Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma.

ABSTRACT: Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in the bone marrow or blood (BCP acute lymphoblastic leukemia [BCP-ALL]) or are less common in extramedullary tissue (BCP lymphoblastic lymphoma [BCP-LBL]). Although both presentations are similar in morphology and immunophenotype, molecular studies have been virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and restriction on small, mostly formalin-fixed paraffin-embedded (FFPE) tissues. Here, to our knowledge, we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n = 97). Whole-exome sequencing indicated a mutational spectrum of BCP-LBL, strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations, and gene expression by RNA sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even in FFPE tissue, and may be relevant to guide treatment.

The Impact of a Creativity Camp Intervention on Depression and Well-Being in Adolescents.

Depression is a serious public health problem that often emerges during adolescence. Many adolescents do not respond to standard treatments, necessitating the development of novel interventions. We conducted a preliminary study to assess the impact of a novel creative arts intervention on depression and well-being in adolescents. In this quasi-experimental study, 69 adolescents 12-17 years completed an 8-day "Creativity Camp" intervention encompassing multiple creative activities. Self-report questionnaires to measure depression, anxiety, and well-being were administered at five time points to allow examination of change across multiple temporal segments: the 2 weeks before the intervention, the 2 weeks during the intervention, the 2 weeks after the intervention, and the 6 months after the intervention. Adolescents were assigned to one of two groups to compare changes observed during the 2 week before-intervention period (Group A) to the changes observed during the intervention period (pre-post intervention) (Group B). According to both parent and child reports, pre-to-post-intervention, mean adolescent depressive symptoms significantly decreased, and these changes were sustained 2 weeks after the intervention and (according to parents) at the 6-month follow-up. There was preliminary evidence for improved well-being at post-intervention. Comparison of Group A's changes during the before-intervention period to Group B's changes pre-post intervention was significant for parent-reported depression. Preliminary evidence suggests that the Creativity Camp intervention may positively impact adolescent depression and well-being. This evidence highlights the importance of investigating and implementing treatment approaches focusing on creative arts for adolescents with depression.

18 F-FDG-PET/CT imaging in diagnostic workup of pediatric precursor B-cell lymphoblastic lymphoma.

18 F-fluorodeoxyglucose-positron emission tomography/computed tomography (PET/CT) imaging is currently not used in standard diagnostics for B-cell precursor lymphoblastic lymphoma (BCP-LBL), and it is unknown whether PET/CT imaging would lead to agreement between detection of lesions with the gold standard imaging methods. Therefore, we performed a retrospective cohort study in which we included 32 pediatric BCP-LBL patients and determined localizations by reviewing local imaging reports. There was a disagreement between protocol-based imaging and PET/CT in 59% of the patients, and the discrepancies mostly comprise of additional lesions detected with PET/CT, typically in lymph node and bone or the absence of bone marrow involvement with PET/CT. If PET/CT was leading in determining definite stage of disease, this would lead to a different stage and therapy branch in 31% and 28% of the patients, respectively.

Association between bacterial resistance profile and the development of intra-abdominal abscesses in pediatric patients with perforated appendicitis: cohort study.

PURPOSE: The objective of this study was to determine the association between the presence of a microorganism resistant to the antibiotic used in empirical therapy and the development of intra-abdominal abscesses in children with perforated appendicitis. METHODS: A prospective cohort study was conducted in patients under 18 years of age who underwent laparoscopic appendectomy between November 1, 2019, and September 30, 2020, in whom perforated appendicitis was documented intraoperatively. Peritoneal fluid samples were taken for bacteria culture purposes, and clinical and microbiological data were collected from all patients. RESULTS: A total of 232 patients were included in the study. The most isolated microorganisms were Escherichia coli (80.14%) and Pseudomonas aeruginosa (7.45%). In addition, 5.31% of E. coli isolates were classified as ESBL-producing organisms. No association was found between a germ resistant to empiric antimicrobial therapy and the development of a postoperative intra-abdominal abscess. Multivariate analysis showed that being a high-risk patient on admission (OR 2.89 (p = 0.01)) was associated with the development of intra-abdominal abscesses postoperatively. CONCLUSION: E. coli was the most commonly isolated microorganism, with a low rate of ESBL-producing isolates. No association between resistance and risk of postoperative intra-abdominal abscess was found. However, it was identified that being a high-risk patient on admission was associated with this complication. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: Level I.

The PxIxIT motif of the RCAN3 inhibits angiogenesis and tumor progression in Triple Negative breast cancer in immunocompetent mice.

RCAN proteins are endogenous regulators of the calcineurin-cytosolic nuclear factor of activated T cells (CN-NFATc) pathway that bind CN through similar conserved motifs PxIxIT and LxVP of the NFATc family. RCAN1 and RCAN3 protein levels were reported to correlate with overall survival of breast cancer patients. We additionally provided supporting results about RCAN3 role on cancer showing that overexpression of the native PxIxIT sequence of RCAN3-derived R3 peptide (PSVVVH, EGFP-R3178-210) dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic mouse model of Triple Negative Breast Cancer (TNBC) in nude mice. On the other hand, RCAN3 protein and its derived peptide EGFP-R3178-210 bind to CN and inhibit NFAT-mediated cytokine gene expression without affecting CN phosphatase activity suggesting that RCAN3 and EGFP-R3178-210 peptide have tumor suppressor and immunosuppressant activity. Due to the known relationship between tumor development and immune system, as well as the relevance of CN-NFATc in the regulation of the immune system, in the present study we decided to assess the effect of EGFP-R3178-210 peptide in an orthotopic syngeneic TNBC mouse model, in order to ensure that the role of RCAN3 as immunosuppressant do not override its tumor suppressor activity. Our results evidence that EGFP-R3178-210 peptide displays an inhibitory potential on tumor growth and tumor angiogenesis similar to those obtained in the previous orthotopic TNBC model. These results highlight the importance of the RCAN3 peptide as a tumor suppressor protein and totally complement our previous results, indicating that this antitumor activity role is maintained in the presence of a complete functional immune system.

Is Dose Volume a Better Predictor of Osteoradionecrosis Risk Than Total Dose for Patients Who Have Received Head and Neck Radiation?

PURPOSE: Oral and maxillofacial surgeons frequently encounter patients who require extractions following exposure to head and neck radiation, and they must assess the risk of extraction and consider alternatives such as deliberate root retention. The purpose of this study was to determine whether dose volume would be a better predictor for osteoradionecrosis (ORN) than total dose. METHODS: This is a retrospective cohort study of patients diagnosed with ORN following head and neck radiation (administered between January 2006 and December 2018) and a comparison group selected based on site and dosage who did not develop ORN. The predictor variables were total radiation dose and mandibular dose volume, and the outcome variable was ORN occurrence. Covariates included age, sex, cancer stage and site, radiation therapy type, smoking status, alcohol use, adjuvant chemotherapy use, medical comorbidities, and concomitant tumor surgery. Logistic regression models were employed and area under receiver operating characteristic curve (AUROC) and model accuracy (Acc) were used to determine the better predictor. RESULTS: A total of 56 patients were included in the study: 27 ORN positive (ORN+) and 29 matched controls who did not develop ORN (ORN-). Most patients were male (76.8%), considered smokers (78.6%), used alcohol (80.4%), were in stage IV (66.1%), received chemotherapy (75.0%), and received intensity modulated radiation therapy radiation (55.4%). The statistical models with V50 Gy (cc) and V65 Gy (cc) dosage variables exhibited greater predictability of ORN occurrence than total dose (AUROC: 0.90 vs 0.76 and model accuracy: 0.82 vs 0.75, respectively). CONCLUSIONS: The results suggest that following head and neck radiation, dose volume may be a better predictor of ORN risk than total dose. This finding is significant, both for the oral and maxillofacial surgeon who is preoperatively assessing ORN risk following radiation exposure, and for the radiation oncologist striving to minimize the risk associated with their treatment.

Introductory patient communication training for medical physics graduate students: Pilot experience.

Despite medical physics becoming a more patient-facing part of the radiation oncology team, medical physics graduate students have no training in patient communication. An introductory patient communication training for medical physics graduate students is presented here. This training exposes participants to foundational concepts and effective communication skills through a lecture and it allows them to apply these concepts through realistic simulated patient interactions. The training was conducted virtually, and eight students participated. The impact of the training was evaluated based on changes in both confidence and competence of the participants' patient communication skills. Participants were asked to fill out a survey to assess their confidence on communicating with patients before and after the training. They also underwent a simulated patient interaction pre- and postlecture. Their performance during these was evaluated by both the simulated patient actors and the participants themselves using a rubric. Each data set was paired and analyzed for significance using a Wilcoxon rank-sum test with an alpha of 0.05. Participants reported significantly higher confidence in their feeling of preparedness to interact with patients (mean = 2.38 vs. 3.88, p = 0.008), comfort interacting independently (mean = 2.00 vs. 4.00, p = 0.002), comfort showing patients they are actively listening (mean = 3.50 vs. 4.50, p = 0.005), and confidence handling challenging patient interactions (mean = 1.88 vs. 3.38, p = 0.01), after the training. Their encounter scores, as evaluated by the simulated patient actors, significantly increased (mean = 77% vs. 91%, p = 0.022). Self-evaluation scores increased, but not significantly (mean = 62% vs. 68%, p = 0.184). The difference between the simulated patient and self-evaluation scores for the postinstruction encounter was statistically significant (p = 0.0014). This patient communication training for medical physics graduate students is effective at increasing both the confidence and the competence of the participants in the subject. We propose that similar trainings be incorporated into medical physics graduate training programs prior to students entering into residency.

Novel application of ultrasound and microwave-assisted methods for aqueous extraction of coconut oil and proteins.

Alternative methods for wet extraction of coconut oil and protein assisted by ultrasound or microwave were developed and compared. Coconut milk was prepared by milling the pulp (5:1 water to coconut pulp ratio), further destabilised at pH 4 and centrifuged to obtain the cream and cream protein fractions (control process). Microwave-assisted treatment applied in milk (1 min, 3 pulses of 20 s; 2.5 GHz; 4.31 kW/kg by pulse) generated a significant increase in cream obtained, and in the coconut oil extraction yield (~ 20%) compared to its control. The ultrasound-assisted treatment (2.5 min; 24 kHz; 0.573 kW/kg, 6.85 W/cm2) also improved oil extraction (10-16%). Moreover, a higher protein yield was achieved in ultrasound treated samples when compared to their control (49.6-86.1%). Large particles of 11 m µ , probably aggregates of particles, and smaller particles of 3.6 m µ , were detected in coconut milk, which were reduced by ultrasound effect. Alternative treatments caused a greater liberation of total phenols in coconut cream. Coconut proteins in water (0.1%) showed high negative electrokinetic potential. The surface pressure of coconut proteins at the air/water interface was not modified by assisted treatments.

Antiproliferative effect of bacterial cyclodipeptides in the HeLa line of human cervical cancer reveals multiple protein kinase targeting, including mTORC1/C2 complex inhibition in a TSC1/2-dependent manner.

Cervix adenocarcinoma rendered by human papillomavirus (HPV) integration is an aggressive cancer that occurs by dysregulation of multiple pathways, including oncogenes, proto-oncogenes, and tumor suppressors. The PI3K/Akt/mTOR pathway, which cross-talks with the Ras-ERK pathway, has been associated with cervical cancers (CC), which includes signaling pathways related to carcinoma aggressiveness, metastasis, recurrence, and drug resistance. Since bacterial cyclodipeptides (CDPs) possess cytotoxic properties in HeLa cells with inhibiting Akt/S6k phosphorylation, the mechanism of CDPs cytotoxicity involved was deepened. Results showed that the antiproliferative effect of CDPs occurred by blocking the PI3K/Akt/mTOR pathway, inhibiting the mTORC1/mTORC2 complexes in a TSC1/TSC2-dependent manner. In addition, the CDPs blocked protein kinases from multiple signaling pathways involved in survival, proliferation, invasiveness, apoptosis, autophagy, and energy metabolism, such as PI3K/Akt/mTOR, Ras/Raf/MEK/ERK1/2, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, Wnt/ß-catenin, HSP27, EMT, CSCs, and receptors, such as EGF/ErbB2/HGF/Met. Thus, the antiproliferative effect of the CDPs made it possible to identify the crosstalk of the signaling pathways involved in HeLa cell malignancy and to suggest that bacterial CDPs may be considered as a potential anti-neoplastic drug in human cervical adenocarcinoma therapy.

Experience with provisional WHO-entities large B-cell lymphoma with IRF4-rearrangement and Burkitt-like lymphoma with 11q aberration in paediatric patients of the NHL-BFM group.

Large B-cell lymphoma with IRF4 rearrangement, and Burkitt-like lymphoma with 11q aberration are two provisional lymphoma entities in the 2017 revision of the WHO classification of lymphoid neoplasms. Despite being more frequent in young patients, knowledge regarding their true incidence and clinical features in unselected cohorts of paediatric and adolescent patients is limited. We screened for both entities among paediatric patients (<18 years of age) in the German NHL-BFM (Non-Hodgkin lymphoma Berlin-Frankfurt-Münster) group. Among follicular lymphomas and diffuse large B-cell lymphomas (DLBCL), 7/34 cases (21%) showed an IRF4 break-apart pattern by fluorescence in situ hybridisation (FISH) and are associated with stages I and II disease (P = 0·043). Among lymphomas morphologically resembling Burkitt lymphoma, DLBCL and high-grade B-cell lymphoma, unclassifiable, 13/102 cases (13%) lacked a MYC break-apart pattern but were positive for 11q proximal gain and telomeric loss by FISH. MYC-negative Burkitt-like lymphomas with the typical 11q gain-loss pattern by FISH were older (P = 0·004), showed less male predominance (P = 0·003), lower stage (P = 0·040), lower serum LDH level (P = 0·01) and less abdominal involvement (P = 0·008) compared to high grade B-cell lymphomas without 11q gain-loss pattern. Both entities showed excellent outcome with overall survival of 100% when managed according to NHL-BFM strategies and may provide candidates for future therapy de-escalation in clinical trials.

Evaluation of 2 Commercial Assays for the Detection of Lymphogranuloma Venereum in Rectal Samples.

BACKGROUND: The early identification of the Chlamydia trachomatis variants that cause lymphogranuloma venereum (LGV) is very important to establish an adequate antibiotic treatment. This identification should be made with molecular techniques that are easy to perform and accessible to most microbiology laboratories. The objective of this study was to evaluate 2 real-time polymerase chain reaction (PCR)-based assay (VIASURE Haemophilus ducreyi + C. trachomatis (LGV) real-time PCR detection kit and the Allplex Genital ulcer Assay) for the detection of LGV in rectal samples. MATERIALS AND METHODS: Prospective study on positive rectal samples for C. trachomatis. All samples were processed in parallel by both tests. As a molecular reference method and to solve possible discrepancies between both assays, a PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1) was used. RESULTS: In total, we detected 157 positive rectal samples for C. trachomatis, of which 36 were identified as LGV by PCR-based restriction fragment length polymorphism analysis. The positive percent agreement, negative percent agreement, and overall percent agreement were 88.9%, 100%, and 97.3%, respectively, for the Allplex Genital ulcer assay and 91.6%, 100%, and 97.1%, respectively, for the VIASURE assay. In the direct comparison between the Seegene assay and the VIASURE assay, we obtained a kappa concordance index of 0.98 between both tests. CONCLUSIONS: According to the results obtained, both tests could be used for the detection of LGV in rectal samples.

Rare non-Hodgkin lymphoma of childhood and adolescence: A consensus diagnostic and therapeutic approach to pediatric-type follicular lymphoma, marginal zone lymphoma, and nonanaplastic peripheral T-cell lymphoma.

Pediatric-type follicular (PTFL), marginal zone (MZL), and peripheral T-cell lymphoma (PTCL) account each for <2% of childhood non-Hodgkin lymphoma. We present clinical and histopathological features of PTFL, MZL, and few subtypes of PTCL and provide treatment recommendations. For localized PTFL and MZL, watchful waiting after complete resection is the therapy of choice. For PTCL, therapy is subtype-dependent and ranges from a block-like anaplastic large cell lymphoma (ALCL)-derived and, alternatively, leukemia-derived therapy in PTCL not otherwise specified and subcutaneous panniculitis-like T-cell lymphoma to a block-like mature B-NHL-derived or, preferentially, ALCL-derived treatment followed by hematopoietic stem cell transplantation in first remission in hepatosplenic and angioimmunoblastic T-cell lymphoma.

Al2O3:Yb3+ integrated microdisk laser label-free biosensor.

Whispering gallery mode resonator lasers hold the promise of an ultralow intrinsic limit of detection. However, the widespread use of these devices for biosensing applications has been hindered by the complexity and lack of robustness of the proposed configurations. In this work, we demonstrate biosensing with an integrated microdisk laser. Al2O3doped with Yb3+ was utilized because of its low optical losses as well as its emission in the range 1020-1050 nm, outside the absorption band of water. Single-mode laser emission was obtained at a wavelength of 1024 nm with a linewidth of 250 kHz while the microdisk cavity was submerged in water. A limit of detection of 300 pM (3.6 ng/ml) of the protein rhS100A4 in urine was experimentally demonstrated, showing the potential of the proposed devices for biosensing.

Impact of use of optical surface imaging on initial patient setup for stereotactic body radiotherapy treatments.

PURPOSE: To evaluate the effectiveness of surface image guidance (SG) for pre-imaging setup of stereotactic body radiotherapy (SBRT) patients, and to investigate the impact of SG reference surface selection on this process. METHODS AND MATERIALS: 284 SBRT fractions (SG-SBRT = 113, non-SG-SBRT = 171) were retrospectively evaluated. Differences between initial (pre-imaging) and treatment couch positions were extracted from the record-and-verify system and compared for the two groups. Rotational setup discrepancies were also computed. The utility of orthogonal kVs in reducing CBCT shifts in the SG-SBRT/non-SG-SBRT groups was also calculated. Additionally, the number of CBCTs acquired for setup was recorded and the average for each cohort was compared. These data served to evaluate the effectiveness of surface imaging in pre-imaging patient positioning and its potential impact on the necessity of including orthogonal kVs for setup. Since reference surface selection can affect SG setup, daily surface reproducibility was estimated by comparing camera-acquired surface references (VRT surface) at each fraction to the external surface of the planning CT (DICOM surface) and to the VRT surface from the previous fraction. RESULTS: The reduction in all initial-to-treatment translation/rotation differences when using SG-SBRT was statistically significant (Rank-Sum test, α = 0.05). Orthogonal kV imaging kept CBCT shifts below reimaging thresholds in 19%/51% of fractions for SG-SBRT/non-SG-SBRT cohorts. Differences in average number of CBCTs acquired were not statistically significant. The reference surface study found no statistically significant differences between the use of DICOM or VRT surfaces. CONCLUSIONS: SG-SBRT improved pre-imaging treatment setup compared to in-room laser localization alone. It decreased the necessity of orthogonal kV imaging prior to CBCT but did not affect the average number of CBCTs acquired for setup. The selection of reference surface did not have a significant impact on initial patient positioning.

A survey of surface imaging use in radiation oncology in the United States.

Surface imaging (SI) has been rapidly integrated into radiotherapy clinics across the country without specific guidelines and recommendations on its commissioning and use aside from vendor-provided information. A survey was created under the auspices of AAPM TG-302 to assess the current status of SI to identify if there is need for formal guidance. The survey was designed to determine the institutional setting of responders, availability and length of its use, commissioning procedures, and clinical applications. This survey was created in REDCap, and approved as IRB exempt to collect anonymized data. Questions were reviewed by multiple physicists to ensure concept validity and piloted by a small group of independent physicists to ensure process validity. All full members of AAPM self-identified as "therapy" or "other" were sent the survey link by email. The survey was active from February to March 2018. Of 3677 members successfully contacted, 439 completed responses; the summary of these responses provides insight on current surface imaging clinical practices, though they should not be assumed to be representative of radiation oncology as a whole. Results showed that 53.3% of respondents have SI in their clinics, mostly in treatment rooms, rarely in simulation rooms. Half of those without SI plan on purchasing it within 3 years. Over 10% have SI but do not use it clinically, 36.8% classify themselves as "expert" users, and 85.5% agreed/strongly agreed that SI guidelines are needed. Initial positioning with SI is most common for breast/chestwall and SRS/SBRT treatments, least common for pediatrics. Use of SI for intra-fraction monitoring follows a similar distribution. Gating with SI is most prevalent for breast/chestwall (66.0%) but also used in SBRT (33.0%), and non-SBRT lung/abdomen (<30%) treatments. SI is a rapidly growing technology in the field with widespread use for several anatomic sites. Guidelines and recommendations on commissioning and clinical use are warranted.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60-250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

S100 to receptor for advanced glycation end-products binding assay: looking for inhibitors.

Secreted by tumor and stromal cells, S100 proteins exert their biological functions via the interaction with surface receptors. The most described receptor is the receptor for advanced glycation end-products (RAGE), thereby participating in the S100-dependent cell migration, invasion, tumor growth, angiogenesis and metastasis. Several approaches have been described for determining this interaction. Here we describe an easy, specific and highly reproducible ELISA-based method, by optimizing several parameters such as the binding and blocking buffer, interaction time and concentrations, directed to screen chemical and biological inhibitors of this interaction for S100A4, S100A7 and S100P proteins. The efficiency of the protocol was validated by using well described neutralizing agents of the RAGE receptor and of the S100A4 activity. The methodology described here will allow future works with other members of the S100 protein family and their receptors.

Assessment of interfractional variation of the breast surface following conventional patient positioning for whole-breast radiotherapy.

The purpose of this study was to quantify the variability of the breast surface position when aligning whole-breast patients to bony landmarks based on MV portal films or skin marks alone. Surface imaging was used to assess the breast surface position of 11 whole-breast radiotherapy patients, but was not used for patient positioning. On filmed fractions, AlignRT v5.0 was used to capture the patient's surface after initial positioning based on skin marks (28 "preshifts" surfaces), and after treatment couch shifts based on MV films (41 "postshifts" surfaces). Translations and rotations based on surface captures were recorded, as well as couch shifts based on MV films. For nonfilmed treatments, "daily" surface images were captured following positioning to skin marks alone. Group mean and systematic and random errors were calculated for all datasets. Pearson correlation coefficients, setup margins, and 95% limits of agreement (LOA) were calculated for preshifts translations and MV film shifts. LOA between postshifts surfaces and the filmed treatment positions were also computed. All the surface captures collected were retrospectively compared to both a DICOM reference surface created from the planning CT and to an AlignRT reference surface. All statistical analyses were performed using the DICOM reference surface dataset. AlignRT reference surface data was only used to calculate the LOA with the DICOM reference data. This helped assess any outcome differences between both reference surfaces. Setup margins for preshifts surfaces and MV films range between 8.3-12.0 mm and 5.4-13.4 mm, respectively. The largest margin is along the left-right (LR) direction for preshift surfaces, and along craniocaudal (CC) for films. LOA ranges between the preshifts surfaces and MV film shifts are large (12.6-21.9 mm); these decrease for postshifts surfaces (9.8-18.4 mm), but still show significant disagreements between the two modalities due to their focus on different anatomical landmarks (patient's topography versus bony anatomy). Pearson's correlation coefficients further support this by showing low to moderate correlations in the anterior-posterior (AP) and LR directions (0.47-0.69) and no correlation along CC (< 0.15). The use of an AlignRT reference surface compared to the DICOM reference surface does not significantly affect the LOA. Alignment of breast patients based solely on bony alignment may lead to interfractional inconsistencies in the breast surface position. The use of surface imaging tools highlights these discrepancies, and allows the radiation oncology team to better assess the possible effects on treatment quality.