Detection of copy number changes in DNA from formalin fixed paraffin embedded tissues using paralogue ratio tests.

The purpose of this study was to evaluate whether paralogue ratio tests (PRT) using real-time PCR can accurately determine the DNA copy number (CN) using formalin fixed paraffin embedded (FFPE) tissue. Histopathology diagnostic archives are an enormous resource of FFPE tissue, but extracted DNA is of poor quality and may be unsuitable for CN assessment, thus representing a missed opportunity for studies of genetic association and somatic change in cancer in large cohorts of easily accessible samples. Assays with paralogues on chromosomes 18 and 20 (18|20 PRT) and chromosomes 13 and X (13|X PRT) were tested using archived FFPE pathology samples with known CN, including tonsil, placentae, and FFPE melanoma cell lines. The assay proved accurate over the dynamic range from 1:1 to 1:3 and gave precise results when repeated four times over several weeks. The precision of the assay was marginally reduced once the CT value for 10 ng of FFPE DNA increased above 30 cycles, reflecting importance of DNA quality. The assays distinguished changes in CN ratio with high sensitivity and specificity. The 13|X PRT could detect cells with distinct genotypes microdissected from within the same FFPE sample. Therefore, PRTs are suitable for analyzing CN in FFPE tissues.

WNT5A expression increases during melanoma progression and correlates with outcome.

PURPOSE: Wnt ligands play a major role in development and are important in cancer. Expression microarray analysis correlates one member of this family, WNT5A, to a subclass of melanomas with increased motility and invasion. There are no large studies of clinical samples primarily addressing the importance of WNT5A in melanoma progression or outcome. Therefore, this study aimed to assess the protein expression of WNT5A during melanoma progression and its effect on outcome. EXPERIMENTAL DESIGN: Expression of WNT5A was determined in a series of 59 primary melanomas with matched metastases. To provide a benchmark of progression against which to assess WNT5A, expression of p16(ink4a) was analyzed, as this has been previously well documented in melanoma. The effect of WNT5A protein expression on outcome was assessed in 102 melanomas. RESULTS: Cytoplasmic WNT5A showed a trend of increasing expression with melanoma progression (P = 0.013), whereas there was diminishing p16(ink4a) expression (P = 0.006). Nevi showed relatively strong WNT5A expression. Strong cytoplasmic WNT5A was an independent risk factor for reduced metastasis-free and overall survival in multivariate analysis (P = 0.001 and 0.003, respectively). CONCLUSION: Cytoplasmic WNT5A increases with melanoma progression and strong expression is associated with poor outcome.

Cutaneous melanoma subtypes show different BRAF and NRAS mutation frequencies.

PURPOSE: BRAF mutations are present in two thirds of cutaneous melanomas and many of the rest have NRAS mutations. However, cutaneous melanoma is a heterogeneous disease with many clinicopathologic subtypes. Of these, the majority fits into four categories: superficial spreading, nodular, lentigo maligna, and acral lentiginous melanoma (ALM). Thus far, there is very limited data combining BRAF and NRAS mutation analysis to explore differences between cutaneous melanoma subtypes. The aim of this study was to address this issue. EXPERIMENTAL DESIGN: The frequency of BRAF and NRAS hotspot mutations, in exons 15 and 2, respectively, was assessed in 59 cutaneous melanomas comprising superficial spreading, nodular, lentigo maligna, and ALM using single-strand conformational polymorphism and RFLP-PCR analysis. RESULTS: Only 2 of 21 (9.5%) ALM showed BRAF exon 15 mutation compared with 9 of 14 (64.3%) superficial spreading malignant melanomas, 4 of 11 (36.4%) nodular melanomas, and 7 of 13 (53.4%) lentigo maligna melanomas (P < 0.01). However, our key finding is that the combined analysis of BRAF exon 15 and NRAS exon 2 showed that there were no significant differences in the overall mutation frequency between subtypes. In particular, 9 of 19 (47.4%) ALM without BRAF exon 15 mutation had an NRAS exon 2 mutation. CONCLUSIONS: We show that the overall BRAF/NRAS frequency in mutation hotspots is not significantly different among cutaneous melanoma subtypes. These data show that mitogen-activated protein kinase pathway activation may be important in all major subtypes of cutaneous melanoma, although the mechanism by which this is achieved varies.

MicroRNA-21 expression and its pathogenetic significance in cutaneous melanoma.

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expression but not with cytoplasmic PTEN expression. An anti-miR-21 preferentially altered nuclear PTEN in melanoma cell lines. The presence of a BRAF or NRAS mutation had no significant effect on miR-21 expression. These data suggest miR-21 may exert an oncogenic effect in melanoma by favouring redistribution of PTEN to the nucleus.

Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma.

Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR-based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%-91%) and a specificity of 88% (95% CI, 84%-92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed.

Improving patient educational literature: an understandable patient package insert for "the pill".

The objective of this project was to develop a simpler, more understandable, and accurate patient package insert (PPI) for inclusion in all packs of oral contraceptives (OCs). The project involved field-testing, using focus groups and semistructured cognitive interviews with family planning patients, a self-administered survey of clinic staff, and written recommendations from oral contraceptive and readability experts. The revision and field-testing of the PPI reduced its length by one third, lowered its reading level from the 10th to 1 2th grade down to 6th grade, included lay as well as medical terminology, and reorganized the information to make it easier to find and easier to use. The revised PPI, as submitted to the FDA in February 2001, could increase patient knowledge of safe and effective pill use and would be a valuable educational tool for providers of oral contraceptives. The FDA is currently updating the OC product labeling to reflect the most recent safety data and will then issue the labeling, including the field-tested patient package insert, for public comment.

Violence against women with physical disabilities.

This study explored risk factors for violence among a sample of adult women with physical disabilities. Fifty-six percent (100) of the 177 women participating in the study indicated a positive history of abuse. Of the women who reported abuse, most reported multiple abuse situations and abusers who were typically their male partners. In addition, only a small proportion of women sought and received adequate help. Women who indicated that they did not seek help were asked why this was the case. Their responses included: feeling that they could handle it themselves, having other sources of support available, being unaware of where to go, feeling embarrassed, feeling guilty about being a burden or that it was their fault, fear that abuser would come after them, fear of not being believed, and, to a lesser extent, concern that the shelter would lack appropriate accommodations. These findings highlight the importance of intervention strategies including advocacy activities for women with disabilities, activities with schools, activities to deter and prevent partner and caregiver violence, community awareness activities, and dissemination activities.

Accurate detection of copy number changes in DNA extracted from formalin-fixed, paraffin-embedded melanoma tissue using duplex ratio tests.

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.

Patient understanding and use of oral contraceptive pills in a southern public health family planning clinic.

OBJECTIVE: To assess patient understanding and use of oral contraceptive pills (OCPs) and determine if these are associated with literacy. METHODS: Four hundred OCP users from a southern public health family planning clinic were orally tested post visit for literacy, demographics, contraceptive knowledge, OCP use, side effects, and adherence. RESULTS: Patients were predominately African American (86%); 78% had completed high school and 42% read below a 9th grade level. Most (94%) understood what to do when they missed one pill, yet few knew the correct action to take after missing two or three pills (19% and 3% respectively); 33% reported missing one or more pills in the past 2 weeks. Literacy was not associated with OCP use, knowledge, or adherence. CONCLUSION: Patients of all literacy levels had limited understanding of OCP side effects and what to do about multiple missed pills. This puts them at risk for misuse.

High BRAF mutation frequency does not characterize all melanocytic tumor types.

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocytic tumors, the majority shows BRAF or NRAS mutation, but it is not known whether these are essential to all forms of melanocytic neoplasia. We screened 79 melanocytic tumors of different types for BRAF and NRAS mutations and looked at MAPK pathway activity using immunohistochemistry in a subset. Significant differences in BRAF exon 15 mutation frequency were found: 14/16 (87.5%) in common acquired naevi (CANs), 9/12 (75%) in CMs, 0/26 in Spitz naevi and 3/25 (12%) in blue naevi (p < 0.01). We looked at whether Spitz and blue naevi showed a compensatory increase in BRAF exon 11 and/or NRAS exons 1 and 2 mutations to account for the low BRAF exon 15 mutation frequency. NRAS mutations were found in only 1/16 (6.3%) Spitz naevi and 0/15 blue naevi. In addition, NRAS mutations were found in 2/11 (18.2%) CANs and 3/12 (25%) CMs. None of the tumors showed BRAF exon 11 mutations. Despite their low combined BRAF and NRAS mutation frequency, Spitz naevi showed strong MAPK pathway activation as measured by cytoplasmic expression of dually phosphorylated ERK1/2, while blue naevi had weak pathway activation. We conclude that BRAF and NRAS mutations are not necessary for melanocytic tumor development and that some types of tumor must arise by alternative mechanisms.

Cumplimiento con el régimen de anticonceptivos orales y su papel en la eficacia del método / Compliance with oral contraceptive regimens and its role in the efficacy

Más de 63 millones de mujeres en todo el mundo toman anticonceptivos orales, conocidos comúnmente como "la píldora". En los Estados Unidos, una de cada cuatro mujeres sexualmente activas y en edad reproductiva usa anticonceptivos orales, con un total de usuarias que excede los 13 millones. Sin embargo, a pesar del volumen de uso en los 30 años transcurridos desde la introducción de los anticonceptivos orales, se ha prestado poca atención al cumplimiento con el régimen y a su papel en la eficacia de dicho método.