieve targeting accuracy are crucial elements of liver SABR. This phase II multi-institutional TROG 17.03 study, Liver Ablative Radiotherapy using Kilovoltage intrafraction monitoring (LARK), aims to investigate and assess the dosimetric impact of the KIM real-time image guidance technology. KIM utilises standard linear accelerator equipment and therefore has the potential to be a widely available real-time image guidance technology for liver SABR. METHODS: Forty-six patients with either hepatocellular carcinoma or oligometastatic disease to the liver suitable for and treated with SABR using Kilovoltage Intrafraction Monitoring (KIM) guidance will be included in the study. The dosimetric impact will be assessed by quantifying accumulated patient dose distribution with or without the KIM intervention. The patient treatment outcomes of local control, toxicity and quality of life will be measured. DISCUSSION: Liver SABR is a highly effective treatment, but precise dose delivery is challenging due to organ motion. Currently, there is a lack of widely available options for performing real-time tumour localisation to assist with accurate delivery of liver SABR. This study will provide an assessment of the impact of KIM as a potential solution for real-time image guidance in liver SABR. TRIAL REGISTRATION: This trial was registered on December 7th 2016 on ClinicalTrials.gov under the trial-ID NCT02984566 .

Stereotactic body radiotherapy in the management of hepatocellular carcinoma: An Australian multi-institutional patterns of practice review.

INTRODUCTION: Stereotactic body radiotherapy (SBRT) is an emerging, therapeutic option in the management of hepatocellular carcinoma (HCC). A multicentre Liver Ablative Stereotactic Radiation (LASR) database was established to provide a collaborative platform for Australian institutions to define the practice of liver SBRT for HCC. This study explores the patterns of SBRT practice amongst Australian institutions. METHODS: This was a multi-institutional retrospective study of patients treated with SBRT for HCC at 10 institutions between January 2013 and December 2019. Patients' demographics, disease characteristics and SBRT details were evaluated. RESULTS: Three hundred and seventeen patients were evaluated with a median age of 67 years (range, 32-90). Liver cirrhosis was present in 88.6%, baseline Child-Pugh score was A5/6 in 85.1% and B7/8 in 13.2%. Median size of HCC treated was 30 mm (range, 10-280). 63.1% had early-stage disease (Barcelona clinic liver cancer (BCLC) stage 0/A) and 36% had intermediate/advanced-stage disease (BCLC B/C). In 2013/2014, six courses of SBRT were delivered, increasing to 108 in 2019. SBRT was prescribed in five fractions for 71.3% of the cohort. The most common dose fractionation schedule was 40 Gy in five fractions (24.3%). Median biologically effective dose (BED10 ) delivered was 85.5 Gy for early-stage and 60 Gy for intermediate/advanced disease, respectively. The most common prescription range was 100-120 Gy BED10 (32.8%). CONCLUSION: SBRT utilisation for HCC is increasing in Australia. There was wide variation in size of tumours and disease stages treated, and prescription patterns. Uniform reporting of clinical and dosimetric details are important in refining the role of liver SBRT.

Results of phase II trial of intensified neoadjuvant treatment with interdigitating radiotherapy and chemotherapy with oxaliplatin, 5-fluorouracil and folinic acid in patients with locally advanced rectal cancer (PROARCT trial).

BACKGROUND AND PURPOSE: The chemotherapy exposure during chemoradiotherapy for rectal cancer is adequate for radiosensitization but suboptimal for treatment of distant micrometastasis. This study aimed to determine tolerability, dose intensity, response, and toxicity of a novel intensified neoadjuvant treatment approach. MATERIALS AND METHODS: Eligible patients were MRI-staged T3-4NxM0 rectal adenocarcinoma. Treatment consisted of FOLFOX chemotherapy given in weeks 1, 6, and 11 with pelvic radiotherapy (25.2 Gy in 3 weeks in 1.8 Gy/fraction with oxaliplatin and 5-FU continuous infusion) given in weeks 3-5, and weeks 8-10. Surgery was performed 4-6 weeks later. The primary endpoint was tolerability defined as the percentage of patients who were able to complete the planned treatment course. Survival rates were estimated using the Kaplan-Meier method. RESULTS: Median age of the 40 patients was 61.5 years. Rectal MRI-stage was T3 in 88%. Overall, 95% completed the regimen. All patients received 50.4 Gy. Relative dose intensity (≥75%) was 92% and 98% for oxaliplatin and 5-FU, respectively. High grade toxicities included neutropenia (25% grade 3; 7.5% grade 4) and diarrhoea (10%). Pathologic CR rate was 20%. Median follow-up was 54 months. The 5-year overall survival, freedom from relapse, locoregional control, and freedom from distant metastasis of the cohort was 82%, 72%, 97% and 72%. CONCLUSIONS: Delivery of intensified neoadjuvant treatment with interdigitating chemotherapy and radiotherapy is feasible with no increase in acute perioperative complications. A larger prospective study is required to further evaluate the potential survival benefit of this design.

Characterising the immune microenvironment in liposarcoma, its impact on prognosis and the impact of radiotherapy.

BACKGROUND AND OBJECTIVES: Limited literature exists examining the immune microenvironment in liposarcoma, particularly with regard to the impact of radiotherapy. A major problem is the lack of scoring system for the tumour-infiltrating lymphocytes (TILs) in sarcoma. This study aims to describe the immune environment pre- and postradiotherapy and identify the optimal immune infiltrate scoring system for sarcoma. METHODS: Thirty-nine paired tissue samples (pre- and postradiotherapy) from patients with liposarcoma were scored by two pathologists for TILs using pre-existing systems (for breast cancer and melanoma) and compared for interobserver reliability. Immunohistochemical staining was performed for various immune markers. RESULTS: The TIL scoring system for breast cancer yielded perfect agreement (κ = 1.000). 21% of patients had increased TILs after radiotherapy, 87.5% of whom had dedifferentiated liposarcoma. Immune suppressor expression was increased frequently after radiotherapy (CD68 increased in 59.4%, PD-L1 increased in 25%). Immune effector expression (CD8) was unchanged in 84.4%. CONCLUSIONS: Breast cancer TIL scoring is reproducible in liposarcoma and has high interobserver reliability. Radiotherapy was observed to have a limited impact on immune effectors but seemed to have more impact in upregulating immune suppressors, suggesting radiotherapy may contribute to disease control through immunomodulatory effects. Dedifferentiated liposarcoma represents a uniquely responsive subtype.

Safety, Efficacy, and Patterns of Failure After Single-Fraction Stereotactic Body Radiation Therapy (SBRT) for Oligometastases.

PURPOSE: Fewer attendances for radiation therapy results in increased efficiency and less foot traffic within a radiation therapy department. We investigated outcomes after single-fraction (SF) stereotactic body radiation therapy (SBRT) in patients with oligometastatic disease. METHODS AND MATERIALS: Between February 2010 and June 2019, patients who received SF SBRT to 1 to 5 sites of oligometastatic disease were included in this retrospective study. The primary objective was to describe patterns of first failure after SBRT. Secondary objectives included overall survival (OS), progression-free survival (PFS), high-grade treatment-related toxicity (Common Terminology Criteria for Adverse Events grade ≥3), and freedom from systemic therapy (FFST). RESULTS: In total, 371 patients with 494 extracranial oligometastases received SF SBRT ranging from 16 Gy to 28 Gy. The most common primary malignancies were prostate (n = 107), lung (n = 63), kidney (n = 52), gastrointestinal (n = 51), and breast cancers (n = 42). The median follow-up was 3.1 years. The 1-, 3-, and 5-year OS was 93%, 69%, and 55%, respectively; PFS was 48%, 19%, and 14%, respectively; and FFST was 70%, 43%, and 35%, respectively. Twelve patients (3%) developed grade 3 to 4 treatment-related toxicity, with no grade 5 toxicity. As the first site of failure, the cumulative incidence of local failure (irrespective of other failures) at 1, 3 and 5 years was 4%, 8%, and 8%, respectively; locoregional relapse at the primary was 10%, 18%, and 18%, respectively; and distant failure was 45%, 66%, and 70%, respectively. CONCLUSIONS: SF SBRT is safe and effective, and a significant proportion of patients remain FFST for several years after therapy. This approach could be considered in resource-constrained or bundled-payment environments. Locoregional failure of the primary site is the second most common pattern of failure, suggesting a role for optimization of primary control during metastasis-directed therapy.

A six-plex droplet digital RT-PCR assay for seasonal influenza virus typing, subtyping, and lineage determination.

BACKGROUND: There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co-circulate in humans. In this study, we report the development of a six-plex droplet digital RT-PCR (ddRT-PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT-PCR platform. METHODS: The six-plex ddRT-PCR used in this study is an amplitude-based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT-PCR methodology was assessed using 55 clinical samples. RESULTS: The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT-PCR assays. CONCLUSIONS: The six-plex ddRT-PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use.

Australasian Gastrointestinal Trials Group (AGITG) and Trans-Tasman Radiation Oncology Group (TROG) Guidelines for Pancreatic Stereotactic Body Radiation Therapy (SBRT).

PURPOSE: Nonrandomized data exploring pancreas stereotactic body radiation therapy (SBRT) has demonstrated excellent local control rates and low toxicity. Before commencing a randomized trial investigating pancreas SBRT, standardization of prescription dose, dose constraints, simulation technique, and clinical target volume delineation are required. METHODS AND MATERIALS: Specialists in radiation oncology, medical oncology, hepatobiliary surgery, and gastroenterology attended 2 consecutive Australasian Gastrointestinal Trials Group workshops in 2017 and 2018. Sample cases were discussed during workshop contact with specifically invited international speakers highly experienced in pancreas SBRT. Furthermore, sample cases were contoured and planned between workshop contact to finalize dose constraints and clinical target volume delineation. RESULTS: Over 2 separate workshops, consensus was reached on dose and simulation technique. The working group recommended a dose prescription of 40 Gy in 5 fractions. Treatment delivery during end-expiratory breath hold with triple-phase contrast enhanced computed tomography was recommended. In addition, dose constraints, stepwise contouring guidelines, and an anatomic atlas for pancreatic SBRT were developed. CONCLUSIONS: Pancreas SBRT is emerging as a promising treatment modality requiring prospective evaluation in randomized studies. This work attempts to standardize dose, simulation technique, and volume delineation to support the delivery of high quality SBRT in a multicenter study.