IL-18-secreting CAR T cells targeting DLL3 are highly effective in small cell lung cancer models.

Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers, including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine IL-18 greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization, and activation of antigen-presenting cells. Additionally, human IL-18-secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion, and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti-PD-1 blockade. All together, these results define DLL3-targeting CAR T cells that produce IL-18 as a potentially promising novel strategy against DLL3-expressing solid tumors.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.

Medicaid payer status is associated with increased 90-day morbidity and resource utilization following primary shoulder arthroplasty: a propensity score-matched analysis.

BACKGROUND: Medicaid payer status has been shown to affect risk-adjusted patient outcomes and health care utilization across multiple medical specialties and orthopedic procedures. However, there is a paucity of data regarding the impact of Medicaid payer status on 90-day morbidity and resource utilization following primary shoulder arthroplasty (reverse total shoulder arthroplasty [rTSA], anatomic total shoulder arthroplasty [aTSA], and hemiarthroplasty [HA]). The purpose of this study was to examine 90-day readmission and reoperation rates, hospital length of stay (LOS), and direct cost following primary shoulder arthroplasty in the Medicaid population. METHODS: The National Readmission Database was queried for all patients undergoing primary aTSA, rTSA, and HA from 2011 to 2016. Medicaid or non-Medicaid payer status was determined. Patient demographic characteristics and comorbidities, along with 90-day readmission, 90-day reoperation, LOS, and inflation-adjusted cost, were queried. Propensity score matching was used to control for baseline differences in cohorts that could be acting as confounders in the exposure-outcome relationship. This was achieved with 1-to-1 propensity score matching between Medicaid and non-Medicaid patients. Odds ratios (ORs) and 95% confidence intervals (CIs) for 90-day readmission and reoperation rates were calculated, and a comparison of LOS and cost was performed between the propensity score-matched cohorts. RESULTS: A total of 4667 Medicaid and 161,147 non-Medicaid patients were identified from the 2011-2016 National Readmission Databases. Propensity score analysis was performed, and 4637 Medicaid patients were matched to 4637 non-Medicaid patients; each group comprised 1504 rTSAs (32.4%), 1934 aTSAs (41.7%), and 1199 HAs (25.9%). Patients with Medicaid payer status yielded significant increases in the 90-day all-cause readmission rate of 11.6% vs. 9.3% (P < .001; OR, 1.28 [95% CI, 1.12-1.46]), 90-day shoulder-related readmission rate of 3.3% vs. 2.3% (P = .004; OR, 1.44 [95% CI, 1.12-1.85]), and 90-day reoperation rate of 2.0% vs. 1.3% (P = .008; OR, 1.54 [95% CI, 1.12-1.94]). Furthermore, there was an increased risk of an extended LOS (ie, LOS > 2 days) (28.4% vs. 25.7%; P = .004; OR, 1.14 [95% CI, 1.04-1.25]) along with increased direct cost (median, $17,612 vs. $16,775; P < .001). DISCUSSION: This study demonstrates that Medicaid payer status is independently associated with increased 90-day readmission and reoperation rates, LOS, and direct cost following primary shoulder arthroplasty. Providers may have a disincentive to treat patient populations who require increased resource utilization following surgery. Risk adjustment models accounting for Medicaid payer status will be necessary to ensure good access to care for this patient population by avoiding penalties for physicians and hospital systems.

Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach Simultaneously Targeting BCMA and GPRC5D to Prevent BCMA Escape-Driven Relapse in Multiple Myeloma.

CAR T-cell therapy for multiple myeloma (MM) targeting B-cell maturation antigen (TNFRSF17; BCMA) induces high overall response rates; however, relapse occurs commonly. Implicated in relapse is a reservoir of MM if cells lacking sufficient BCMA surface expression (antigen escape). We demonstrate that simultaneous targeting of an additional antigen-here, G protein-coupled receptor class-C group-5 member-D (GPRC5D)-can prevent BCMA escape-mediated relapse in a model of MM. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include (1) parallel-produced and pooled mono-targeted CAR T-cells, (2) bicistronic constructs expressing distinct CARs from a single vector, and (3) a dual-scFv "single-stalk" CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen-expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions.

Quadriceps Strength Deficits After a Femoral Nerve Block Versus Adductor Canal Block for Anterior Cruciate Ligament Reconstruction: A Prospective, Single-Blinded, Randomized Trial.

BACKGROUND: Peripheral nerve blocks, particularly femoral nerve blocks (FNBs), are commonly performed for anterior cruciate ligament (ACL) reconstruction. However, associated quadriceps muscle weakness after FNBs is well described and may occur for up to 6 months postoperatively. The adductor canal block (ACB) has emerged as a viable alternative to the FNB, theoretically causing less quadriceps weakness during the immediate postoperative period, as it bypasses the majority of the motor fibers of the femoral nerve that branch off proximal to the adductor canal. PURPOSE/HYPOTHESIS: This study sought to identify if a difference in quadriceps strength exists after an ACB or FNB for ACL reconstruction beyond the immediate postoperative period. Beyond the immediate postoperative period, we anticipated no difference in quadriceps strength between patients who received ACBs or FNBs for ACL reconstruction. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A total of 102 patients undergoing primary ACL reconstruction using a variety of graft types were enrolled between November 2015 and April 2016. All patients were randomized to receive an ACB or FNB before surgery, and the surgeon was blinded to the block type. All patients underwent aggressive rehabilitation without functional bracing postoperatively. The time to the first straight-leg raise was reported by the patient. Isokinetic strength testing was performed at 3 and 6 months postoperatively. RESULTS: Data for 73 patients were analyzed. There was no significant difference in patient demographics of age, body mass index, sex, or tourniquet time between the FNB (n = 35) and ACB (n = 38) groups. The mean time to the first straight-leg raise was similar, at 13.1 ± 1.0 hours for the FNB group and 15.5 ± 1.2 hours for the ACB group (P = .134). The mean extension torque at 60 deg/s increased significantly for both the ACB (53.7% ± 3.4% to 68.3% ± 2.9%; P = .008) and the FNB (53.3% ± 3.3% to 68.5% ± 4.1%; P = .006) groups from 3 to 6 months postoperatively. There was also no significant difference in mean extension torque at 60 deg/s or 180 deg/s between the FNB and ACB groups at 3 and 6 months. There were no significant differences in postoperative complications (infection, arthrofibrosis, retear) between groups. CONCLUSION: Although prior studies have shown immediate postoperative benefits of ACBs compared with FNBs, with a faster return of quadriceps strength, in the current study there was no statistically or clinically significant difference in quadriceps strength at 3 and 6 months postoperatively in patients who received ACBs or FNBs for ACL reconstruction.

Red Flags for Low Back Pain Are Not Always Really Red: A Prospective Evaluation of the Clinical Utility of Commonly Used Screening Questions for Low Back Pain.

BACKGROUND: Low back pain has a high prevalence and morbidity, and is a source of substantial health-care spending. Numerous published guidelines support the use of so-called red flag questions to screen for serious pathology in patients with low back pain. This paper examines the effectiveness of red flag questions as a screening tool for patients presenting with low back pain to a multidisciplinary academic spine center. METHODS: We conducted a retrospective review of the cases of 9,940 patients with a chief complaint of low back pain. The patients completed a questionnaire that included several red flag questions during their first physician visit. Diagnostic data for the same clinical episode were collected from medical records and were corroborated with imaging reports. Patients who were diagnosed as having a vertebral fracture, malignancy, infection, or cauda equina syndrome were classified as having a red flag diagnosis. RESULTS: Specific individual red flags and combinations of red flags were associated with an increased probability of underlying serious spinal pathology, e.g., recent trauma and an age of >50 years were associated with vertebral fracture. The presence or absence of other red flags, such as night pain, was unrelated to any particular diagnosis. For instance, for patients with no recent history of infection and no fever, chills, or sweating, the presence of night pain was a false-positive finding for infection >96% of the time. In general, the absence of red flag responses did not meaningfully decrease the likelihood of a red flag diagnosis; 64% of patients with spinal malignancy had no associated red flags. CONCLUSIONS: While a positive response to a red flag question may indicate the presence of serious disease, a negative response to 1 or 2 red flag questions does not meaningfully decrease the likelihood of a red flag diagnosis. Clinicians should use caution when utilizing red flag questions as screening tools.

Side population analysis using a violet-excited cell-permeable DNA binding dye.

Hoechst 33342 side population (SP) analysis is a common method for identifying stem cells in mammalian hematopoietic and nonhematopoietic tissues. Although widely employed for stem cell analysis, this method requires an ultraviolet (UV) laser to excite Hoechst 33342. Flow cytometers equipped with UV sources are not common because of the cost of both the laser and optics that can transmit light UV light. Violet laser sources are inexpensive and are now common fixtures on flow cytometers, but have been previously shown to provide insufficient Hoechst dye excitation for consistent resolution of SP cells. One solution to this problem is to identify additional fluorescent substrates with the same pump specificity as Hoechst 33342, but with better violet excitation characteristics. DyeCycle Violet reagent has emission characteristics similar to those of Hoechst 33342, but with a longer wavelength excitation maxima (369 nm). When this dye is loaded into hematopoietic cells, a sharply resolved side population was also observed, similar in appearance to that seen with Hoechst 33342. Unlike Hoechst SP, DCV SP was similar in appearance with both violet and UV excitation. DCV SP could be inhibited fumitremorgin C, and showed the same membrane pump specificity as Hoechst 33342. Simultaneous immunophenotyping with stem cell markers in mouse bone marrow demonstrated that DCV SP was restricted to the stem cell lineage(-) Sca-1(+) c-kit(+) cells population, as is Hoechst SP. Pending confirmation by functional analysis of DCV SP cells, these results suggest that DCV efflux identified approximately the same stem cell population as did Hoechst 33342 efflux. Substituting DCV for Hoechst 33342 in the SP technique may, therefore, allow side population analysis on flow cytometers with violet lasers.

Proton magnetic resonance spectroscopy of brain tumors correlated with pathology.

RATIONALE AND OBJECTIVES: Evaluate proton magnetic resonance spectroscopy ((1)H-MRS) for assessing and grading brain tumors. MATERIALS AND METHODS: The research was done at Detroit Medical Center in a 1.5-T Siemens MR magnet using single-voxel or multivoxel MRS. This study consisted of 27 patients: 10 females and 17 males ages 22-83 years (average age 43.8). The data were recorded for three peaks-N-acetyl aspartate (NAA), choline (Cho), creatine (Cr)-which were used to calculate the ratios Cho/NAA and Cho/Cr. RESULTS: Abnormal spectra were seen in 25 patients and normal spectra in 2. In 16 patients with brain astrocytoma of various grades, the pathology grading was correlated with Cho/NAA and Cho/Cr. These values were 6.53 and 3.35 for nine patients with Grade 4 astrocytoma; 1.85 and 1.62 for three patients with Grade 3 astrocytoma; 2.21 and 1.50 for three patients with Grade 2 astrocytoma; and 1.45 and 1.49 for one patient with Grade 1 astrocytoma. The remaining nine patients with abnormal spectra were also correlated with pathology. CONCLUSION: MRS ratios can be used to differentiate malignant and nonmalignant lesions from normal brain tissue. In general, high-grade astrocytoma have higher Cho/NAA and Cho/Cr ratios compared with low-grade astrocytoma.