Tumor-derived hypoxic small extracellular vesicles promote endothelial cell migration and tube formation via ALS2/Rab5/ß-catenin signaling.

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.

Effect of Preoperative Bilateral Ultrasound-guided Quadratus Lumborum Nerve Block on Quality of Recovery after Minimally Invasive Hysterectomy in an Enhanced Recovery after Surgery (ERAS) Setting.

STUDY OBJECTIVE: To assess the effect of preoperative bilateral ultrasound-guided quadratus lumborum nerve block (QLB) on quality of recovery after minimally invasive hysterectomy, in an enhanced recovery after surgery setting. DESIGN: Randomized, controlled, double-blinded trial (Canadian Task Force level I). SETTING: University-affiliated tertiary medical center. PATIENTS: All women undergoing an elective robotic or laparoscopic hysterectomy. Women with chronic pain, chronic anticoagulation, and body mass index >50 kg/m2 were excluded. INTERVENTION: Patients were randomized with a 1:1 allocation, to one of the following 2 arms, and stratified based on robotic versus laparoscopic approach. 1. QLB: QLB (bupivacaine) + sham local trocar sites infiltration (normal saline) 2. Local infiltration: sham QLB (normal saline) + local infiltration (bupivacaine) MEASUREMENTS AND MAIN RESULTS: The primary outcome was defined as the quality of recovery score based on the validated questionnaire Quality of Recovery, completed 24 hours postoperatively. Secondary outcomes included dynamic pain scores, accumulated opioid consumption up to 24 hours, postoperative nausea and vomiting, surgical complications, length of hospital stay, time to first pain medication administration in the postanesthesia care unit, and adverse events. A total of 76 women were included in the study. Demographic characteristics were similar in both groups. Median age was 44 years (interquartile range 39-50), 47% of the participants were African American, and mean body mass index was 32.8 kg/m2 (standard deviation [SD] 8.1). The mean Quality of Recovery score was 179.1 (SD ± 10.3) in the QLB and 175.6 (SD ± 9.7) for the local anesthesia group (p = .072). All secondary outcomes were comparable between groups. CONCLUSIONS: QLBs do not significantly improve quality of recovery after elective robotic or laparoscopic hysterectomy compared with local anesthetic port site infiltration.

Glucagon-Like Peptide-1 Receptor Agonist Use and Residual Gastric Content Before Anesthesia.

Importance: Glucagon-like peptide-1 receptor agonist (GLP-1 RA) use is rapidly increasing in the US, driven by its expanded approval for weight management in addition to hyperglycemia management in patients with type 2 diabetes. The perioperative safety of these medications, particularly with aspiration risk under anesthesia, is uncertain. Objective: To assess the association between GLP-1 RA use and prevalence of increased residual gastric content (RGC), a major risk factor for aspiration under anesthesia, using gastric ultrasonography. Design, Setting, and Participants: This cross-sectional study prospectively enrolled patients from a large, tertiary, university-affiliated hospital from June 6 through July 12, 2023. Participants followed preprocedural fasting guidelines before an elective procedure under anesthesia. Patients with altered gastric anatomy (eg, from previous gastric surgery), pregnancy, recent trauma (<1 month), or an inability to lie in the right lateral decubitus position for gastric ultrasonography were excluded. Exposure: Use of a once-weekly GLP-1 RA. Main Outcomes and Measures: The primary outcome was the presence of increased RGC, defined by the presence of solids, thick liquids, or more than 1.5 mL/kg of clear liquids on gastric ultrasonography. Analysis was adjusted for confounders using augmented inverse probability of treatment weighting, a propensity score-based technique. Secondarily, the association between the duration of drug interruption and the prevalence of increased RGC was explored. Results: Among the 124 participants (median age, 56 years [IQR, 46-65 years]; 75 [60%] female), the prevalence of increased RGC was 56% (35 of 62) in patients with GLP-1 RA use (exposure group) compared with 19% (12 of 62) in patients who were not taking a GLP-1 RA drug (control group). After adjustment for confounding, GLP-1 RA use was associated with a 30.5% (95% CI, 9.9%-51.2%) higher prevalence of increased RGC (adjusted prevalence ratio, 2.48; 95% CI, 1.23-4.97). There was no association between the duration of GLP-1 RA interruption and the prevalence of increased RGC (adjusted odds ratio, 0.86; 95% CI, 0.65-1.14). Conclusions and Relevance: Use of a GLP-1 RA was independently associated with increased RGC on preprocedural gastric ultrasonography. The findings suggest that the preprocedural fasting duration suggested by current guidelines may be inadequate in this group of patients at increased risk of aspiration under anesthesia.

Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K/Akt/mTOR signaling pathway.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

Ultrasound-Guided Percutaneous Intercostal Cryoneurolysis for Acute-on-Chronic Pain in CLOVES Syndrome.

Cryoneurolysis is an analgesic method that has been shown to provide extended pain relief in postoperative patients. However, to date, this method has not been described in nonsurgical inpatients with chronic pain experiencing an acute exacerbation. This analgesic modality has the potential to provide pain relief for patients whose expected duration of severe acute pain would outlast that of other regional anesthetic techniques while avoiding opioid escalation and facilitating discharge. We present a patient with acute exacerbation of chronic pain from breast ulcerations caused by congenital lipomatous overgrowth, vascular malformations, epidermal nevis, spinal/skeletal anomalies/scoliosis (CLOVES) syndrome that was successfully treated as an inpatient with a portable cryoneurolysis device.  This is the first reported use of cryoneurolysis in an inpatient setting to treat acute-on-chronic pain in a nonsurgical patient. The authors recommend regional anesthesiologists and acute pain specialists to utilize this technique to provide analgesia in patients with complex pain to facilitate hospital throughput.

Characterizing the interplay of treatment parameters and complexity and their impact on performance on an IROC IMRT phantom using machine learning.

AIM OF THE STUDY: To elucidate the important factors and their interplay that drive performance on IMRT phantoms from the Imaging and Radiation Oncology Core (IROC). METHODS: IROC's IMRT head and neck phantom contains two targets and an organ at risk. Point and 2D dose are measured by TLDs and film, respectively. 1,542 irradiations between 2012-2020 were retrospectively analyzed based on output parameters, complexity metrics, and treatment parameters. Univariate analysis compared parameters based on pass/fail, and random forest modeling was used to predict output parameters and determine the underlying importance of the variables. RESULTS: The average phantom pass rate was 92% and has not significantly improved over time. The step-and-shoot irradiation technique had significantly lower pass rates that significantly affected other treatment parameters' pass rates. The complexity of plans has significantly increased with time, and all aperture-based complexity metrics (except MCS) were associated with the probability of failure. Random forest-based prediction of failure had an accuracy of 98% on held-out test data not used in model training. While complexity metrics were the most important contributors, the specific metric depended on the set of treatment parameters used during the irradiation. CONCLUSION: With the prevalence of errors in radiotherapy, understanding which parameters affect treatment delivery is vital to improve patient treatment. Complexity metrics were strongly predictive of irradiation failure; however, they are dependent on the specific treatment parameters. In addition, the use of one complexity metric is insufficient to monitor all aspects of the treatment plan.

Mechanical Disturbance of Osteoclasts Induces ATP Release That Leads to Protein Synthesis in Skeletal Muscle through an Akt-mTOR Signaling Pathway.

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X7 receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.

The current status and shortcomings of stereotactic radiosurgery.

Background: Stereotactic radiosurgery (SRS) is a common treatment for intracranial lesions. This work explores the state of SRS treatment delivery to characterize current treatment accuracy based on treatment parameters. Methods: NCI clinical trials involving SRS rely on an end-to-end treatment delivery on a patient surrogate (credentialing phantom) from the Imaging and Radiation Oncology Core (IROC) to test their treatment accuracy. The results of 1072 SRS phantom irradiations between 2012 and 2020 were retrospectively analyzed. Univariate analysis and random forest models were used to associate irradiation conditions with phantom performance. The following categories were evaluated in terms of how they predicted outcomes: year of irradiation, TPS algorithm, machine model, energy, and delivered field size. Results: Overall, only 84.6% of irradiations have met the IROC/NCI acceptability criteria. Pass rate has remained constant over time, while dose calculation accuracy has slightly improved. Dose calculation algorithm (P < .001), collimator (P = .024), and field size (P < .001) were statistically significant predictors of pass/fail. Specifically, pencil beam algorithms and cone collimators were more likely to be associated with failing phantom results. Random forest modeling identified the size of the field as the most important factor for passing or failing followed by algorithm. Conclusion: Constant throughout this retrospective study, approximately 15% of institutions fail to meet IROC/NCI standards for SRS treatment. In current clinical practice, this is particularly associated with smaller fields that yielded less accurate results. There is ongoing need to improve small field dosimetry, beam modeling, and QA to ensure high treatment quality, patient safety, and optimal clinical trials.

The Use of Peripheral Nerve Blockade in Laparoscopic and Robotic Surgery: Is There a Benefit?

PURPOSE OF REVIEW: The purpose of this review is to synthesize recent literature investigating the use of regional anesthesia for minimally invasive surgery. RECENT FINDINGS: Recent studies investigating the use of newer peripheral nerve blocks such as erector spinae plane (ESP) and quadratus lumborum (QL) block are very limited. Evidence supporting the use of peripheral nerve blockade in laparoscopic or robotic surgery is very limited and of low-moderate quality. While transverse abdominal plane (TAP) block may decrease opioid and pain scores after laparoscopic cholecystectomy, bariatric surgery, and colorectal surgery, the benefit of the block in the presence of multimodal analgesia remains to be clarified. Unilateral paravertebral block may be beneficial for percutaneous nephrolithotomy. ESP and rectus sheath blockade may enhance analgesia in laparoscopic surgery, but the magnitude of this benefit may not be clinically relevant. Limited evidence supports the use of QL block in laparoscopic urologic surgery. There is insufficient recent evidence to support the use of TAP or QL block for laparoscopic gynecologic surgery.

Interscalene Block for Analgesia in Orthopedic Treatment of Shoulder Trauma: Single-Dose Liposomal Bupivacaine versus Perineural Catheter.

BACKGROUND: Interscalene brachial plexus block is frequently utilized to provide perioperative analgesia to patients undergoing shoulder surgery to optimize recovery, minimize opioid consumption, and decrease overall hospital length of stay. The use of an indwelling perineural interscalene catheter provides extended analgesia and is efficacious in managing severe postoperative pain following major shoulder surgery. Currently, the only alternative to perineural catheters for extended analgesia with interscalene block involves the perineural infiltration of liposomal bupivacaine. However, there is limited published data regarding the overall analgesic effectiveness of using interscalene liposomal bupivacaine in the setting of shoulder surgery. METHODS: We performed a retrospective review of 43 patients in the acute trauma setting who underwent major shoulder surgery and received extended analgesia via perioperative interscalene brachial plexus block with either an indwelling continuous catheter or single-dose liposomal bupivacaine to determine if comparable analgesia can be achieved. The primary outcomes of interest were postoperative pain scores and opioid consumption. Due to the ability to titrate and bolus local anesthetic infusions to a desired clinical effect, we hypothesized that opioid consumption and pain scores would be lower when using the continuous catheter technique. RESULTS: After statistical analysis, our results demonstrated no significant difference between the two techniques in regards to opioid consumption as well as numeric pain scores during the 48-hour postoperative period, but did note a higher rate of complications with patients who received perineural interscalene continuous catheters. Secondary outcomes showed an increase in time required to complete the regional block procedure with the use of indwelling catheters. CONCLUSION: Interscalene brachial plexus block with liposomal bupivacaine may be a viable alternative to indwelling continuous catheters for providing extended analgesia in patients undergoing major shoulder surgery.

Lumbosacral paraspinal interfascial plane blocks for pain control after feminizing genital gender affirmation surgery.

Aim: Feminizing genital gender affirmation surgery (fgGAS) is increasing in prevalence in the USA. Management of postoperative pain following fgGAS is challenging. We report a series of patients where post-fgGAS pain was adequately controlled with paraspinal blocks. Materials & methods: This is a case series of three patients who received bilateral lumbar and sacral erector spinae plane blocks after fgGAS. Block techniques, medications administered, opioid requirements and pain scores were reviewed. Results: Erector spinae plane blocks provided adequate analgesia for 24-48 h following the block. Conclusion: Currently, there are two regional anesthetic techniques described for the treatment of postoperative pain after fgGAS. We describe two additional approaches as options for improved pain management in this patient population.

Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

Upper extremity regional anesthesia techniques: A comprehensive review for clinical anesthesiologists.

Surgeries and chronic pain states of the upper extremity are quite common and pose unique challenges for the clinical anesthesiology and pain specialists. Most innervation of the upper extremity involves the brachial plexus. The four most common brachial plexus blocks performed in clinical setting include the interscalene, supraclavicular, infraclavicular, and axillary brachial plexus blocks. These blocks are most commonly performed with the use of ultrasound-guided techniques, whereby analgesia is achieved by anesthetizing the brachial plexus at different levels such as the roots, divisions, cords, and branches. Additional regional anesthetic techniques for upper extremity surgery include wrist, intercostobrachial, and digital nerve blocks, which are most frequently performed using landmark anatomical techniques. This review provides a comprehensive summary of each of these blocks including anatomy, best practice techniques, and potential complications.

Human Papillomavirus Infection in Female Sex Workers: A Case Control Study.

BACKGROUND: To determine the association of infection with human papillomavirus (HPV) and the occupation of female sex worker; and the correlation of infection with HPV with sociodemographic, clinical and behavioral characteristics of female sex workers. METHODS: We performed a case-control study of 217 female sex workers and 354 women without sex work in Durango City, Mexico. We determined the prevalence of infection with HPV in cervical samples of women using polymerase chain reaction, and HPV genotypes were determined using line probe assay. Bivariate and multivariate analyses were used to assess the association between the characteristics of women and infection. RESULTS: Twelve (5.5%) of the 217 sex workers, and 10 (2.8%) of the 354 control women were positive for HPV DNA (age-adjusted OR = 1.51; 95% CI: 0.62 - 3.68; P = 0.36). Six (50.0%) of the 12 HPV DNA positive sex workers had infections with high-risk genotypes (16, 31, 33, 35, 51, 58). Seven (70%) of the 10 HPV DNA positive control women had infections with high-risk genotypes (16, 18, 56, 58, and 66). The frequency of high risk genotypes in the control women was equal with that found in the female sex workers (P = 0.41). Logistic regression analysis showed that the variable alcohol consumption was associated with HPV infection (OR = 4.0; 95% CI: 1.0 - 16.0; P = 0.04). CONCLUSIONS: No association between HPV infection and female sex work was found in our setting. High risk HPV genotypes were prevalent among the women studied. Results can be used for the design of preventive measures against HPV infection.

Assessment of a multimodal analgesia protocol to allow the implementation of enhanced recovery after cardiac surgery: Retrospective analysis of patient outcomes.

STUDY OBJECTIVE: To investigate the impact of utilizing a multimodal analgesia protocol to allow the implementation of Enhanced Recovery after Cardiac Surgery (ERACS) in patients requiring cardio-pulmonary bypass. DESIGN: Retrospective analysis of patients treated with the proposed ERACS bundle in comparison to matched controls. SETTING: Single-center study. PATIENTS: A total of 50 patients undergoing elective cardiac surgery limited to on pump coronary artery bypass graft. MEASUREMENTS: Perioperative outcomes of 25 patients that underwent ERACS protocol and 25 controls were measured. In-operating room (OR) extubation, total intubation time, total intra-OP fentanyl given, total post-OP morphine equivalent given, intensive care unit (ICU) length of stay (LOS), hospital LOS and post-OP complications were examined. MAIN RESULTS: The ERACS group and control group were equivalent with regards to age, gender, comorbidities, ASA classification and type of surgery. Mean cardiac bypass time and mean aortic clamp time were similar. Extubation in the OR was achieved for 12 patients in the ERACS group compared to 1 in the control group. Post-operative opioid consumption was lower in ERACS group (27.3 vs. 51.7 morphine equivalents, p = 0.006). Although ICU LOS and hospital LOS were shorter in the ERACS group, this did not reach significance. CONCLUSIONS: The ERACS group showed a significant decrease in opioid use and increased incidence of successful in OR extubation.

Erector spinae block for postoperative analgesia following axillary hidradenitis suppurativa resection: a case report.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease of the terminal hair follicle of the apocrine gland-bearing skin, presenting with inflamed nodules, abscesses, and sinus tracts. Autoimmune, genetic, and infectious factors have been implicated in its pathogenesis. HS can be managed medically using topical and systemic antibiotics and with tumor necrosis factor antibody therapy in severe cases. Surgical treatment can range from local excision, deroofing or excision of sinus tracts, or wide radical excision of severe lesions. Lesions can be severely painful on their own or following surgical resection. Patients may require opioid therapy due to pain from the lesions themselves or following painful surgical resection. Erector spinae block (ESB) is a recently developed plane block used for the management of pain located in the chest wall or the abdominal wall. Ultrasound guidance is used to guide placement of the needle tip between the transverse process at the desired dermatomal level and the erector spinae muscle group. Traditional descriptions of the ESB have been at the level of the fifth transverse process to treat chest wall pain; in this case report, we describe a novel use of ESB at the level of the second transverse process to treat axillary pain after surgical debridement of axillary HS.

Results From the Imaging and Radiation Oncology Core Houston's Anthropomorphic Phantoms Used for Proton Therapy Clinical Trial Credentialing.

PURPOSE: The purpose of this study was to summarize the findings of anthropomorphic proton phantom irradiations analyzed by the Imaging and Radiation Oncology Core Houston QA Center (IROC Houston). METHODS AND MATERIALS: A total of 103 phantoms were irradiated by proton therapy centers participating in clinical trials. The anthropomorphic phantoms simulated heterogeneous anatomy of a head, liver, lung, prostate, and spine. Treatment plans included those for scattered, uniform scanning, and pencil beam scanning beam delivery modalities using 5 different treatment planning systems. For every phantom irradiation, point doses and planar doses were measured using thermoluminescent dosimeters (TLD) and film, respectively. Differences between measured and planned doses were studied as a function of phantom, beam delivery modality, motion, repeat attempt, treatment planning system, and date of irradiation. RESULTS: The phantom pass rate (overall, 79%) was high for simple phantoms and lower for phantoms that introduced higher levels of difficulty, such as motion, multiple targets, or increased heterogeneity. All treatment planning systems overestimated dose to the target, compared to TLD measurements. Errors in range calculation resulted in several failed phantoms. There was no correlation between treatment planning system and pass rate. The pass rates for each individual phantom are not improving over time, but when individual institutions received feedback about failed phantom irradiations, pass rates did improve. CONCLUSIONS: The proton phantom pass rates are not as high as desired and emphasize potential deficiencies in proton therapy planning and/or delivery. There are many areas for improvement with the proton phantom irradiations, such as treatment planning system dose agreement, range calculations, accounting for motion, and irradiation of multiple targets.