S100 Protein Expression in Primary and Metastatic Neuroendocrine Neoplasms: A Specific Marker of Pancreatic Origin.

Neuroendocrine neoplasms can arise in a wide variety of anatomic sites including the gastrointestinal tract, pancreas, and lung, among others. Here, we report on the expression of S100 protein in a tissue microarray composed of 919 distinct primary and metastatic neuroendocrine neoplasms from 548 patients. S100 protein is a commonly used marker in many laboratories for the identification of neural and melanocytic neoplasms and occasionally used in the workup for neuroendocrine neoplasms when the diagnosis of paraganglioma is being considered. We show that strong S100 protein expression is highly specific to well-differentiated neuroendocrine tumors of pancreatic origin. This finding suggests potential diagnostic utility of this marker in cases of tumors of unknown origin, and emphasizes that S100 protein expression should not be an unexpected finding in neuroendocrine tumors of pancreatic origin.

Lessons learned from the use of COVID-19 convalescent plasma at Kaiser Permanente.

Background: In April 2020, the Mayo Clinic helped establish the US Food and Drug Administration Expanded Access Protocol for COVID-19 (coronavirus disease 2019) convalescent plasma (CCP). The effectiveness of CCP in the published literature is contradictory because some retrospective studies showed benefit in reducing mortality and severe illness, whereas prospective randomized controlled trials demonstrated no benefit of CCP. Objectives: To discuss (1) the implementation of CCP across Kaiser Permanente Southern California between April 2020 and April 2021, (2) retrospective multivariable analysis of 2,831 patients with COVID-19 who were transfused with CCP compared with 18,475 patients with COVID-19 who did not receive CCP, (3) how to reconcile contradictory published data regarding the efficacy of CCP, and (4) guidance regarding the future use of convalescent plasma in a large community hospital setting. Methods: Multivariable analysis was controlled for demographic characteristics, level of oxygen delivery, intensive care unit stay, selected laboratory findings, and other concurrent treatment-related variables. Tubing segments from 151 CCP units transfused between October 2020 and April 2021 were retrospectively tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike protein receptor-binding domain IgG. Multivariable analysis showed that CCP transfusion did not affect mortality rates at 30 days and 5 months (odds ratio, 1.04, 95% CI, 0.87-1.25, and hazard ratio, 1.05, 95% CI, 0.93-1.19). Conclusions: If convalescent plasma is offered as a therapeutic in a future viral pandemic, we recommend (1) transfusing only those patients who are negative for neutralizing antibodies, (2) transfusing very early during the disease course, (3) only using convalescent plasma with known levels of neutralizing antibodies, or (4) alternatively providing fractionated hyperimmune globulin.

PAM staining intensity of primary neuroendocrine neoplasms is a potential prognostic biomarker.

Neuroendocrine neoplasms (NENs) are rare epithelial tumors with heterogeneous and frequently unpredictable clinical behavior. Available biomarkers are insufficient to guide individual patient prognosis or therapy selection. Peptidylglycine α-amidating monooxygenase (PAM) is an enzyme expressed by neuroendocrine cells that participates in hormone maturation. The objective of this study was to assess the distribution, clinical associations and survival implications of PAM immunoreactivity in primary NENs. Of 109 primary NENs, 7% were PAM-negative, 25% were PAM-low and 68% were PAM-high. Staining intensity was high in small bowel (p = 0.04) and low in stomach (p = 0.004) NENs. PAM staining was lower in higher grade tumors (p < 0.001) and patients who died (p < 0.001) but did not vary by tumor size or stage at surgery. In patients who died, time to death was shorter in patients with reduced PAM immunoreactivity: median times to death were 11.3 (PAM-negative), 29.4 (PAM-low) and 61.7 (PAM-high) months. Lower PAM staining was associated with increased risk of death after adjusting for disease stage [PAM negative, HR = 13.8 (CI: 4.2-45.5)]. PAM immunoreactivity in primary NENs is readily assessable and a potentially useful stage-independent predictor of survival.

Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Big data modeling to predict platelet usage and minimize wastage in a tertiary care system.

Maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care in modern health care systems. However, daily blood product use is difficult to anticipate. Platelet products are the most variable in daily usage, have short shelf lives, and are also the most expensive to produce, test, and store. Due to the combination of absolute need, uncertain daily demand, and short shelf life, platelet products are frequently wasted due to expiration. Our aim is to build and validate a statistical model to forecast future platelet demand and thereby reduce wastage. We have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-mo period. Using a convex statistical formulation, we have found that platelet usage is highly dependent on weekday/weekend pattern, number of patients with various abnormal complete blood count measurements, and location-specific hospital census data. We incorporated these relationships in a mathematical model to guide collection and ordering strategy. This model minimizes waste due to expiration while avoiding shortages; the number of remaining platelet units at the end of any day stays above 10 in our model during the same period. Compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5 to 3.2%. Extrapolating our results to the ∼2 million units of platelets transfused annually within the United States, if implemented successfully, our model can potentially save ∼80 million dollars in health care costs.

Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease.

BACKGROUND: Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD. METHODS: Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified. RESULTS: Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases. CONCLUSIONS: The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

Comparison of treatment outcomes between intracapsular and total tonsillectomy for pediatric obstructive sleep apnea.

BACKGROUND: Intracapsular tonsillectomy (IT) has been advocated as a treatment for pediatric obstructive sleep apnea (OSA). However, evidence in the literature utilizing polysomnography (PSG) is limited. OBJECTIVE: To examine the experience at a tertiary children's hospital to evaluate the effectiveness and risks of intracapsular tonsillectomy compared to total tonsillectomy (TT) for treating pediatric OSA. METHODS: A retrospective study was undertaken of pediatric tonsillectomy cases performed for OSA at a tertiary children's hospital from 2005 to 2010. Patients with recurrent tonsillitis, craniofacial abnormalities, chromosomal abnormalities, neuromuscular disease, and congenital malformations were excluded. Main outcome measures were apnea-hypopnea index (AHI), minimum oxygen saturation (minO2), and surgical complications. RESULTS: Of the 1583 patients reviewed in this study, there were 75 IT and 93 TT patients with pre- and post-operative PSG results. The IT patients were younger, had lower BMI, larger tonsil size, lower pre-operative (AHI) and lower post-operative AHI (p < 0.05). There was a similar percentage of patients that showed improvement in AHI and minimum oxygen saturation between the IT and TT groups. There were statistically similar average change in AHI and minimum oxygen saturation between the IT and TT groups at 5.6 ± 8.6 and 8.6 ± 12.9, respectively (p = 0.8) as well as similar improvement in minimum oxygen saturation between the two groups at 3.3% ± 4.3% and 3.0% ± 5.2%, respectively (p = 0.66). Of TT patients, 2.9% experienced post-operative bleeding with 1.6% requiring OR for control of hemorrhage. Of IT patients, 2.2% were found to have tonsillar regrowth with 2.0% returning to the OR for secondary tonsillectomy. CONCLUSIONS: Intracapsular tonsillectomy, like total tonsillectomy, is effective in improving polysomnogram results in appropriately selected children. Intracapsular tonsillectomy is a suitable option for the surgical treatment of pediatric OSA consequent to its demonstrated efficacy in relieving OSA and its favorable safety profile.

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Mechanical properties of porcine cartilage after uniform RF heating.

BACKGROUND AND OBJECTIVES: Thermally mediated modalities of cartilage reshaping utilize localized heating of cartilage combined with mechanical deformation to achieve new geometries. We sought to determine the steady state elastic modulus of thermally modified cartilage without deformation, as this provides a constraint in mechanical models of the shape change process. STUDY DESIGN/MATERIALS AND METHODS: The main objective of this study was to characterize the steady state elastic modulus of porcine septal cartilage after uniform heating with radiofrequency (RF) to peak temperatures of 50 ± 5, 65 ± 5, and 85 ± 5°C. The cartilage was divided into three equally sized regions, designated as anterior, middle and posterior. Each region was then sectioned into two specimens with the proximal component serving as a paired control. RESULTS: The data confirm that there is high baseline variability in control steady state elastic moduli between animals. Also, the control values confirm a decreasing steady state elastic modulus from anterior to posterior. There is no statistical significance (P > 0.05) found between the elastic moduli of control and treated samples. CONCLUSIONS: Although shape change and retention have been fairly well characterized, little is known about the specific relation between steady state elastic modulus of cartilage and maximum treatment temperature. We determined that the difference of steady state elastic modulus between control and treated porcine septal samples was not statistically significant after uniform heating with RF to peak temperatures of 50 ± 5, 65 ± 5, and 85 ± 5°C. Ultimately, the results of this study do not pertain to the regions of heated cartilage that are shaped to hold a new form; however, it does show that the regions that are not mechanically deformed do return to the original pre-treatment elastic modulus. This is still useful information that may be used in finite element models to predict changes in internal stress distributions of cartilage after laser reshaping.

Model for estimating the threshold mechanical stability of structural cartilage grafts used in rhinoplasty.

OBJECTIVES/HYPOTHESIS: Characterizing the mechanical properties of structural cartilage grafts used in rhinoplasty is valuable because softer engineered tissues are more time- and cost-efficient to manufacture. The aim of this study is to quantitatively identify the threshold mechanical stability (e.g., Young's modulus) of columellar, L-strut, and alar cartilage replacement grafts. STUDY DESIGN: Descriptive, focus group survey. METHODS: Ten mechanical phantoms of identical size (5 x 20 x 2.3 mm) and varying stiffness (0.360 to 0.85 MPa in 0.05 MPa increments) were made from urethane. A focus group of experienced rhinoplasty surgeons (n = 25, 5 to 30 years in practice) were asked to arrange the phantoms in order of increasing stiffness. Then, they were asked to identify the minimum acceptable stiffness that would still result in favorable surgical outcomes for three clinical applications: columellar, L-strut, and lateral crural replacement grafts. Available surgeons were tested again after 1 week to evaluate intra-rater consistency. RESULTS: For each surgeon, the threshold stiffness for each clinical application differed from the threshold values derived by logistic regression by no more than 0.05 MPa (accuracy to within 10%). Specific thresholds were 0.56, 0.59, and 0.49 MPa for columellar, L-strut, and alar grafts, respectively. For comparison, human nasal septal cartilage is approximately 0.8 MPa. CONCLUSIONS: There was little inter- and intra-rater variation of the identified threshold values for adequate graft stiffness. The identified threshold values will be useful for the design of tissue-engineered or semisynthetic cartilage grafts for use in structural nasal surgery.

Viability of human septal cartilage after 1.45 microm diode laser irradiation.

BACKGROUND AND OBJECTIVES: Chondrocyte viability following laser irradiation and reshaping has not been established for human nasal septal cartilage. Knowledge of the relationship between thermal injury and laser dosimetry is needed in order to optimize septal laser cartilage reshaping. The objective of this study was to determine the depth and width of thermal injury in human septal cartilage following laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Excess fresh nasal septal cartilage sections from rhinoplasty or septoplasty operations were irradiated using a 1.45 microm diode laser 1.25-3.6 W (2.8 mm spot diameter) with 1 second fixed exposure time, and then at exposure times of 1-4 seconds for a fixed power of 1.25 W. An infrared camera recorded surface temperature profiles during irradiation, and the temperature data were incorporated into a rate process model to numerically estimate thermal damage. Calcein AM and ethidium homodimer-1 fluorescent dyes combined with confocal laser microscopy (CLM) were used to measure thermal damage. RESULTS: CLM demonstrated clear demarcation between dead and living cells following irradiation. The extent of non-viable chondrocyte distributions increased with power and exposure time. The maximum depths of injury were 1,012 and 1,372 microm after 3.6 W 1 second and 1.25 W 4 seconds irradiation respectively. The damage predictions made by the rate process model underestimated thermal injury when compared with CLM measurements. CONCLUSIONS: The assay system identified regions of non-viable chondrocytes in human septal cartilage and defined how thermal injury varies with dosimetry when using a 1.45 microm diode laser.

Temperature dependent change in equilibrium elastic modulus after thermally induced stress relaxation in porcine septal cartilage.

BACKGROUND AND OBJECTIVES: Laser cartilage reshaping (LCR) is a promising method for the in situ treatment of structural deformities in the nasal septum, external ear and trachea. Laser heating leads to changes in cartilage mechanical properties and produces relaxation of internal stress allowing formation of a new stable shape. While some animal and preliminary human studies have demonstrated clinical feasibility of LCR, application of the method outside specialized centers requires a better understanding of the evolution of cartilage mechanical properties with temperature. The purpose of this study was to (1) develop a method for reliable evaluation of mechanical changes in the porcine septal cartilage undergoing stress relaxation during laser heating and (2) model the mechanical changes in cartilage at steady state following laser heating. STUDY DESIGN/MATERIALS AND METHODS: Rectangular cartilage specimens harvested from porcine septum were heated uniformly by a radio-frequency (RF) electric field (500 kHz) for 8 and 12 seconds to maximum temperatures from 50 to 90 degrees C. Cylindrical samples were fashioned from the heated specimens and their equilibrium elastic modulus was measured in a step unconfined compression experiment. Functional dependencies of the elastic modulus and maximum temperature were interpolated from the measurements. Profiles of the elastic modulus produced after 8 and 12 seconds of laser irradiation (Nd:YAG, lambda = 1.34 microm, spot diameter 4.8 mm, laser power 8 W) were calculated from interpolation functions and surface temperature histories measured with a thermal camera. The calculated elastic modulus profiles were incorporated into a numerical model of uniaxial unconfined compression of laser irradiated cylindrical samples. The reaction force to a 0.1 compressive strain was calculated and compared with the reaction force obtained in analogous mechanical measurements experiment. RESULTS: RF heating of rectangular cartilage sample produces a spatially uniform temperature field (temperature variations < or = 4 degrees C) in a central region of the sample which is also large enough for reliable mechanical testing. Output power adjustment of the RF generator allows production of temperature histories that are very similar to those produced by laser heating at temperatures above 60 degrees C. This allows creation of RF cartilage samples with mechanical properties similar to laser irradiated cartilage, however with a spatially uniform temperature field. Cartilage equilibrium elastic modulus as a function of peak temperature were obtained from the mechanical testing of RF heated samples. In the temperature interval from 60 to 80 degrees C, the equilibrium modulus decreased from 0.08+/- 0.01 MPa to 0.016+/-0.007 MPa, respectively. The results of the numerical simulation of uniaxial compression of laser heated samples demonstrate good correlation with experimentally obtained reaction force. CONCLUSIONS: The thermal history and corresponding thermally induced modification of mechanical properties of laser irradiated septal cartilage can be mimicked by heating tissue samples with RF electric current with the added advantage of a uniform temperature profile. The spatial distribution of the mechanical properties obtained in septal cartilage after laser irradiation could be computed from mechanical testing of RF heated samples and used for numerical simulation of LCR procedure. Generalization of this methodology to incorporate orthogonal mechanical properties may aid in optimizing clinical laser cartilage reshaping procedures.

Analysis of Nd:YAG laser-mediated thermal damage in rabbit nasal septal cartilage.

BACKGROUND AND OBJECTIVES: Laser cartilage reshaping (LCR) involves the use of photo-thermal heating to reshape cartilage. Its clinical relevance depends on the ability to minimize thermal injury in irradiated regions. The present study seeks to understand the safety of LCR by determining shape change and resultant tissue viability as a function of laser dosimetry. STUDY DESIGN/MATERIALS AND METHODS: Rabbit nasal septal cartilage were irradiated using a Nd:YAG laser (lambda = 1.32 microm, 5.4 mm spot diameter) with different exposure times of 4, 6, 8, 10, 12, and 16 seconds and powers of 4, 6, and 8 W. Temperature on the cartilage surface in the laser-irradiated region was collected using infrared thermography, this data was then used to predict tissue damage via a rate process model. A Live/Dead viability assay combined with fluorescent confocal microscopy was used to measure the amount of thermal damage generated in the irradiated specimens. RESULTS: Considerable thermal injury occurred at and below the laser-reshaping parameters that produced clinically relevant shape change using the present Nd:YAG laser. Confocal microscopy identified dead cells spanning the entire cross-sectional thickness of the cartilage specimen (about 500 microm thick) at laser power density and exposure times above 4 W and 6 seconds; damage increased with time and irradiance. The damage predictions made by the rate process model compared favorably with measured data. CONCLUSIONS: These results demonstrate that significant thermal damage is concurrent with clinically relevant shape change. This contradicts previous notions that there is a privileged laser dosimetry parameter where clinically relevant shape change and tissue viability coexist.