A Pilot Study of Neoadjuvant Nivolumab, Ipilimumab, and Intralesional Oncolytic Virotherapy for HER2-negative Breast Cancer.

Purpose: Neoadjuvant combination immune checkpoint blockade and intralesional oncolytic virotherapy have the potential to activate antitumor responses in patients with breast cancer. Experimental Design: Eligibility for this pilot phase I trial included patients with localized HER2-negative breast cancer who received systemic nivolumab and ipilimumab and intratumor talimogene laherparepvec (T-VEC; NCT04185311). The primary objective was to evaluate the safety and adverse event profile of immunotherapy combined with T-VEC in patients with localized, HER2-negative breast cancer. Results: Six patients were enrolled, 4 having relapses after prior neoadjuvant chemotherapy and 2 who were previously untreated. Toxicities included 1 patient having grade 3 hypotension and type 1 diabetes mellitus, 3 patients with hypothyroidism, and all patients having constitutional symptoms known to be associated with the administration of T-VEC. One patient had a pathologic complete response, 3 patients had pathologic partial responses, 1 showed no significant response, and 1 had disease progression. Biopsies demonstrated increased immune cell infiltration in samples from patients who responded to therapy. Conclusions: This triple immunotherapy regimen provided responses in patients with advanced or relapsed HER2-negative breast cancer, at the expense of long-term toxicities. Significance: Systemic immune checkpoint blockade with a programmed death receptor 1 and a CTL antigen-4 blocking antibody, combined with intralesional oncolytic virotherapy, is a chemotherapy-free combination aimed at inducing an antitumor immune response locally and systemic immunity.

Quantitative Nodal Burden and Mortality Across Solid Cancers.

BACKGROUND: Nodal staging systems vary substantially across solid tumors, implying heterogeneity in the behavior of nodal variables in various contexts. We hypothesized, in contradiction to this, that metastatic lymph node (LN) number is a universal and dominant predictor of outcome across solid tumors. METHODS: We performed a retrospective cohort analysis of 1 304 498 patients in the National Cancer Database undergoing surgery between 2004 and 2015 across 16 solid cancer sites. Multivariable Cox regression analyses were constructed using restricted cubic splines to model the association between nodal number and mortality. Recursive partitioning analysis (RPA) was used to derive nodal classification systems for each solid cancer based on metastatic LN count. The reproducibility of these findings was assessed in 1 969 727 patients from the Surveillance, Epidemiology, and End Results registry. Two-sided tests were used for all statistical analyses. RESULTS: Consistently across disease sites, mortality risk increased continuously with increasing number of metastatic LNs (P < .001 for all spline segments). Each RPA-derived nodal classification system produced multiple prognostic groups spanning a wide spectrum of mortality risk (P < .001). Multivariable models using these RPA-derived nodal classifications demonstrated improved concordance with mortality compared with models using American Joint Committee on Cancer staging in sites where nodal classification is not based on metastatic LN count. Each RPA-derived nodal classification system was reproducible in a large validation cohort for all-cause and cause-specific mortality (P < .001). High quantitative nodal burden was the single strongest tumor-intrinsic variable associated with mortality in 12 of 16 disease sites. CONCLUSIONS: Quantitative metastatic LN burden is a fundamental driver of mortality across solid cancers and should serve as a foundation for pathologic nodal staging across solid tumors.

Development and Validation of a Modified Pathologic Nodal Classification System for Cutaneous Melanoma.

Importance: Given the evolving patterns of lymph node evaluation for cutaneous melanoma, it is unclear whether the current nodal classification system will continue to accurately reflect prognosis in the modern era. Existing nodal staging for cutaneous melanoma was developed primarily for patients undergoing completion lymph node dissection (CLND) for node-positive disease and does not produce groups with continuously increasing mortality. Objective: To develop and validate a modified nodal classification system for cutaneous melanoma. Design, Setting, and Participants: This retrospective cohort analysis included 105 785 patients with cutaneous melanoma undergoing surgery and nodal evaluation from January 1, 2004, to December 31, 2015, in the National Cancer Database. Extent of lymph node dissection was available for patients diagnosed in 2012 and onward. Multivariable models were generated with number of positive lymph nodes modeled using restricted cubic splines. A modified nodal classification system was derived using recursive partitioning analysis (RPA). The proposed lymph node classification system was validated in 85 499 patients from the Surveillance, Epidemiology, and End Results (SEER-18) database. Data were analyzed from April 9, 2020, to May 28, 2021. Main Outcomes and Measures: Overall survival. Results: Among the 105 785 patients included in the analysis (62 496 men [59.1%]; mean [SD] age, 59.9 [15.5] years), number of positive lymph nodes (hazard ratio [HR] per lymph node for 0 to 2 positive lymph nodes, 2.48 [95% CI, 2.37-2-61; P < .001]; HR per lymph node for ≥3 positive lymph nodes, 1.10 [95% CI 1.07-1.13; P < .001]), clinically detected metastases (HR, 1.35; 95% CI, 1.27-1.42; P < .001), and in-transit metastases (HR, 1.48; 95% CI, 1.34-1.65; P < .001) were independently associated with mortality. An RPA-derived system using these variables demonstrated continuously increasing mortality for each proposed lymph node classification group, with HRs of 1.83 (95% CI, 1.76-1.91) for N1a, 2.72 (95% CI, 2.58-2.86) for N1b, 3.79 (95% CI, 3.51-4.08) for N2a, 4.56 (95% CI, 4.22-4.92) for N2b, 6.15 (95% CI, 5.59-6.76) for N3a, and 8.25 (95% CI, 7.64-8.91) for N3b in the proposed system (P < .001). By contrast, the current American Joint Committee on Cancer (AJCC) nodal classification system produced a more haphazard mortality profile, with HRs of 1.83 (95% CI, 1.76-1.91) for N1a, 3.81 (95% CI, 3.53-4.12) for N1b, 2.59 (95% CI, 2.30-2.93) for N1c, 2.71 (95% CI, 2.56-2.87) for N2a, 4.51 (95% CI, 4.17-4.87) for N2b, 3.44 (95% CI, 2.60-4.55) for N2c, 6.06 (95% CI, 5.51-6.67) for N3a, 8.15 (95% CI, 7.54-8.81) for N3b, and 6.90 (95% CI, 5.60-8.49) for N3c. As a sensitivity analysis, the proposed system continued to accurately stratify patients when excluding those undergoing CLND for microscopic lymph node metastases. This system was validated for overall survival and cause-specific mortality in SEER-18. Last, a new overall staging system for node-positive patients was developed by RPA and demonstrated improved concordance vs the AJCC, 8th edition system (C statistic, 0.690 [95% CI, 0.689-0.691] vs 0.666 [95% CI, 0.666-0.668]). Conclusions and Relevance: The findings of this cohort study suggest that a modified nodal classification system can accurately stratify mortality risk in cutaneous melanoma in an era of increasing use of sentinel lymph node biopsy without CLND and should be considered for future staging systems.

Microstructure and nanomechanical properties of the exoskeleton of an ironclad beetle (Zopherus haldemani).

This study examined natural composite structures within the remarkably strong exoskeleton of the southwestern ironclad beetle (Z. haldemani). Structural and nanomechanical analyses revealed that the exoskeleton's extraordinary resistance to external forces is provided by its exceptional thickness and multi-layered structure, in which each layer performed a distinct function. In detail, the epicuticle, the outmost layer, comprised 3%-5% of the overall thickness with reduced Young's moduli of 2.2-3.2 GPa, in which polygonal-shaped walls (2-3µm in diameter) were observed on the surface. The next layer, the exocuticle, consisted of 17%-20% of the total thickness and exhibited the greatest Young's moduli (∼15 GPa) and hardness (∼800 MPa) values. As such, this layer provided the bulk of the mechanical strength for the exoskeleton. While the endocuticle spanned 70%-75% of the total thickness, it contained lower moduli (∼8-10 GPa) and hardness (∼400 MPa) values than the exocuticle. Instead, this layer may provide flexibility through its specifically organized chitin fiber layers, known as Bouligand structures. Nanoindentation testing further reiterated that the various fibrous layer orientations resulted in different elastic moduli throughout the endocuticle's cross-section. Additionally, this exoskeleton prevented delamination within the composite materials by overlapping approximately 5%-19% of each fibrous stack with neighboring layers. Finally, the innermost layer, the epidermis contributing 5%-7 % of the total thickness, contains attachment sites for muscle and soft tissue that connect the exoskeleton to the beetle. As such, it is the softest region with reduced Young's modulus of ∼2-3 GPa and hardness values of ∼290 MPa. These findings can be applied to the development of innovative, fiber-reinforced composite materials.

Flow Rate and Raspberry Pi-based Paper Microfluidic Blood Coagulation Assay Device.

Monitoring blood coagulation in response to an anticoagulant (heparin) and its reversal agent (protamine) is essential during and after surgery, especially with cardiopulmonary bypass (CPB). A current clinical standard is the use of activated clotting time (ACT), where the mechanical movement of a plunger through a whole blood-filled channel is monitored to evaluate the endpoint time of coagulation. As a rapid, simple, low-volume, and cost-effective alternative, we have developed a paper microfluidic assay and Raspberry Pi-based device with the aim of quantifying the extent of blood coagulation in response to varying doses of heparin and protamine. The flow rate of blood through the paper microfluidic channel is automatically monitored using Python-coded edge detection algorithm. For each set of assay, 8 µL of fresh human whole blood (untreated and undiluted) from human subjects is loaded onto each of 8 sample pads, which have been preloaded with varying amounts of heparin or protamine. Total assay time is 3-5 minutes including the time for sample loading and incubation.

Outcomes of patients with AL amyloidosis and low serum free light chain levels at diagnosis.

Serum free light chains (sFLC) are independent prognostic markers of disease in light chain (AL) amyloidosis, and are used in the haematologic response criteria for treatment. However, up to 20% of patients have low sFLCs at diagnosis, with a difference between involved and uninvolved free light chains (dFLC) of less than 50 mg/L, making responses to treatment difficult to evaluate. In order to characterize this distinct subgroup of patients, we retrospectively analyzed 123 AL amyloidosis patients with dFLC <50 mg/L who were diagnosed between 2002 and 2013. The majority (n = 117) were treated for their AL amyloidosis, with over half (n = 68) receiving high-dose melphalan and autologous stem cell transplantation as first-line therapy. Overall they had a prolonged median survival of 9.2 years with less cardiac involvement (30%) and more renal involvement (76%). We also evaluated the newly proposed low dFLC partial response (PR) criteria, defined as a dFLC <10 mg/L if the initial dFLC 20-50 mg/L. The 14 patients with low dFLC PR had improved survival and organ responses compared with patients with no haematologic response. However, one-third of patients (n = 41) had an initial dFLC <20 mg/L so could not be evaluated. More sensitive methods of monitoring response to treatment for this subgroup population are still needed.

Modified High-Dose Melphalan and Autologous Stem Cell Transplantation for Immunoglobulin Light Chain Amyloidosis.

High-dose melphalan and autologous stem cell transplantation (HDM/SCT) have been used in patients with immunoglobulin light chain (AL) amyloidosis for over 2 decades now with durable responses, prolonged survival, and decreasing treatment-related mortality. Historically, patients with poorer baseline functional status, advanced age, renal compromise, and cardiac involvement have been treated with a risk-adapted modified conditioning dose of melphalan (mHDM) of 100 to 140 mg/m2 before SCT. In part because of these baseline characteristics, patients receiving mHDM/SCT have had poorer outcomes compared with patients receiving full-dose melphalan at 200 mg/m2. With the advent of novel therapeutic agents such as proteasome inhibitors, immunomodulatory agents, and monoclonal antibodies for the treatment of AL amyloidosis, it is imperative to understand the long-term effects of mHDM/SCT. Here we report the long-term outcomes of 334 patients with AL amyloidosis treated with mHDM/SCT. Median overall survival was 6.1 years and median event-free survival 4.3 years, with median overall survival reaching 13.4 years for patients who had achieved a hematologic complete response (CR). Overall hematologic response rate was 69%, and treatment-related mortality was 3% after 2010. Thus, mHDM/SCT leads to prolonged survival and favorable outcomes, especially if a hematologic CR is achieved.

Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells.

Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 µg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 µg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 µg/mL of CD80+VEGF and 0.1 µg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 µg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 µg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having spontaneous OSA, such as the dog, and on a human OSA cell line model.

Prehospital rapid sequence intubation improves functional outcome for patients with severe traumatic brain injury: a randomized controlled trial.

OBJECTIVE: To determine whether paramedic rapid sequence intubation in patients with severe traumatic brain injury (TBI) improves neurologic outcomes at 6 months compared with intubation in the hospital. BACKGROUND: Severe TBI is associated with a high rate of mortality and long-term morbidity. Comatose patients with TBI routinely undergo endo-tracheal intubation to protect the airway, prevent hypoxia, and control ventilation. In many places, paramedics perform intubation prior to hospital arrival. However, it is unknown whether this approach improves outcomes. METHODS: In a prospective, randomized, controlled trial, we assigned adults with severe TBI in an urban setting to either prehospital rapid sequence intubation by paramedics or transport to a hospital emergency department for intubation by physicians. The primary outcome measure was the median extended Glasgow Outcome Scale (GOSe) score at 6 months. Secondary end-points were favorable versus unfavorable outcome at 6 months, length of intensive care and hospital stay, and survival to hospital discharge. RESULTS: A total of 312 patients with severe TBI were randomly assigned to paramedic rapid sequence intubation or hospital intubation. The success rate for paramedic intubation was 97%. At 6 months, the median GOSe score was 5 (interquartile range, 1-6) in patients intubated by paramedics compared with 3 (interquartile range, 1-6) in the patients intubated at hospital (P = 0.28).The proportion of patients with favorable outcome (GOSe, 5-8) was 80 of 157 patients (51%) in the paramedic intubation group compared with 56 of 142 patients (39%) in the hospital intubation group (risk ratio, 1.28; 95% confidence interval, 1.00-1.64; P = 0.046). There were no differences in intensive care or hospital length of stay, or in survival to hospital discharge. CONCLUSIONS: In adults with severe TBI, prehospital rapid sequence intubation by paramedics increases the rate of favorable neurologic outcome at 6 months compared with intubation in the hospital.

An extensive genetic program occurring during postnatal growth in multiple tissues.

Mammalian somatic growth is rapid in early postnatal life but then slows and eventually ceases in multiple tissues. We hypothesized that there exists a postnatal gene expression program that is common to multiple tissues and is responsible for this coordinate growth deceleration. Consistent with this hypothesis, microarray analysis identified more than 1600 genes that were regulated with age (1 vs. 4 wk) coordinately in kidney, lung, and heart of male mice, including many genes that regulate proliferation. As examples, we focused on three growth-promoting genes, Igf2, Mest, and Peg3, that were markedly down-regulated with age. In situ hybridization revealed that expression occurred in organ-specific parenchymal cells and suggested that the decreasing expression with age was due primarily to decreased expression per cell rather than a decreased number of expressing cells. The declining expression of these genes was slowed during hypothyroidism and growth inhibition (induced by propylthiouracil at 0-5 wk of age) in male rats, suggesting that the normal decline in expression is driven by growth rather than by age per se. We conclude that there exists an extensive genetic program occurring during postnatal life. Many of the involved genes are regulated coordinately in multiple organs, including many genes that regulate cell proliferation. At least some of these are themselves apparently regulated by growth, suggesting that, in the embryo, a gene expression pattern is established that allows for rapid somatic growth of multiple tissues, but then, during postnatal life, this growth leads to negative-feedback changes in gene expression that in turn slow and eventually halt somatic growth, thus imposing a fundamental limit on adult body size.