Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.

Dexamethasone alone and in combination with desipramine, phenytoin, valproic acid or levetiracetam interferes with 5-ALA-mediated PpIX production and cellular retention in glioblastoma cells.

Extent of resection of glioblastoma (GBM) correlates with overall survival. Fluorescence-guided resection (FGR) using 5-aminolevulinic acid (5-ALA) can improve the extent of resection. Unfortunately not all patients given 5-ALA accumulate sufficient quantities of protoporphyrin IX (PpIX) for successful FGR. In this study, we investigated the effects of dexamethasone, desipramine, phenytoin, valproic acid, and levetiracetam on the production and accumulation of PpIX in U87MG cells. All of these drugs, except levetiracetam, reduce the total amount of PpIX produced by GBM cells (p < 0.05). When dexamethasone is mixed with another drug (desipramine, phenytoin, valproic acid or levetiracetam) the amount of PpIX produced is further decreased (p < 0.01). However, when cells are analyzed for PpIX cellular retention, dexamethasone accumulated significantly more PpIX than the vehicle control (p < 0.05). Cellular retention of PpIX was not different from controls in cells treated with dexamethasone plus desipramine, valproic acid or levetiracetam, but was significantly less for dexamethasone plus phenytoin (p < 0.01). These data suggest that medications given before and during surgery may interfere with PpIX accumulation in malignant cells. At this time, levetiracetam appears to be the best medication in its class (anticonvulsants) for patients undergoing 5-ALA-mediated FGR.

Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique.

Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.

Leptin promotes glioblastoma.

The hormone leptin has a variety of functions. Originally known for its role in satiety and weight loss, leptin more recently has been shown to augment tumor growth in a variety of cancers. Within gliomas, there is a correlation between tumor grade and tumor expression of leptin and its receptor. This suggests that autocrine signaling within the tumor microenvironment may promote the growth of high-grade gliomas. Leptin does this through stimulation of cellular pathways that are also advantageous for tumor growth and recurrence: antiapoptosis, proliferation, angiogenesis, and migration. Conversely, a loss of leptin expression attenuates tumor growth. In animal models of colon cancer and melanoma, a decline in the expression and secretion of leptin resulted in a reduction of tumor growth. In these models, positive mental stimulation through environmental enrichment decreased leptin secretion and improved tumor outcome. This review explores the link between leptin and glioblastoma.

Prevalence of HCV risk factors in Hispanic-American sub populations.

To assess the prevalence of HCV risk factors among Hispanic-American subpopulations in Philadelphia. Patients from four primary care practices in Philadelphia were enrolled. Demographics and HCV risk factors were ascertained using a self-administered questionnaire. Five hundred and three patients who identified themselves as Hispanic or Latino were included in the study. Approximately half were born in Puerto Rico or mainland US and the remaining participants were born in 19 other countries. One quarter or less of individuals born in these countries reported having a HCV risk factor. In comparison, 45% of individuals born in Puerto Rico and mainland US reported having a HCV risk factor. With each year individuals born outside the US live in the US, odds of having a risk factor increased by approximately 7% (P = 0.014). US born Hispanics are more likely to have a HCV risk factor than Hispanics born outside the US. Furthermore, the prevalence of risk factors increase among Hispanic immigrants after living in the US. These findings have a direct public health impact by providing rationale to focus HCV prevention efforts on recent immigrants.

Hepatitis C risk assessment, testing and referral for treatment in urban primary care: role of race and ethnicity.

AIM: To determine rates of hepatitis C (HCV) risk factor ascertainment, testing, and referral in urban primary care practices, with particular attention to the effect of race and ethnicity. METHODS: Retrospective chart review from four primary care sites in Philadelphia; two academic primary care practices and two community clinics was performed. Demographics, HCV risk factors, and other risk exposure information were collected. RESULTS: Four thousand four hundred and seven charts were reviewed. Providers documented histories of injection drug use (IDU) and transfusion for less than 20% and 5% of patients, respectively. Only 55% of patients who admitted IDU were tested for HCV. Overall, minorities were more likely to have information regarding a risk factor documented than their white counterparts (79% vs 68%, P < 0.0001). Hispanics were less likely to have a risk factor history documented, compared to blacks and whites (P < 0.0001). Overall, minorities were less likely to be tested for HCV than whites in the presence of a known risk factor (23% vs 35%, P = 0.004). Among patients without documentation of risk factors, blacks and Hispanics were more likely to be tested than whites (20% and 24%, vs 13%, P < 0.005, respectively). CONCLUSION: (1) Documentation of an HCV risk factor history in urban primary care is uncommon, (2) Racial differences exist with respect to HCV risk factor ascertainment and testing, (3) Minority patients, positive for HCV, are less likely to be referred for subspecialty care and treatment. Overall, minorities are less likely to be tested for HCV than whites in the presence of a known risk factor.

How to reach sexual minority youth in the health care setting: the teens offer guidance.

PURPOSE: To explore factors sexual minority youth believe make them feel safe in a health care setting. METHODS: Participants in three urban programs serving lesbian/gay/bisexual/transgendered and questioning (LGBTQ) youth engaged in a four-stage process to generate, prioritize, and explain their own ideas. In Stage III, 94 youth, aged 14 to 23 years, completed a survey comprised of the 34 highest rated items generated in earlier stages. Using a Likert scale, they answered, "How important are each of the following ideas in making you feel safe as an LGBTQ youth when you go for health care?" In Stage IV, youth discussed the results in focus groups. The Marginal Homogeneity Test divided the items into priority ranks and the Kruskal-Wallis test explored subgroup differences in item ratings. RESULTS: The 34 items were divided into six ranks. Five items shared the top rank: the clinician maintaining privacy, demonstrating cleanliness, offering respect, being well-educated, and being honest. The second rank was shared by the following: the clinician not talking down to patients, being a good listener, not downplaying patients' fears, being professional, holding a nonjudgmental stance of the LGBTQ lifestyle, and not assuming every LGBTQ youth has HIV. Interspersed among other ranks were items specific to the needs of sexual minority youth: the clinician not assuming LGBTQ sexual behavior was painful or dangerous; the clinician being educated about the gay lifestyle; clinician sensitivity to the needs of same-sex partners; staff sensitivity to the needs of closeted youth; having a choice of an LGBTQ provider; and the clinician not assuming heterosexuality. Youth who had not publicly disclosed their sexuality rated health information being offered in a private place higher (p =.01). CONCLUSIONS: LGBTQ youth value the same clinician characteristics desired by all adolescents: privacy, cleanliness, honesty, respect, competency, and a nonjudgmental stance. They clearly describe what attracts them (e.g., clinicians educated about their lifestyle) and what offends them (e.g., equating their sexuality with HIV). Clinicians need to achieve and convey a higher comfort level in addressing the special needs of sexual minority youth.