Same-day post-therapy imaging with a new generation whole-body digital SPECT/CT in assessing treatment response to [177Lu]Lu-PSMA-617 in metastatic castration-resistant prostate cancer.

PURPOSE: Lutetium-177 [177Lu]Lu-PSMA-617 radioligand therapy (RLT) represents a significant advancement for metastatic castration-resistant prostate cancer (mCRPC), demonstrating improvements in radiographic progression free survival (rPFS) and overall survival (OS) with a low rate of associated side effects. Currently, most post-therapy SPECT/CT is conducted at 24 h after infusion. This study examines the clinical utility of a next-generation multi-detector Cadmium-Zinc-Telluride (CZT) SPECT/CT system (StarGuide) in same-day post-infusion assessment and early treatment response to [177Lu]Lu-PSMA-617. METHODS: In this retrospective study, 68 men with progressive mCRPC treated with [177Lu]Lu-PSMA-617 at our center from June 2022 to June 2023 were evaluated. Digital whole-body SPECT/CT imaging was performed after [177Lu]Lu-PSMA-617infusion (mean ± SD: 1.8 ± 0.6 h, range 1.1-4.9 h). Quantitative analysis of [177Lu]Lu-PSMA-617 positive lesions was performed in patients who underwent at least 2 post-therapy SPECT/CT, using liver parenchyma uptake as reference. Metrics including [177Lu]Lu-PSMA-617 positive total tumor volume (Lu-TTV), SUVmax and SUVmean were calculated. These quantitative metrics on post-infusion SPECT/CT images after cycles 1, 2 and 3 were correlated with overall survival (OS), prostate specific antigen-progression free survival (PSA-PFS) as defined by prostate cancer working group 3 (PCWG3), and PSA decrease over 50% (PSA50) response rates. RESULTS: 56 patients (means age 76.2 ± 8.1 years, range: 60-93) who underwent at least 2 post-therapy SPECT/CT were included in the image analysis. The whole-body SPECT/CT scans (~ 12 min per scan) were well tolerated, with 221 same-day scans performed (89%). At a median of 10-months follow-up, 33 (58.9%) patients achieved PSA50 after [177Lu]Lu-PSMA-617 treatment and median PSA-PFS was 5.0 months (range: 1.0-15 months) while median OS was not reached. Quantitative analysis of SPECT/CT images showed that 37 patients (66%) had > 30% reduction in Lu-TTV, associated with significantly improved overall survival (median not reached vs. 6 months, P = 0.008) and PSA-PFS (median 6 months vs. 1 months, P < 0.001). However, changes in SUVmax or SUVmean did not correlate with PSA-PFS or OS. CONCLUSION: We successfully implemented same-day post-therapy SPECT/CT after [177Lu]Lu-PSMA-617 infusions. Quantitation of 1-2 h post-therapy SPECT/CT images is a promising method for assessing treatment response. However, the approach is currently limited by its suboptimal detection of small tumor lesions and the necessity of incorporating a third-cycle SPECT/CT to mitigate the effects of any potential treatment-related flare-up. Further investigation in a larger patient cohort and prospective validation is essential to confirm these findings and to explore the role of SPECT/CT as a potential adjunct to PSMA PET/CT in managing mCRPC.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest.

BACKGROUND: Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. METHODS: To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. RESULTS: AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. CONCLUSION: VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

An enteroendocrine-microbial axis in the large intestine controls host metabolism.

Nutrient handling is an essential function of the gastrointestinal tract. Most nutrient absorption occurs in the small intestine and is coordinated by hormone-producing intestinal epithelial cells known as enteroendocrine cells (EECs)1. In contrast, the colon mostly reclaims water and electrolytes, and handles the influx of microbially-derived metabolites, including short chain fatty acids (SCFA)2-4. Hormonal responses of small intestinal EECs have been extensively studied but much less in known about the role of colonic EECs in metabolic regulation. To address this core question, we investigated a mouse model deficient in colonic EECs. We found that colonic EEC deficiency leads to hyperphagia and obesity. Surprisingly, colonic EEC deficiency results in altered microbiota composition and metabolism, which we found through antibiotic treatment and transfer to germ free recipients, to be both necessary and sufficient for the development of obesity. Moreover, studying stool and blood metabolomes, we found that differential glutamate production by intestinal microbiota corresponds to increase appetite due to EEC loss. Finally, we show that colonic glutamate administration can directly increase food intake and activate appetite centers in the central nervous system. These observations shed light on an unanticipated host-microbiota axis in the colon, part of a larger gut-brain axis, that regulates host metabolism and body weight.

The association of leukocyte immunoglobulin-like receptor subfamily B-4 expression in acute myeloid leukemia and central nervous system involvement.

Central nervous system (CNS) involvement in patients with acute myeloid leukemia (AML) varies, ranging from 0.6%-46%. Leukocyte immunoglobulin-like receptor B4 (LILRB4) has been shown to be critical in orchestration of infiltration of AML cells into the CNS in animal models, however it is unknown if an association exists between LILRB4 and CNS involvement (CNS+) in human patients with AML. LILRB4 was measured by flow cytometry in a heterogeneous population of fifty-six AML patients. Patients were then followed clinically for the development of CNS + . LILRB4 was positive in 91 % of patients with CNS + compared to 38 % without CNS involvement (p < 0.002). In logistic analysis: age, BMI, serum albumin and positive LILRB4 were predictive for CNS+ [OR, 95 % CI, p-value]: 0.95, 0.92-0.99, p < 0.01; 0.85, 0.73-0.998, p < 0.05; 0.23, 0.066-0.78, p < 0.02; 16.46, 1.93-140.2, p < 0.02, respectively. This finding of the association of LILRB4 with CNS + in combination with earlier findings suggests that LILRB4 has a mechanistic role in infiltration of the CNS and may provide insight into the pathogenesis of AML seeding the CNS. Moreover, this proof of concept and the findings in the present study may lead to the development of innovative and novel therapies to improve the lives of patients with AML.

Thrombin Generation in a patient with Triple Positive Antiphospholipid Syndrome Treated with Three Different Anticoagulants.

INTRODUCTION: Venous and arterial thrombosis is one of the hallmarks of Antiphospholipid Antibody Syndrome (APS). The traditional treatment for individuals with APS and venous thrombosis has been vitamin K antagonists. However, with the widespread use of direct oral anticoagulants (DOACs) there has been conflicting evidence regarding their safety and failure rate as alternatives to warfarin. Reasons for this failure remain elusive. We utilized the thrombin generation assay (TGA) to investigate the anticoagulation efficacy of three different agents in a patient with triple-positive APS to acquire a better understanding of the pathophysiology of APS. METHODS: Blood samples were obtained from a single patient with APS at five distinct time points while on three different anticoagulants: rivaroxaban, warfarin, and enoxaparin. The effects of these anticoagulants on TG potential were evaluated using the TGA. RESULTS: In the presence of thrombomodulin, rivaroxaban had the highest endogenous thrombin potential, thrombin peak, velocity index, and thrombin inactivation velocity (821.9 nMmin, 121.5 nM, 36.44 nM/min, 7.19 nM/min) when compared to warfarin (121-367 nMmin, 13.85-121.5 nM, 3.02-3.85 nM/min, 0.64-4.55 nM/min) and enoxaparin (242-378.8 nM min, 21.33-23.78 nM, 2.87-3.85 nM/min, 0.747-0.784 nM/min). This trend was also observed in the absence of thrombomodulin. CONCLUSIONS: These results suggest that patients with APS treated with rivaroxaban may be at greater risk for thrombosis compared to warfarin or enoxaparin. The findings may provide insight into the recent studies in patients with triple positive APS randomized to different anticoagulants demonstrating high rates of thrombosis with rivaroxaban. Further studies are necessary to elucidate the clinical significance.

The association of lymphotoxin-beta receptor with the subsequent diagnosis of incident gastrointestinal cancer: results from the Dallas Heart Study.

BACKGROUND: Lymphotoxin-beta receptor (LTßR) is an immunological protein associated with inflammation, and from preclinical studies is implicated in tumorigenesis. The epidemiological relationships with cancer are unknown, hence this study investigated their associations. METHODS: From a multiethnic population-based cohort, 3,032 participants without a prevalent cancer (a diagnosis prior to or within one year of enrollment) at baseline underwent measurement of plasma LTßR. These participants were followed for incident cancer using the Texas Cancer Registry (TCR). RESULTS: Over a median follow-up of 12.1 years, 178 participants developed incident cancer, of which 30 participants developed incident gastrointestinal (GI) cancer. Median plasma LTßR (1.10 vs. 1.00 ng/mL, P<0.02) levels were higher in individuals with overall incident cancer compared to those without cancer. After adjustments for age, sex, and race/ethnicity, these relationships were no longer significant. When analyses were stratified by cancer type, LTßR was positively associated with GI cancer after adjustments: HR, 95% CI per 1-standard deviation increase in concentration 2.64 (1.23-5.68), P=0.013. LTßR stratified by quartiles was significantly associated temporally with the risk of incident GI cancer, log-rank: P=0.011. The median interval to incident GI cancer diagnosis was 5.9 years. CONCLUSIONS: Increased plasma levels of LTßR are associated with the development of GI cancer. The antecedent findings years prior to a subsequent diagnosis of incident GI cancer suggest a role for LTßR in the pathogenesis of GI cancer. Further studies are needed to determine if LTßR can serve as an immune biomarker for GI cancer, in particular hepatocellular and colorectal cancers.

Docetaxel and mitoxantrone before radical prostatectomy in men with high-risk prostate cancer: 10-year follow-up and immune correlates.

The aims of this study were to report the clinical outcomes in a cohort of men with high-risk prostate cancer treated with neoadjuvant docetaxel and mitoxantrone 10 years after treatment, identify pretreatment clinical parameters that may be predictors of recurrence, and describe tumor-infiltrating leukocytes present in radical prostatectomy specimens. We conducted a phase I/II study of neoadjuvant docetaxel and mitoxantrone before radical prostatectomy in high-risk localized prostate cancer to determine the feasibility of this combination and predictors of prostate cancer recurrence after cytotoxic chemotherapy. After 10 years of follow-up, 34 (63%) of 54 participants experience a recurrence. In univariate analysis, prostate-specific antigen (PSA) density (P=0.01), pathological stage (P=0.03), lymph node status (P<0.0001), seminal vesicle invasion (P=0.003), and tissue vascular endothelial growth factor (VEGF) expression (P=0.016) were significantly associated with recurrence. In multivariate analysis, only lymph node status, PSA density, and VEGF expression were significant predictors of disease recurrence. We used a tissue microarray for the first 50 participants to characterize the tumor-infiltrating lymphocytes and evaluate them for association with recurrence. We measured CD3, CD4, CD8, FoxP3, CD20, CD15, CD68, and CD163 by immunohistochemistry in both tumor and normal prostate specimens, but did not find an association between immunophenotype and recurrence. There was a significantly different density of CD68 and CD163 cells between normal and tumor tissue. Lymph node status, PSA density, and tissue VEGF expression predict recurrence after chemotherapy for high-risk prostate cancer. Additional studies are needed to determine the potential benefit of chemotherapy in the neoadjuvant setting.

Discordant Mutations in Paired Primary and Metastatic Endometrial Adenocarcinomas Identified by Semiconductor-Based Sequencing for Rapid Cancer Genotyping.

Although endometrial adenocarcinoma is usually treated with surgery, patients with metastatic disease have a poor prognosis. To address the need for better treatment options, molecularly targeted drug therapies are being developed. These targeted therapies rely on accurate mutational profiling of the tumor, which is most often performed on DNA from the primary tumor. Our objective was to compare mutational concordance in primary tumors with their metastases. We genotyped 11 pairs of primary and metastatic endometrial adenocarcinomas using DNA from formalin-fixed paraffin-embedded tissue blocks and semiconductor-based next-generation sequencing. Five of these cases had multiple metastases for comparison. We sequenced 37 known cancer genes that are targets for new drug therapies. A total of 62 mutations were identified in 16 of these 37 genes. The most common mutations were in PIK3CA and PTEN. Overall, there was a 53% discordance in mutations between primary tumors and their paired (33 of 62). The absence of mutations in metastases (25 of 33, 76%) compared with the primary neoplasm was more common than gain of mutations (8 of 33, 24%). There was a 15% discordance rate between paired metastases within individuals (6 of 40), which was significantly less frequent than the rate between primary tumors and their metastases (Fisher exact P value <.0001). Although the sample size is relatively small, our data suggest it may be prudent to test metastases, rather than the primary neoplasm, when using molecularly targeted drug therapies, because isolated metastases may lack mutations detected in the heterogeneous mixture of the tumor's origin.

Biochemical stabilization of glucagon at alkaline pH.

BACKGROUND: For patients with type 1 diabetes mellitus, a bihormonal artificial endocrine pancreas system utilizing glucagon and insulin has been found to stabilize glycemic control. However, commercially available formulations of glucagon cannot currently be used in such systems because of physical instability characterized by aggregation and chemical degradation. Storing glucagon at pH 10 blocks protein aggregation but results in chemical degradation. Reductions in pH minimize chemical degradation, but even small reductions increase protein aggregation. We hypothesized that common pharmaceutical excipients accompanied by a new excipient would inhibit glucagon aggregation at an alkaline pH. METHODS AND RESULTS: As measured by tryptophan intrinsic fluorescence shift and optical density at 630 nm, protein aggregation was indeed minimized when glucagon was formulated with curcumin and albumin. This formulation also reduced chemical degradation, measured by liquid chromatography with mass spectrometry. Biological activity was retained after aging for 7 days in an in vitro cell-based bioassay and also in Yorkshire swine. CONCLUSIONS: Based on these findings, a formulation of glucagon stabilized with curcumin, polysorbate-80, l-methionine, and albumin at alkaline pH in glycine buffer may be suitable for extended use in a portable pump in the setting of a bihormonal artificial endocrine pancreas.

Mechanisms of glucagon degradation at alkaline pH.

Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.