Phase II study of pembrolizumab and lenvatinib in advanced well-differentiated neuroendocrine tumors.

BACKGROUND: Immune checkpoint inhibitors (CPIs) have not been shown to be active in well-differentiated neuroendocrine tumors (NETs), with response rates <5%. Lenvatinib is a multitargeted tyrosine kinase inhibitor which binds to vascular endothelial growth factor and fibroblast growth factor receptors and has demonstrated efficacy in pancreatic and gastrointestinal NETs [44% and 16% objective radiographic response rate (ORR), respectively]. The combination of antiangiogenic and CPI therapies can be synergistic. We therefore evaluated the combination of lenvatinib and pembrolizumab in well-differentiated gastrointestinal (GI) and thoracic NETs. PATIENTS AND METHODS: A prospective, phase II trial evaluated patients with advanced GI/thoracic NETs (pancreatic NETs were excluded due to high response rate of lenvatinib monotherapy in this patient population), with evidence of progression within 8 months of study entry and at least two prior lines of systemic therapy. Patients received lenvatinib 20 mg daily and pembrolizumab 200 mg intravenously every 3 weeks until unacceptable toxicity or progression of disease. Primary endpoint was objective response rate, and an interim analysis was planned once 20 patients were enrolled. Four ORRs were required to continue enrollment. RESULTS: Twenty patients were enrolled on protocol from April 2021 to January 2022 (nine small intestine, five lung, two thymic, two unknown primary, one cecal, one presacral primaries). Two patients (10%) achieved a partial response (atypical lung and small intestinal primaries). Median progression-free survival (PFS) was 8 months (95% confidence interval 5.8-10.2 months). Twelve (60%) patients experienced probably or definitely associated grade 3 adverse events (10 hypertension). Fourteen patients (70%) required dose reductions or discontinued one of the medications. Two patients discontinued treatment before radiographic assessment. CONCLUSIONS: The combination of pembrolizumab and lenvatinib did not show sufficient response in patients with NETs to warrant continued enrollment on trial.

Efficacy of ipilimumab and nivolumab in patients with high-grade neuroendocrine neoplasms.

BACKGROUND: Dual checkpoint inhibitor therapy with anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated protein 4 therapy has shown promising results in patients with high-grade neuroendocrine neoplasms (NENs), demonstrating varying response rates of 9%-44%. More data are needed to evaluate the true response in a real-world cohort of patients. PATIENTS AND METHODS: We conducted a retrospective study of all patients with high-grade NENs treated at the Moffitt Cancer Center and Mayo Clinic between September 2017 and July 2020 who received combination therapy with ipilimumab and nivolumab. RESULTS: Thirty-four patients met the eligibility criteria. Patients had received an average of two prior lines of therapy, including at least one cytotoxic chemotherapy regimen. Twenty-seven (79.4%) patients had poorly differentiated neuroendocrine carcinomas, and seven (20.6%) had well-differentiated high-grade neuroendocrine tumors. The most common primary site (10, 29.4%) was pancreas; other primary sites of disease included colon (n = 5), endometrium (n = 3), anorectum (n = 2), esophagus (n = 2), cervix (n = 1), stomach (n = 1), small intestine (n = 1), and unknown primary (n = 9). Five patients (14.7%) exhibited a best response of partial response as per RECIST 1.1 criteria, 9 (26.5%) stable disease, and 17 (50%) progressive disease: 3 patients did not have a follow-up scan as they discontinued treatment shortly after initiation due to clinical progression. The objective response rate was 14.7%, and disease control rate was 41.2%. Median progression-free survival was 1 month [95% confidence interval (CI), 0.54-1.46 months]; median overall survival (OS) from time of treatment initiation was 5.0 months (95% CI, 4.07-5.93 months), and median OS from diagnosis was 14.0 months (95% CI, 11.79-16.21 months). The median duration of treatment was 1 month (range 0-10 months). Twenty-eight patients discontinued treatment for progression, four patients for toxicity, and two remain on treatment at the time of data cut-off. Twelve patients (35%) experienced grade 3 and 4 treatment-emergent toxicities. CONCLUSIONS: The ipilimumab and nivolumab regimen has modest activity in aggressive and heavily pretreated high-grade NENs who have progressed on prior cytotoxic chemotherapy.

Analysis of potential response predictors to capecitabine/temozolomide in metastatic pancreatic neuroendocrine tumors.

The capecitabine and temozolomide (CAPTEM) regimen is active in the treatment of metastatic pancreatic neuroendocrine tumors (pNETs), with response rates ranging from 30 to 70%. Small retrospective studies suggest that O(6)-methylguanine DNA methyltransferase (MGMT) deficiency predicts response to temozolomide. High tumor proliferative activity is also commonly perceived as a significant predictor of response to cytotoxic chemotherapy. It is unclear whether chromosomal instability (CIN), which correlates with alternative lengthening of telomeres (ALT), is a predictive factor. In this study, we evaluated 143 patients with advanced pNET who underwent treatment with CAPTEM for radiographic and biochemical response. MGMT expression (n=52), grade (n=128) and ALT activation (n=46) were investigated as potential predictive biomarkers. Treatment with CAPTEM was associated with an overall response rate (ORR) of 54% by RECIST 1.1. Response to CAPTEM was not influenced by MGMT expression, proliferative activity or ALT pathway activation. Based on these results, no biomarker-driven selection criteria for use of the CAPTEM regimen can be recommended at this time.

Pelvic squamous cell carcinoma of unknown primary: a case report and review of the literature.

Retroperitoneal squamous cell carcinoma (SCC) of unknown primary is very rare with variable survival rates. Standard optimal therapeutic management is not yet established.

A phase II study of axitinib in advanced neuroendocrine tumors.

Neuroendocrine tumors (NETs) are highly vascular neoplasms overexpressing vascular endothelial growth factor (VEGF) as well as VEGF receptors (VEGFR). Axitinib is a potent, selective inhibitor of VEGFR-1, -2 and -3, currently approved for the treatment of advanced renal cell carcinoma. We performed an open-label, two-stage design, phase II trial of axitinib 5mg twice daily in patients with progressive unresectable/metastatic low-to-intermediate grade carcinoid tumors. The primary end points were progression-free survival (PFS) and 12-month PFS rate. The secondary end points included time to treatment failure (TTF), overall survival (OS), overall radiographic response rate (ORR), biochemical response rate and safety. A total of 30 patients were enrolled and assessable for toxicity; 22 patients were assessable for response. After a median follow-up of 29months, we observed a median PFS of 26.7months (95% CI, 11.4-35.1), with a 12-month PFS rate of 74.5% (±10.2). The median OS was 45.3 months (95% CI, 24.4-45.3), and the median TTF was 9.6months (95% CI, 5.5-12). The best radiographic response was partial response (PR) in 1/30 (3%) and stable disease (SD) in 21/30 patients (70%); 8/30 patients (27%) were unevaluable due to early withdrawal due to toxicity. Hypertension was the most common toxicity that developed in 27 patients (90%). Grade 3/4 hypertension was recorded in 19 patients (63%), leading to treatment discontinuation in six patients (20%). Although axitinib appears to have an inhibitory effect on tumor growth in patients with advanced, progressive carcinoid tumors, the high rate of grade 3/4 hypertension may represent a potential impediment to its use in unselected patients.

Phase II clinical trial of pasireotide long-acting repeatable in patients with metastatic neuroendocrine tumors.

Pasireotide long-acting repeatable (LAR) is a novel somatostatin analog (SSA) with avid binding affinity to somatostatin receptor subtypes 1, 2, 3 (SSTR1,2,3) and 5 (SSTR5). Results from preclinical studies indicate that pasireotide can inhibit neuroendocrine tumor (NET) growth more robustly than octreotide in vitro. This open-label, phase II study assessed the clinical activity of pasireotide in treatment-naïve patients with metastatic grade 1 or 2 NETs. Patients with metastatic pancreatic and extra-pancreatic NETs were treated with pasireotide LAR (60 mg every 4 weeks). Previous systemic therapy, including octreotide and lanreotide, was not permitted. Tumor assessments were performed every 3 months using Response Evaluation Criteria in Solid Tumors (RECIST) criteria. The primary endpoint was progression-free survival (PFS). The secondary endpoints included overall survival (OS), overall radiographic response rate (ORR), and safety. Twenty-nine patients were treated with pasireotide LAR (60 mg every 4 weeks) and 28 were evaluable for response. The median PFS was 11 months. The most favorable effect was observed in patients with low hepatic tumor burden, normal baseline chromogranin A, and high tumoral SSTR5 expression. Median OS has not been reached; the 30-month OS rate was 70%. The best radiographic response was partial response in one patient (4%), stable disease in 17 patients (60%), and progressive disease in ten patients (36%). Although grade 3/4 toxicities were rare, pasireotide LAR treatment was associated with a 79% rate of hyperglycemia including 14% grade 3 hyperglycemia. Although pasireotide appears to be an effective antiproliferative agent in the treatment of advanced NETs, the high incidence of hyperglycemia raises concerns regarding its suitability as a first-line systemic agent in unselected patients. SSTR5 expression is a potentially predictive biomarker for response.

Measurement of muscle exudate protein composition as an indicator of beef tenderness.

UNLABELLED: The objective of this study was to determine the relationship between the protein composition of muscle exudates and meat tenderness in beef. Frozen, intact beef strip loins (n = 24) were each divided into 3 equal portions (anterior, middle, and posterior). Steaks were removed from each portion, individually vacuum packaged, thawed at 4 °C, and aged for 0, 7, or 14 d. After the designated aging period, exudate was collected from the packaging and 1 steak from each strip loin portion was utilized for shear force measurements. Muscle exudates were analyzed for protein content (biuret assay) and composition (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Shear force decreased (P < 0.0001) with aging from 0 to 14 d. The protein concentrations of the muscle exudates were not influenced by the aging period and were not related to the amount of exudate expressed. Electrophoretic analyses of the muscle exudates indicated that with aging the relative abundance of 4 proteins decreased (P < 0.01) and 10 proteins increased (P < 0.05) within the protein profiles of the exudates. The relative abundance of the 167, 97, and 47 kDa proteins in exudates at day 0 were significantly correlated (|r| = 0.57 to 0.77) to shear force at day 14. These data demonstrate that exudate protein composition changes with postmortem aging and beef tenderness. PRACTICAL APPLICATION: This research showed that the protein profiles of exudates that accumulate on the surface and in the packaging of beef change with meat aging and tenderness. These data suggest that muscle exudates may be a good source of protein markers that are useful in the development of rapid, noninvasive methodologies for predicting beef tenderness.

Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing.

UNLABELLED: Capillary electrophoresis (CE) and reversed-phase high performance liquid chromatography (RP-HPLC) analyses were utilized to detect differences in the sarcoplasmic protein fractions of beef strip loins subjected to aging and hydrodynamic pressure processing (HDP) treatments. At 48 h postmortem, strip loins (n = 12) were halved and subjected to control or HDP treatments. Following treatment, each half was divided into 3 portions which were aged for 0, 5, and 8 d. After each aging period, steaks were removed for Warner-Bratzler shear force (WBSF) analysis and for the extraction of sarcoplasmic proteins which were analyzed by CE and RP-HPLC. Aging by HDP interactions were not detected using either separation technique. With CE analysis, no HDP effects were observed; however, the relative peak area of 8 protein peaks ranging in size from 17 to >200 kDa were influenced by postmortem aging. Separation of proteins by RP-HPLC demonstrated that HDP influenced the relative size of 2 protein peaks while postmortem aging effects were observed in 6 peaks. Alterations in the sarcoplasmic protein fractions detected by both CE and RP-HPLC were correlated to WBSF measurements. Overall, data demonstrate that HDP has minimal effects on sarcoplasmic proteins and that aging related changes in the water soluble protein fractions of muscle may be useful as indirect indicators of beef tenderness. PRACTICAL APPLICATION: Using 2 different postmortem tenderization techniques, aging and hydrodynamic pressure processing, this study demonstrates that postmortem changes in the soluble protein fraction of beef may be useful as potential indicators of meat tenderness.

Use of gelatin gels as a reference material for performance evaluation of meat shear force measurements.

UNLABELLED: Establishing standards for meat tenderness based on Warner-Bratzler shear force (WBSF) is complicated by the lack of methods for certifying WBSF testing among texture systems or laboratories. The objective of this study was to determine the suitability of using gelatin gels as reference materials for performance testing of texture measurement systems. Three replications of 5 gels (15%, 20%, 25%, 30%, and 35% gelatin) were prepared, vacuum packaged, and stored at 4 °C until use. Three randomly selected strips from each gel were subjected to WBSF testing on 4 instruments (A, B, C, or D) on days 1 and 8. Additional strips from each gel were subjected to WBSF testing on instruments A and C on day 29. Regression line estimates for each set of gels were analyzed. Gel WBSF values ranged from 10 to 177 N. The WBSF by gel concentration response was highly linear (P<0.0001) for all replications, instruments, and days of analysis. R2-values across all sets of gels ranged from 0.9562 to 0.9998. On days 1 and 8, instruments A and D exhibited higher slope (P<0.0001) and lower intercept (P<0.0001) estimates than instruments B and C. Regression line parameters (slope, intercept, and R2-values) were not influenced (P>0.05) by length of gel storage (1, 8, and 29 d). Data from this study suggest that gelatin gels can be used for evaluating WBSF values from various instruments and for validating the performance of meat shear force testing. PRACTICAL APPLICATION: Validating the performance of meat shear force testing is vital to establishing meat tenderness standards. The gelatin gel standards developed in this study exhibited a highly linear, repeatable relationship with shear force and were found to be stable for at least a month. These gel standards would provide a tool for the meat industry to harmonize shear force measurements across laboratories and various texture measuring instruments.

Distribution of penicillin G residues in culled dairy cow muscles: implications for residue monitoring.

The U.S. Food and Drug Administration sets tolerances for veterinary drug residues in muscle but does not specify which type of muscle should be analyzed. To determine if antibiotic residue levels are dependent upon muscle type, seven culled dairy cows were dosed with penicillin G (Pen G) from 1 to 3 days and then sacrificed on day 1, 2, or 5 of withdrawal. A variety (9-15) of muscle samples were collected, along with liver and kidney samples. In addition, corresponding muscle juice samples were prepared. All samples were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine Pen G levels. Results showed that Pen G residue levels can vary between and within different muscles, although no reproducible pattern was identified between cows or withdrawal times. Muscle juice appeared to be a promising substitute for muscle as a matrix for screening purposes. Because of the potential for variation within muscles, all samples taken need to be large enough to be representative.

Cross-contamination of lettuce (Lactuca sativa L.) with Escherichia coli O157:H7 via contaminated ground beef.

A lettuce outbreak strain of E. coli O157:H7 was used to quantitate the pathogen's survival in ground beef and its transfer to hands, cutting board surfaces, and lettuce. Overnight storage of inoculated beef at 4 degrees C resulted in no pathogen growth, while room-temperature storage allowed multiplication. Hamburger patty formation allowed the transfer of bacteria to hands. Contaminated fingers subsequently transferred the pathogen to lettuce during handling. E. coli was transferred from hamburgers to cutting board surfaces; overnight storage of boards decreased the numbers of recoverable pathogens by approximately 1 log CFU. A 15-s water rinse failed to remove significant numbers of pathogens from cutting boards whether it was applied immediately after contamination or following overnight room-temperature storage. Three lettuce leaves were successively applied to a single contaminated cutting board area both immediately after contamination and after overnight room-temperature storage of contaminated boards. Another set of leaves was pressed onto boards immediately following contamination and was then stored overnight at 4 degrees C before pathogen enumeration. The numbers of pathogens transferred to the first pressed leaves were larger than those transferred to the second or third leaves. There were no significant differences in the numbers of pathogensrecovered from leaves pressed immediately after contamination whether pathogens were enumerated immediately or following overnight storage at 4 degrees C. However, fewer pathogens were transferred to leaves pressed to boards stored overnight at room temperature prior to contact with lettuce. Twenty-five lettuce pieces were successively pressed onto one area on a board containing 1.25 x 10(2) CFU of E. coli. Pathogens were transferred to 46% of the leaves, including the 25th exposed leaf.

Reduction of spoilage microorganisms in fresh beef using hydrodynamic pressure processing.

Hydrodynamic pressure processing (HDP) was investigated as a technology to reduce spoilage microorganisms found in fresh beef. In two separate studies (studies 1 and 2), retail ground beef and beef roasts were purchased (day 0). The roasts were divided into stew pieces (30 to 40 g). All meat samples, including control samples, were stored at 5 degrees C for 20 h in a plastic film. After storage, designated samples were treated with HDP In study 3, ground beef was treated with HDP (day 0) and stored aerobically (5 degrees C) for 14 days with control samples. Each meat type was vacuum-packaged for HDP (100 g binary explosive, steel shock wave container). The pHs and the aerobic plate counts (log10 CFU/g) were measured on day 0 (studies I and 2) and on days 0, 7, and 14 (study 3) for control samples and for HDP-treated samples. There was no pH difference between control and HDP-treated meat types (studies 1 and 2); HDP reduced bacteria in both meat types in study 1 (2 log) and study 2 (1.5 log) on day 0. In study 3, there was a significant difference (P < 0.05) in pH between control meat (8.2) and HDP-treated meat (5.6) after storage. There was an immediate reduction (1.5 log) of microorganisms following HDP (day 0) and a 4.5-log difference between control samples (9 log) and HDP-treated samples (4.5) after 14 days of storage. With HDP, it is possible to reduce spoilage microorganisms found in or on different meat types (ground beef versus stew pieces), which could extend the shelf life of meat products.

Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors.

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.

Evaluation of a transdermal fentanyl system in yucatan miniature pigs.

Of 18 pigs used on a coronary stent experimental protocol, 6 each received a transdermal fentanyl patch to document the patterns of transdermal fentanyl absorption in swine. This approach was taken to reduce animal use and potentially refine the surgical regimen. The objective of the fentanyl portion of the study was to demonstrate that transdermal fentanyl may be useful in the management of postoperative analgesia in swine. This study sought to document that demonstrable levels of fentanyl are achievable in swine plasma via a transdermal system and to compare the magnitude of these levels to data in other species. This study does not directly correlate plasma fentanyl levels with analgesic efficacy. Plasma fentanyl concentration peaked within 42 h in five pigs and within 48 h in the remaining pig. All pigs had similar absorption patterns; the only difference was in magnitude. One pig reached 0.99 ng/ml at 42 h; the next highest concentration was 0.77 ng/ml at 48 h in a different animal. The peak concentration in the others ranged from 0.38 to 0.71 ng/ml.

Anatomic modeling from unstructured samples using variational implicit surfaces.

We describe the use of variational implicit surfaces (level sets of an embedded generating function modeled using radial basis interpolants) in anatomic modeling. This technique allows the practitioner to employ sparsely and unevenly sampled data to represent complex biological surfaces, including data acquired as a series of non-parallel image slices. The method inherently accommodates interpolation across irregular spans. In addition, shapes with arbitrary topology are easily represented without interpolation or aliasing errors arising from discrete sampling. To demonstrate the medical use of variational implicit surfaces, we present the reconstruction of the inner surfaces of blood vessels from a series of endovascular ultrasound images.

Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2.

Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.

Reducing complications from alcohol use during pregnancy through screening.

Prenatal providers are reluctant to discuss alcohol use in the clinical setting, even though heavy alcohol use is associated with fetal alcohol syndrome (FAS) and fetal alcohol effects (FAE), sometimes known as alcohol-related neurodevelopmental disorder. Fourteen percent to 20% of pregnant women report drinking some alcohol during pregnancy. Approximately 0.2% to 1% meet the criteria for heavy drinking. Reducing drinking during pregnancy has the potential to reduce the risk for FAS and FAE. Routine screening for alcohol use during pregnancy followed by referrals for those considered to be at risk is recommended. Women are often more receptive to intervention during pregnancy, as they focus on positive health behaviors. A number of brief screening tools designed for use on a routine basis are reviewed. Physicians who learn to comfortably discuss alcohol use during pregnancy can help substantially reduce the impact of these disorders.

Production of 6-deoxy-13-cyclopropyl-erythromycin B by Saccharopolyspora erythraea NRRL 18643.

Cyclopropane carboxylic acid was fed to Saccharopolyspora erythraea NRRL 18643 (6-deoxyerythromycin producer), resulting in the production of 6-deoxy-13-cyclopropyl-erythromycin B. These studies provide further evidence that deoxyerythronolide B synthase has a relaxed specificity for the starter unit.

Evaluation of fibrin sealants in cutaneous wound closure.

Human fibrin sealant (HFS) and bovine fibrin sealant (BFS) were delivered as preformulated fibrinogen-thrombin mixtures that are light activated. These formulations were evaluated in the healing of incised cutaneous wounds in beagle dogs. Four groups were differentiated by sealant type and study duration with group: BFS for 10 days, HFS for 10 days, BFS for 30 days, and HFS for 30 days. Healing was evaluated by noting incidences of open wounds, laser Doppler perfusion imaging (LDPI), planimetry, breaking strength, and histopathology. In the absence of tension, both sealants tended to hold wound edges together; however, HFS tended to be better than its controls and BFS. Both sealants augmented suture closure, necessitating fewer sutures for wound closure. At 5 and 30 days BFS wounds had more perfusion than HFS wounds, indicating more inflammation. At 10 and 30 days BFS wounds had larger scar areas than their controls, while scar areas of HFS wounds were smaller than either BFS wounds or controls. Breaking strengths indicated that HFS wounds were stronger than their controls and BFS wounds. Histologically, mild to moderate chronic-active inflammation was observed in wounds receiving either sealant, and this persisted longer in BFS wounds. Overall, HFS had positive qualities, thus showing potential for functional and cosmetic wound closure.