Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34⁺ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8⁺ and CD4⁺ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8⁺ and CD4⁺ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34⁺ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.     

Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets

In prostate cancer, multiple metastases from the same patient share similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns supporting their clonal origins. Whether actionable targets such as tyrosine kinases are also similarly expressed and activated in anatomically distinct metastatic lesions of the same patient is not known. We evaluated active kinases using phosphotyrosine peptide enrichment and quantitative mass spectrometry to identify druggable targets in metastatic castration-resistant prostate cancer obtained at rapid autopsy. We identified distinct phosphopeptide patterns in metastatic tissues compared with treatment-naive primary prostate tissue and prostate cancer cell line-derived xenografts. Evaluation of metastatic castration-resistant prostate cancer samples for tyrosine phosphorylation and upstream kinase targets revealed SRC, epidermal growth factor receptor (EGFR), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and MAPK1/3 and other activities while exhibiting intrapatient similarity and interpatient heterogeneity. Phosphoproteomic analyses and identification of kinase activation states in metastatic castration-resistant prostate cancer patients have allowed for the prioritization of kinases for further clinical evaluation.Mutational and copy number analyses from epithelial tumors have identified several activating tyrosine kinase mutations and amplifications, such as epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma and erythroblastic leukemia viral oncogene homolog 2 (ERBB2 or HER2/neu) gene amplification in breast cancer (1). The dependence on these tyrosine kinases for tumor growth and survival has led to successful clinical treatment with tyrosine kinase inhibitors (TKIs) (2, 3). However, recent genomic analyses of prostate adenocarcinoma revealed that activating tyrosine kinase mutations or amplifications are very rare (1, 4–6).Despite the scarcity of tyrosine kinase amplifications or activating mutations in prostate cancer, tyrosine kinase expression and activity has been shown to play an important role in disease progression. For example, coexpression of wild-type SRC tyrosine kinase and androgen receptor (AR) can synergistically drive the formation of mouse prostate adenocarcinoma (7). Evaluation of nontyrosine-kinase–initiated mouse models of prostate cancer further identified activation of the nonreceptor tyrosine kinases SRC, ABL1, and Janus kinase 2 (JAK2) (8). We also observed increased tyrosine phosphorylation in nearly 50% of castration-resistant prostate cancer (CRPC) tissues examined compared with hormone-naïve prostate cancer (8). These studies suggest that comprehensive evaluation of metastatic CRPC samples for tyrosine kinase activity may lead to the identification of new drug targets.Studies in melanoma and breast cancer have revealed that despite heterogeneity in primary, localized disease, metastases seem to arise from a single precursor cell (9, 10). The multifocal nature of organ-confined prostate cancer poses a question as to the clonality of metastatic disease (11). Investigation into clonality in metastatic CRPC has found that tumors isolated from anatomically different lesions in the same patient bear similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns from multiple metastatic lesions supporting their clonal origins (6, 12–14). In addition, these studies found a remarkable amount of interpatient heterogeneity, suggesting that personalized medicine approaches may be necessary to efficiently target metastatic lesions. Previous observations of intrapatient similarity hold promise with regard to treatment strategies for metastatic CRPC patients by means of systematically attacking the cancer cell clone contributing to disease.This led us to investigate whether actionable targets such as tyrosine kinases also maintain similar activation patterns across anatomically distinct metastases from the same patient. With access to rare metastatic CRPC tissue from the University of Michigan’s Rapid Autopsy Program (15), we evaluated global tyrosine phosphorylation patterns in lethal metastatic CRPC patients. Phosphotyrosine peptide enrichment and quantitative mass spectrometry (MS) identified diverse phosphorylation events in the metastatic tissues compared with naive primary prostate tissue and prostate cancer cell line-derived xenografts. Validation of activated kinases that were identified via either MS or kinase–substrate relationships revealed intrapatient similarity and interpatient heterogeneity across a large panel of targets. Interestingly, these kinase activities are a result not of mutation (6) but rather of pathway activation within the tumors themselves. In summary, the observation that similar tyrosine kinase activities are present in most if not all anatomically disparate metastatic lesions from the same patient reveals that (i) CRPC lesions may be clonal in origin and (ii) kinase activation patterns observed in these lesions should be prioritized for further evaluation as new targeted therapeutic strategies.     

The Diagnostic Use of Radioactive Isotopes

Dentists and their medical colleagues must approach old patients as they would younger ones. They should not accept loss of teeth as a physiologic process associated with aging but should strive to prevent dental caries and periodontal diseases, the major causes of loss of teeth.

Communities should provide equipment through hospitals or clinics for dental care of home bound patients. Even the edentulous patient should have regular oral examinations to prevent some of the deaths caused by oral cancer.     

Influence of Adiposity on Insulin Resistance and Glycemia Markers Among U.K. Children of South Asian,Black African-Caribbean,and White European Origin: Child Heart and Health Study in England

OBJECTIVE

Ethnic differences in type 2 diabetes risk between South Asians and white Europeans originate before adult life and are not fully explained by higher adiposity levels in South Asians. Although metabolic sensitivity to adiposity may differ between ethnic groups, this has been little studied in childhood. We have therefore examined the associations among adiposity, insulin resistance, and glycemia markers in children of different ethnic origins.

RESEARCH DESIGN AND METHODS

Cross-sectional study of 4,633 9- to 10-year-old children (response rate 68%) predominantly of South Asian, black African-Caribbean, and white European origin (n = 1,266, 1,176, and 1,109, respectively) who had homeostasis model assessments of insulin resistance (HOMA-IR), glycemia markers (HbA_1c and fasting glucose), and adiposity (BMI, waist circumference, skinfold thicknesses, and bioimpedance [fat mass]).

RESULTS

All adiposity measures were positively associated with HOMA-IR in all ethnic groups, but associations were stronger among South Asians compared to black African-Caribbeans and white Europeans. For a 1-SD increase in fat mass percentage, percentage differences in HOMA-IR were 37.5% (95% CI 33.3–41.7), 29.7% (25.8–33.8), and 27.0% (22.9–31.2), respectively (P interaction < 0.001). All adiposity markers were positively associated with HbA_1c in South Asians and black African-Caribbeans but not in white Europeans; for a 1-SD increase in fat mass percentage, percentage differences in HbA_1c were 0.04% (95% CI 0.03–0.06), 0.04% (0.02–0.05), and 0.02% (−0.00 to 0.04), respectively (P interaction < 0.001). Patterns for fasting glucose were less consistent.

CONCLUSIONS

South Asian children are more metabolically sensitive to adiposity. Early prevention or treatment of childhood obesity may be critical for type 2 diabetes prevention, especially in South Asians.The prevalence of type 2 diabetes has been rising, both in the U.K. population (1) and worldwide (2,3). In the U.K., there are marked ethnic differences in the risks of type 2 diabetes, which are particularly high among South Asians and to a lesser extent black African-Caribbeans (4); increased type 2 diabetes risks are also apparent in these ethnic groups in the U.S. (5,6). Recent evidence suggests that ethnic differences in risks of type 2 diabetes are apparent in childhood, with higher levels of insulin resistance (a key precursor of type 2 diabetes), glycated hemoglobin (HbA_1c), and (less consistently) fasting blood glucose concentrations observed in U.K. South Asians and to a lesser extent black African-Caribbeans compared with white Europeans by the age of 10 years (7).Excess body fat (adiposity) is an important independent risk factor for the development of type 2 diabetes and insulin resistance both in adults (8,9) and children (10). However, the role of adiposity in these ethnic differences in type 2 diabetes is complex. In South Asian adults, it is well-recognized that BMI underestimates adiposity (11) and that body fat levels (particularly of central body fat) are higher than those of white Europeans (10,12–14). However, in most studies, adjusting for the higher body fat levels (usually assessed using skinfold thickness or waist and hip circumferences) has not accounted for the higher risks of diabetes and insulin resistance observed in South Asians (13,15–17). It has also been observed that the risks of diabetes, insulin resistance, and cardiovascular disease emerge at lower levels of adiposity (particularly BMI) in South Asian populations and that their associations with adiposity are stronger than those in white Europeans (13,17). We have previously shown in U.K. South Asian children that higher insulin and HbA_1c levels do not appear to be explained by higher adiposity levels (7,18). In a preliminary study, we also showed that insulin resistance in U.K. South Asian children may be more sensitive to adiposity than in white Europeans (18). However, this latter observation needs to be substantiated in larger scale studies in which insulin resistance, glycemia marker levels, and a range of adiposity markers are assessed in children from the relevant ethnic groups.We have therefore examined the cross-sectional associations among adiposity, insulin resistance, and glycemia markers in a study of ∼5,000 children of South Asian and white European origin aged 9 to 10 years; we also report on associations among children of black African-Caribbean origin. We hypothesized that metabolic sensitivity to adiposity would be greater in South Asians than white Europeans. Several adiposity markers were assessed, including measures based on skinfold thickness and bioimpedance, which provide robust measures of adiposity in this multiethnic population (19), as well as BMI and waist circumference. We have also included data on leptin, an adipokine with circulating levels that are strongly correlated with total body fat percentage (20). Because other studies have suggested that lower lean mass may be an important determinant of insulin resistance in young South Asian men (21), we also report on the influence of fat-free mass (FFM) on insulin resistance and glycemia.     

Prognostic implications of microRNA-21 overexpression in pancreatic ductal adenocarcinoma: an international multicenter study of 686 patients

Despite progress in genomic characterization, no single prognostic marker that can be evaluated using an easy-to-perform and relatively inexpensive method is available for pancreatic ductal adenocarcinoma (PDAC). MicroRNAs, which are stable, tumor- and tissue-specific molecules, are potentially ideal biomarkers, and we established an inter-laboratory validated method to investigate miR-21 as a prognostic biomarker in PDAC. The study samples of PDAC patients were recruited from a test cohort of Glasgow (n = 189) and three validation cohorts of Pisa (n = 69), Sydney (n = 249), and International Cancer Genome Consortium (ICGC) (n = 249). Tissue microarrays were used for miR-21 staining by chromogenic in situ hybridization (CISH). The patients were subdivided into no/low and high miR-21 staining groups using a specific histoscore. Furthermore, miR-21 staining was evaluated against clinicopathological variables and follow-up data by Fisher/log-rank test and Cox proportional models. The prognostic variables found to be significant in univariate analysis (P value < 0.10) were included in multivariate analysis in a backward-stepwise fashion. MiR-21 expression was cytoplasmic, with more consistent staining in the malignant ductal epithelium than in the stroma. The expression of miR-21 was significantly associated with tumor size and lymph node metastasis, whereas no association was observed with other clinicopathological variables. High miR-21 staining (histoscore ≥ 45 [median score]) was an independent predictor of survival in the Glasgow test cohort (HR 2.37, 95% CI: 1.42-3.96, P < 0.0001) and three validation cohorts (Pisa, HR 2.03, 95% CI: 1.21-3.39, P = 0.007; Sydney, HR 2.58, 95% CI (1.21-3.39), P < 0.0001; and ICGC, HR 3.34, 95% CI: 2.07-5.84, P = 0.002) when adjusted for clinical variables in a multivariate model. In comparison to the patients with low miR-21, the patients with high miR-21 expression had significant increase in OS as they benefit from gemcitabine-based adjuvant chemotherapy (Glasgow 16.5 months [with chemotherapy] vs 10.5 months [without chemotherapy]); Sydney 25.0 vs 10.6; ICGC 25.2 vs 11.9. These results indicated that miR-21 is a predictor of survival, prompting prospective trials. Evaluation of miR-21 offers new opportunities for the stratification of patients with PDAC and might facilitate the implementation of clinical management and therapeutic interventions for this devastating disease.     

Variations in the first heart sound in complete A-V block

Four patients with complete atrioventricular block have been studied stethographically, and the loudness of the first heart sound related to the preceding P wave. Deductions have been drawn which emphasize the importance of vibrations of valvular origin in the production of the first heart sound.